anno_start	anno_end	anno_text	entity_type	sentence	section
17	34	Crystal Structure	evidence	Biochemistry and Crystal Structure of Ectoine Synthase: A Metal-Containing Member of the Cupin Superfamily	TITLE
38	54	Ectoine Synthase	protein_type	Biochemistry and Crystal Structure of Ectoine Synthase: A Metal-Containing Member of the Cupin Superfamily	TITLE
58	74	Metal-Containing	protein_state	Biochemistry and Crystal Structure of Ectoine Synthase: A Metal-Containing Member of the Cupin Superfamily	TITLE
89	106	Cupin Superfamily	protein_type	Biochemistry and Crystal Structure of Ectoine Synthase: A Metal-Containing Member of the Cupin Superfamily	TITLE
0	7	Ectoine	chemical	Ectoine is a compatible solute and chemical chaperone widely used by members of the Bacteria and a few Archaea to fend-off the detrimental effects of high external osmolarity on cellular physiology and growth.	ABSTRACT
84	92	Bacteria	taxonomy_domain	Ectoine is a compatible solute and chemical chaperone widely used by members of the Bacteria and a few Archaea to fend-off the detrimental effects of high external osmolarity on cellular physiology and growth.	ABSTRACT
103	110	Archaea	taxonomy_domain	Ectoine is a compatible solute and chemical chaperone widely used by members of the Bacteria and a few Archaea to fend-off the detrimental effects of high external osmolarity on cellular physiology and growth.	ABSTRACT
0	16	Ectoine synthase	protein_type	Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction.	ABSTRACT
18	22	EctC	protein_type	Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction.	ABSTRACT
51	58	ectoine	chemical	Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction.	ABSTRACT
117	157	N-gamma-acetyl-L-2,4-diaminobutyric acid	chemical	Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction.	ABSTRACT
168	173	water	chemical	Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction.	ABSTRACT
13	30	crystal structure	evidence	However, the crystal structure of ectoine synthase is not known and a clear understanding of how its fold contributes to enzyme activity is thus lacking.	ABSTRACT
34	50	ectoine synthase	protein_type	However, the crystal structure of ectoine synthase is not known and a clear understanding of how its fold contributes to enzyme activity is thus lacking.	ABSTRACT
10	26	ectoine synthase	protein_type	Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form.	ABSTRACT
49	65	marine bacterium	taxonomy_domain	Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form.	ABSTRACT
66	89	Sphingopyxis alaskensis	species	Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form.	ABSTRACT
91	93	Sa	species	Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form.	ABSTRACT
163	167	EctC	protein	Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form.	ABSTRACT
199	216	crystal structure	evidence	Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form.	ABSTRACT
224	227	apo	protein_state	Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form.	ABSTRACT
0	19	Structural analysis	experimental_method	Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily.	ABSTRACT
36	38	Sa	species	Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily.	ABSTRACT
39	43	EctC	protein	Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily.	ABSTRACT
71	88	cupin superfamily	protein_type	Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily.	ABSTRACT
0	4	EctC	protein	EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure.	ABSTRACT
13	18	dimer	oligomeric_state	EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure.	ABSTRACT
26	38	head-to-tail	protein_state	EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure.	ABSTRACT
80	97	crystal structure	evidence	EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure.	ABSTRACT
4	13	interface	site	The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer.	ABSTRACT
21	26	dimer	oligomeric_state	The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer.	ABSTRACT
81	105	hydrophobic interactions	bond_interaction	The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer.	ABSTRACT
122	133	beta-sheets	structure_element	The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer.	ABSTRACT
146	153	monomer	oligomeric_state	The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer.	ABSTRACT
32	48	ectoine synthase	protein_type	We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme.	ABSTRACT
83	88	metal	chemical	We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme.	ABSTRACT
100	146	metal depletion and reconstitution experiments	experimental_method	We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme.	ABSTRACT
160	164	EctC	protein	We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme.	ABSTRACT
180	194	iron-dependent	protein_state	We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme.	ABSTRACT
14	18	EctC	protein	We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency.	ABSTRACT
71	111	N-gamma-acetyl-L-2,4-diaminobutyric acid	chemical	We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency.	ABSTRACT
117	124	ectoine	chemical	We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency.	ABSTRACT
199	239	N-alpha-acetyl-L-2,4-diaminobutyric acid	chemical	We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency.	ABSTRACT
292	312	catalytic efficiency	evidence	We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency.	ABSTRACT
0	42	Structure-guided site-directed mutagenesis	experimental_method	Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants.	ABSTRACT
94	125	evolutionarily highly conserved	protein_state	Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants.	ABSTRACT
145	164	EctC protein family	protein_type	Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants.	ABSTRACT
206	223	iron-binding site	site	Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants.	ABSTRACT
296	300	EctC	protein	Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants.	ABSTRACT
37	41	iron	chemical	An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel.	ABSTRACT
130	141	active site	site	An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel.	ABSTRACT
176	180	EctC	protein	An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel.	ABSTRACT
181	193	cupin barrel	structure_element	An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel.	ABSTRACT
0	7	Ectoine	chemical	Ectoine [(S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] and its derivative 5-hydroxyectoine [(4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] are such compatible solutes.	INTRO
9	68	(S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid	chemical	Ectoine [(S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] and its derivative 5-hydroxyectoine [(4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] are such compatible solutes.	INTRO
89	105	5-hydroxyectoine	chemical	Ectoine [(S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] and its derivative 5-hydroxyectoine [(4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] are such compatible solutes.	INTRO
107	180	(4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid	chemical	Ectoine [(S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] and its derivative 5-hydroxyectoine [(4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] are such compatible solutes.	INTRO
5	42	marine and terrestrial microorganisms	taxonomy_domain	Both marine and terrestrial microorganisms produce them widely in response to osmotic or temperature stress.	INTRO
13	20	ectoine	chemical	Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1).	INTRO
61	87	L-aspartate-ß-semialdehyde	chemical	Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1).	INTRO
146	180	L-2,4-diaminobutyrate transaminase	protein_type	Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1).	INTRO
182	186	EctB	protein_type	Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1).	INTRO
202	239	2,4-diaminobutyrate acetyltransferase	protein_type	Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1).	INTRO
241	245	EctA	protein_type	Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1).	INTRO
266	282	ectoine synthase	protein_type	Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1).	INTRO
284	288	EctC	protein_type	Synthesis of ectoine occurs from the intermediate metabolite L-aspartate-ß-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB; EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), and ectoine synthase (EctC; EC 4.2.1.108) (Fig 1).	INTRO
4	11	ectoine	chemical	The ectoine derivative 5-hydroxyectoine, a highly effective stress protectant in its own right, is synthesized by a substantial subgroup of the ectoine producers.	INTRO
23	39	5-hydroxyectoine	chemical	The ectoine derivative 5-hydroxyectoine, a highly effective stress protectant in its own right, is synthesized by a substantial subgroup of the ectoine producers.	INTRO
144	151	ectoine	chemical	The ectoine derivative 5-hydroxyectoine, a highly effective stress protectant in its own right, is synthesized by a substantial subgroup of the ectoine producers.	INTRO
45	52	ectoine	chemical	This stereospecific chemical modification of ectoine (Fig 1) is catalyzed by the ectoine hydroxylase (EctD) (EC 1.14.11), a member of the non-heme containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily.	INTRO
81	100	ectoine hydroxylase	protein_type	This stereospecific chemical modification of ectoine (Fig 1) is catalyzed by the ectoine hydroxylase (EctD) (EC 1.14.11), a member of the non-heme containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily.	INTRO
102	106	EctD	protein_type	This stereospecific chemical modification of ectoine (Fig 1) is catalyzed by the ectoine hydroxylase (EctD) (EC 1.14.11), a member of the non-heme containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily.	INTRO
138	219	non-heme containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily	protein_type	This stereospecific chemical modification of ectoine (Fig 1) is catalyzed by the ectoine hydroxylase (EctD) (EC 1.14.11), a member of the non-heme containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily.	INTRO
46	54	ectoines	chemical	The remarkable function preserving effects of ectoines for macromolecules and cells, frequently also addressed as chemical chaperones, led to a substantial interest in exploiting these compounds for biotechnological purposes and medical applications.	INTRO
24	31	ectoine	chemical	Biosynthetic routes for ectoine and 5-hydroxyectoine.	FIG
36	52	5-hydroxyectoine	chemical	Biosynthetic routes for ectoine and 5-hydroxyectoine.	FIG
14	21	ectoine	chemical	Scheme of the ectoine and 5-hydroxyectoine biosynthetic pathway.	FIG
26	42	5-hydroxyectoine	chemical	Scheme of the ectoine and 5-hydroxyectoine biosynthetic pathway.	FIG
17	33	ectoine synthase	protein_type	Here we focus on ectoine synthase (EctC), the key enzyme of the ectoine biosynthetic route (Fig 1).	INTRO
35	39	EctC	protein	Here we focus on ectoine synthase (EctC), the key enzyme of the ectoine biosynthetic route (Fig 1).	INTRO
64	71	ectoine	chemical	Here we focus on ectoine synthase (EctC), the key enzyme of the ectoine biosynthetic route (Fig 1).	INTRO
12	29	characterizations	experimental_method	Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out.	INTRO
33	50	ectoine synthases	protein_type	Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out.	INTRO
60	73	extremophiles	taxonomy_domain	Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out.	INTRO
74	92	Halomonas elongata	species	Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out.	INTRO
94	123	Methylomicrobium alcaliphilum	species	Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out.	INTRO
129	149	Acidiphilium cryptum	species	Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out.	INTRO
164	183	nitrifying archaeon	taxonomy_domain	Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out.	INTRO
184	208	Nitrosopumilus maritimus	species	Biochemical characterizations of ectoine synthases from the extremophiles Halomonas elongata, Methylomicrobium alcaliphilum, and Acidiphilium cryptum, and from the nitrifying archaeon Nitrosopumilus maritimus have been carried out.	INTRO
74	110	N-γ-acetyl-L-2,4-diaminobutyric acid	chemical	Each of these enzymes catalyzes as their main activity the cyclization of N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA), the reaction product of the 2,4-diaminobutyrate acetyltransferase (EctA), to ectoine with the concomitant release of a water molecule (Fig 1).	INTRO
112	121	N-γ-ADABA	chemical	Each of these enzymes catalyzes as their main activity the cyclization of N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA), the reaction product of the 2,4-diaminobutyrate acetyltransferase (EctA), to ectoine with the concomitant release of a water molecule (Fig 1).	INTRO
152	189	2,4-diaminobutyrate acetyltransferase	protein_type	Each of these enzymes catalyzes as their main activity the cyclization of N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA), the reaction product of the 2,4-diaminobutyrate acetyltransferase (EctA), to ectoine with the concomitant release of a water molecule (Fig 1).	INTRO
191	195	EctA	protein_type	Each of these enzymes catalyzes as their main activity the cyclization of N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA), the reaction product of the 2,4-diaminobutyrate acetyltransferase (EctA), to ectoine with the concomitant release of a water molecule (Fig 1).	INTRO
201	208	ectoine	chemical	Each of these enzymes catalyzes as their main activity the cyclization of N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA), the reaction product of the 2,4-diaminobutyrate acetyltransferase (EctA), to ectoine with the concomitant release of a water molecule (Fig 1).	INTRO
243	248	water	chemical	Each of these enzymes catalyzes as their main activity the cyclization of N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA), the reaction product of the 2,4-diaminobutyrate acetyltransferase (EctA), to ectoine with the concomitant release of a water molecule (Fig 1).	INTRO
19	23	EctC	protein	In side reactions, EctC can promote the formation of the synthetic compatible solute 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC) through the cyclic condensation of two glutamine molecules and it also possesses a minor hydrolytic activity for ectoine and synthetic ectoine derivatives with either reduced or expanded ring sizes.	INTRO
85	129	5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate	chemical	In side reactions, EctC can promote the formation of the synthetic compatible solute 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC) through the cyclic condensation of two glutamine molecules and it also possesses a minor hydrolytic activity for ectoine and synthetic ectoine derivatives with either reduced or expanded ring sizes.	INTRO
131	135	ADPC	chemical	In side reactions, EctC can promote the formation of the synthetic compatible solute 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC) through the cyclic condensation of two glutamine molecules and it also possesses a minor hydrolytic activity for ectoine and synthetic ectoine derivatives with either reduced or expanded ring sizes.	INTRO
176	185	glutamine	chemical	In side reactions, EctC can promote the formation of the synthetic compatible solute 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC) through the cyclic condensation of two glutamine molecules and it also possesses a minor hydrolytic activity for ectoine and synthetic ectoine derivatives with either reduced or expanded ring sizes.	INTRO
250	257	ectoine	chemical	In side reactions, EctC can promote the formation of the synthetic compatible solute 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC) through the cyclic condensation of two glutamine molecules and it also possesses a minor hydrolytic activity for ectoine and synthetic ectoine derivatives with either reduced or expanded ring sizes.	INTRO
272	279	ectoine	chemical	In side reactions, EctC can promote the formation of the synthetic compatible solute 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC) through the cyclic condensation of two glutamine molecules and it also possesses a minor hydrolytic activity for ectoine and synthetic ectoine derivatives with either reduced or expanded ring sizes.	INTRO
84	100	ectoine synthase	protein_type	Although progress has been made with respect to the biochemical characterization of ectoine synthase, a clear understanding of how its structure contributes to its enzyme activity and reaction mechanism is still lacking. With this in mind, we have biochemically characterized the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa).	INTRO
135	144	structure	evidence	Although progress has been made with respect to the biochemical characterization of ectoine synthase, a clear understanding of how its structure contributes to its enzyme activity and reaction mechanism is still lacking. With this in mind, we have biochemically characterized the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa).	INTRO
248	275	biochemically characterized	experimental_method	Although progress has been made with respect to the biochemical characterization of ectoine synthase, a clear understanding of how its structure contributes to its enzyme activity and reaction mechanism is still lacking. With this in mind, we have biochemically characterized the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa).	INTRO
280	296	ectoine synthase	protein_type	Although progress has been made with respect to the biochemical characterization of ectoine synthase, a clear understanding of how its structure contributes to its enzyme activity and reaction mechanism is still lacking. With this in mind, we have biochemically characterized the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa).	INTRO
319	335	marine bacterium	taxonomy_domain	Although progress has been made with respect to the biochemical characterization of ectoine synthase, a clear understanding of how its structure contributes to its enzyme activity and reaction mechanism is still lacking. With this in mind, we have biochemically characterized the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa).	INTRO
336	359	Sphingopyxis alaskensis	species	Although progress has been made with respect to the biochemical characterization of ectoine synthase, a clear understanding of how its structure contributes to its enzyme activity and reaction mechanism is still lacking. With this in mind, we have biochemically characterized the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa).	INTRO
361	363	Sa	species	Although progress has been made with respect to the biochemical characterization of ectoine synthase, a clear understanding of how its structure contributes to its enzyme activity and reaction mechanism is still lacking. With this in mind, we have biochemically characterized the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa).	INTRO
48	64	ectoine synthase	protein_type	We demonstrate here for the first time that the ectoine synthase is a metal-dependent enzyme, with iron as the most likely physiologically relevant co-factor.	INTRO
70	75	metal	chemical	We demonstrate here for the first time that the ectoine synthase is a metal-dependent enzyme, with iron as the most likely physiologically relevant co-factor.	INTRO
99	103	iron	chemical	We demonstrate here for the first time that the ectoine synthase is a metal-dependent enzyme, with iron as the most likely physiologically relevant co-factor.	INTRO
4	8	EctC	protein	The EctC protein forms a dimer in solution and our structural analysis identifies it as a member of the cupin superfamily.	INTRO
25	30	dimer	oligomeric_state	The EctC protein forms a dimer in solution and our structural analysis identifies it as a member of the cupin superfamily.	INTRO
51	70	structural analysis	experimental_method	The EctC protein forms a dimer in solution and our structural analysis identifies it as a member of the cupin superfamily.	INTRO
104	121	cupin superfamily	protein_type	The EctC protein forms a dimer in solution and our structural analysis identifies it as a member of the cupin superfamily.	INTRO
8	26	crystal structures	evidence	The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase.	INTRO
56	58	Sa	species	The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase.	INTRO
59	63	EctC	protein	The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase.	INTRO
157	182	site-directed mutagenesis	experimental_method	The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase.	INTRO
205	236	evolutionarily highly conserved	protein_state	The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase.	INTRO
266	278	EctC protein	protein_type	The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase.	INTRO
337	351	catalytic core	site	The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase.	INTRO
359	375	ectoine synthase	protein_type	The two crystal structures that we report here for the (Sa)EctC protein (with resolutions of 1.2 Å and 2.0 Å, respectively), and data derived from extensive site-directed mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase.	INTRO
0	14	Overproduction	experimental_method	Overproduction, purification and oligomeric state of the ectoine synthase in solution	RESULTS
16	28	purification	experimental_method	Overproduction, purification and oligomeric state of the ectoine synthase in solution	RESULTS
57	73	ectoine synthase	protein_type	Overproduction, purification and oligomeric state of the ectoine synthase in solution	RESULTS
15	49	biochemical and structural studies	experimental_method	We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product.	RESULTS
57	73	ectoine synthase	protein_type	We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product.	RESULTS
79	92	S. alaskensis	species	We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product.	RESULTS
95	97	Sa	species	We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product.	RESULTS
98	102	EctC	protein	We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product.	RESULTS
120	147	marine ultra-microbacterium	taxonomy_domain	We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product.	RESULTS
192	209	crystal structure	evidence	We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product.	RESULTS
217	236	ectoine hydroxylase	protein_type	We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product.	RESULTS
238	242	EctD	protein_type	We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product.	RESULTS
244	259	in complex with	protein_state	We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium, from which we recently also determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product.	RESULTS
46	59	S. alaskensis	species	We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography.	RESULTS
60	64	ectC	gene	We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography.	RESULTS
73	80	E. coli	species	We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography.	RESULTS
149	178	Strep-tag II affinity peptide	experimental_method	We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography.	RESULTS
209	211	Sa	species	We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography.	RESULTS
212	216	EctC	protein	We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography.	RESULTS
217	229	Strep-Tag-II	experimental_method	We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography.	RESULTS
241	264	affinity chromatography	experimental_method	We expressed a codon-optimized version of the S. alaskensis ectC gene in E. coli to produce a recombinant protein with a carboxy-terminally attached Strep-tag II affinity peptide to allow purification of the (Sa)EctC-Strep-Tag-II protein by affinity chromatography.	RESULTS
5	7	Sa	species	The (Sa)EctC protein was overproduced and isolated with good yields (30–40 mg L-1 of culture) and purity (S2a Fig).	RESULTS
8	12	EctC	protein	The (Sa)EctC protein was overproduced and isolated with good yields (30–40 mg L-1 of culture) and purity (S2a Fig).	RESULTS
13	42	size-exclusion chromatography	experimental_method	Conventional size-exclusion chromatography (SEC) has already shown that (Sa)EctC preparations produced in this fashion are homogeneous and that the protein forms dimers in solution.	RESULTS
44	47	SEC	experimental_method	Conventional size-exclusion chromatography (SEC) has already shown that (Sa)EctC preparations produced in this fashion are homogeneous and that the protein forms dimers in solution.	RESULTS
73	75	Sa	species	Conventional size-exclusion chromatography (SEC) has already shown that (Sa)EctC preparations produced in this fashion are homogeneous and that the protein forms dimers in solution.	RESULTS
76	80	EctC	protein	Conventional size-exclusion chromatography (SEC) has already shown that (Sa)EctC preparations produced in this fashion are homogeneous and that the protein forms dimers in solution.	RESULTS
162	168	dimers	oligomeric_state	Conventional size-exclusion chromatography (SEC) has already shown that (Sa)EctC preparations produced in this fashion are homogeneous and that the protein forms dimers in solution.	RESULTS
0	38	High performance liquid chromatography	experimental_method	High performance liquid chromatography coupled with multi-angle light-scattering detection (HPLC-MALS) experiments carried out here confirmed that the purified (Sa)EctC protein was mono-disperse and possessed a molecular mass of 33.0 ± 2.3 kDa (S2b Fig).	RESULTS
52	90	multi-angle light-scattering detection	experimental_method	High performance liquid chromatography coupled with multi-angle light-scattering detection (HPLC-MALS) experiments carried out here confirmed that the purified (Sa)EctC protein was mono-disperse and possessed a molecular mass of 33.0 ± 2.3 kDa (S2b Fig).	RESULTS
92	101	HPLC-MALS	experimental_method	High performance liquid chromatography coupled with multi-angle light-scattering detection (HPLC-MALS) experiments carried out here confirmed that the purified (Sa)EctC protein was mono-disperse and possessed a molecular mass of 33.0 ± 2.3 kDa (S2b Fig).	RESULTS
161	163	Sa	species	High performance liquid chromatography coupled with multi-angle light-scattering detection (HPLC-MALS) experiments carried out here confirmed that the purified (Sa)EctC protein was mono-disperse and possessed a molecular mass of 33.0 ± 2.3 kDa (S2b Fig).	RESULTS
164	168	EctC	protein	High performance liquid chromatography coupled with multi-angle light-scattering detection (HPLC-MALS) experiments carried out here confirmed that the purified (Sa)EctC protein was mono-disperse and possessed a molecular mass of 33.0 ± 2.3 kDa (S2b Fig).	RESULTS
89	91	Sa	species	This value corresponds very well with the theoretically calculated molecular mass of an (Sa)EctC dimer (molecular mass of the monomer, including the Strep-tag II affinity peptide: 16.3 kDa).	RESULTS
92	96	EctC	protein	This value corresponds very well with the theoretically calculated molecular mass of an (Sa)EctC dimer (molecular mass of the monomer, including the Strep-tag II affinity peptide: 16.3 kDa).	RESULTS
97	102	dimer	oligomeric_state	This value corresponds very well with the theoretically calculated molecular mass of an (Sa)EctC dimer (molecular mass of the monomer, including the Strep-tag II affinity peptide: 16.3 kDa).	RESULTS
126	133	monomer	oligomeric_state	This value corresponds very well with the theoretically calculated molecular mass of an (Sa)EctC dimer (molecular mass of the monomer, including the Strep-tag II affinity peptide: 16.3 kDa).	RESULTS
149	178	Strep-tag II affinity peptide	experimental_method	This value corresponds very well with the theoretically calculated molecular mass of an (Sa)EctC dimer (molecular mass of the monomer, including the Strep-tag II affinity peptide: 16.3 kDa).	RESULTS
30	35	dimer	oligomeric_state	Such a quaternary assembly as dimer has also been reported for the EctC proteins from H. elongata and N. maritimus.	RESULTS
67	80	EctC proteins	protein_type	Such a quaternary assembly as dimer has also been reported for the EctC proteins from H. elongata and N. maritimus.	RESULTS
86	97	H. elongata	species	Such a quaternary assembly as dimer has also been reported for the EctC proteins from H. elongata and N. maritimus.	RESULTS
102	114	N. maritimus	species	Such a quaternary assembly as dimer has also been reported for the EctC proteins from H. elongata and N. maritimus.	RESULTS
30	46	ectoine synthase	protein_type	Biochemical properties of the ectoine synthase	RESULTS
4	8	EctA	protein	The EctA-produced substrate of the ectoine synthase, N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA) (Fig 1), is commercially not available.	RESULTS
35	51	ectoine synthase	protein_type	The EctA-produced substrate of the ectoine synthase, N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA) (Fig 1), is commercially not available.	RESULTS
53	89	N-γ-acetyl-L-2,4-diaminobutyric acid	chemical	The EctA-produced substrate of the ectoine synthase, N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA) (Fig 1), is commercially not available.	RESULTS
91	100	N-γ-ADABA	chemical	The EctA-produced substrate of the ectoine synthase, N-γ-acetyl-L-2,4-diaminobutyric acid (N-γ-ADABA) (Fig 1), is commercially not available.	RESULTS
31	38	ectoine	chemical	We used alkaline hydrolysis of ectoine and subsequent chromatography on silica gel columns to obtain N-γ-ADABA in chemically highly purified form (S1a Fig).	RESULTS
101	110	N-γ-ADABA	chemical	We used alkaline hydrolysis of ectoine and subsequent chromatography on silica gel columns to obtain N-γ-ADABA in chemically highly purified form (S1a Fig).	RESULTS
42	51	N-γ-ADABA	chemical	This procedure also yielded the isomer of N-γ-ADABA, N-α-acetyl-L-2,4-diaminobutyric acid (N-α-ADABA) (S1b Fig).	RESULTS
53	89	N-α-acetyl-L-2,4-diaminobutyric acid	chemical	This procedure also yielded the isomer of N-γ-ADABA, N-α-acetyl-L-2,4-diaminobutyric acid (N-α-ADABA) (S1b Fig).	RESULTS
91	100	N-α-ADABA	chemical	This procedure also yielded the isomer of N-γ-ADABA, N-α-acetyl-L-2,4-diaminobutyric acid (N-α-ADABA) (S1b Fig).	RESULTS
0	9	N-α-ADABA	chemical	N-α-ADABA has so far not been considered as a substrate for EctC, but microorganisms that use ectoine as a nutrient produce it as an intermediate during catabolism.	RESULTS
60	64	EctC	protein	N-α-ADABA has so far not been considered as a substrate for EctC, but microorganisms that use ectoine as a nutrient produce it as an intermediate during catabolism.	RESULTS
70	84	microorganisms	taxonomy_domain	N-α-ADABA has so far not been considered as a substrate for EctC, but microorganisms that use ectoine as a nutrient produce it as an intermediate during catabolism.	RESULTS
94	101	ectoine	chemical	N-α-ADABA has so far not been considered as a substrate for EctC, but microorganisms that use ectoine as a nutrient produce it as an intermediate during catabolism.	RESULTS
6	15	N-γ-ADABA	chemical	Using N-γ-ADABA as the substrate, we initially evaluated a set of biochemical parameters of the recombinant (Sa)EctC protein.	RESULTS
109	111	Sa	species	Using N-γ-ADABA as the substrate, we initially evaluated a set of biochemical parameters of the recombinant (Sa)EctC protein.	RESULTS
112	116	EctC	protein	Using N-γ-ADABA as the substrate, we initially evaluated a set of biochemical parameters of the recombinant (Sa)EctC protein.	RESULTS
0	13	S. alaskensis	species	S. alaskensis, from which the studied ectoine synthase was originally derived, is a microorganism that is well-adapted to a life in permanently cold ocean waters.	RESULTS
38	54	ectoine synthase	protein_type	S. alaskensis, from which the studied ectoine synthase was originally derived, is a microorganism that is well-adapted to a life in permanently cold ocean waters.	RESULTS
84	97	microorganism	taxonomy_domain	S. alaskensis, from which the studied ectoine synthase was originally derived, is a microorganism that is well-adapted to a life in permanently cold ocean waters.	RESULTS
69	71	Sa	species	Consistent with the physicochemical attributes of this habitat, the (Sa)EctC protein was already enzymatically active at 5°C, had a temperature optimum of 15°C and was able to function over a broad range of temperatures (S3a Fig).	RESULTS
72	76	EctC	protein	Consistent with the physicochemical attributes of this habitat, the (Sa)EctC protein was already enzymatically active at 5°C, had a temperature optimum of 15°C and was able to function over a broad range of temperatures (S3a Fig).	RESULTS
97	117	enzymatically active	protein_state	Consistent with the physicochemical attributes of this habitat, the (Sa)EctC protein was already enzymatically active at 5°C, had a temperature optimum of 15°C and was able to function over a broad range of temperatures (S3a Fig).	RESULTS
16	24	alkaline	protein_state	It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0).	RESULTS
77	94	ectoine synthases	protein_type	It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0).	RESULTS
104	117	halo-tolerant	protein_state	It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0).	RESULTS
118	129	H. elongata	species	It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0).	RESULTS
162	173	alkaliphile	taxonomy_domain	It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0).	RESULTS
174	189	M. alcaliphilum	species	It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0).	RESULTS
219	229	acidophile	taxonomy_domain	It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0).	RESULTS
230	250	Acidiphilium cryptum	species	It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0).	RESULTS
291	295	EctC	protein	It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0).	RESULTS
309	321	N. maritimus	species	It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0).	RESULTS
328	338	neutral pH	protein_state	It possessed an alkaline pH optimum of 8.5 (S3b Fig), a value similar to the ectoine synthases from the halo-tolerant H. elongata (pH optimum of 8.5 to 9.0), the alkaliphile M. alcaliphilum (pH optimum of 9.0), and the acidophile Acidiphilium cryptum (pH optimum of 8.5 to 9.0), whereas the EctC protein from N. maritimus has a neutral pH optimum (pH 7.0).	RESULTS
100	102	Sa	species	The salinity of the assay buffer had a significant influence on the maximal enzyme activity of the (Sa)EctC protein.	RESULTS
103	107	EctC	protein	The salinity of the assay buffer had a significant influence on the maximal enzyme activity of the (Sa)EctC protein.	RESULTS
26	30	NaCl	chemical	An increase in either the NaCl or the KCl concentration led to an approximately 5-fold enhancement of the ectoine synthase activity.	RESULTS
38	41	KCl	chemical	An increase in either the NaCl or the KCl concentration led to an approximately 5-fold enhancement of the ectoine synthase activity.	RESULTS
106	122	ectoine synthase	protein_type	An increase in either the NaCl or the KCl concentration led to an approximately 5-fold enhancement of the ectoine synthase activity.	RESULTS
32	34	Sa	species	The maximum enzyme activity of (Sa)EctC occurred around 250 mM NaCl or KCl, respectively.	RESULTS
35	39	EctC	protein	The maximum enzyme activity of (Sa)EctC occurred around 250 mM NaCl or KCl, respectively.	RESULTS
63	67	NaCl	chemical	The maximum enzyme activity of (Sa)EctC occurred around 250 mM NaCl or KCl, respectively.	RESULTS
71	74	KCl	chemical	The maximum enzyme activity of (Sa)EctC occurred around 250 mM NaCl or KCl, respectively.	RESULTS
1	3	Sa	species	(Sa)EctC is a highly salt-tolerant enzyme since it exhibited substantial enzyme activity even at NaCl and KCl concentrations of 1 M in the assay buffer (S3c and S3d Fig).	RESULTS
4	8	EctC	protein	(Sa)EctC is a highly salt-tolerant enzyme since it exhibited substantial enzyme activity even at NaCl and KCl concentrations of 1 M in the assay buffer (S3c and S3d Fig).	RESULTS
97	101	NaCl	chemical	(Sa)EctC is a highly salt-tolerant enzyme since it exhibited substantial enzyme activity even at NaCl and KCl concentrations of 1 M in the assay buffer (S3c and S3d Fig).	RESULTS
106	109	KCl	chemical	(Sa)EctC is a highly salt-tolerant enzyme since it exhibited substantial enzyme activity even at NaCl and KCl concentrations of 1 M in the assay buffer (S3c and S3d Fig).	RESULTS
19	23	EctC	protein	The stimulation of EctC enzyme activity by salts has previously also been observed for other ectoine synthases.	RESULTS
93	110	ectoine synthases	protein_type	The stimulation of EctC enzyme activity by salts has previously also been observed for other ectoine synthases.	RESULTS
4	20	ectoine synthase	protein_type	The ectoine synthase is a metal-containing protein	RESULTS
26	50	metal-containing protein	protein_type	The ectoine synthase is a metal-containing protein	RESULTS
53	57	EctC	protein	Considerations based on bioinformatics suggests that EctC belongs to the cupin superfamily.	RESULTS
73	90	cupin superfamily	protein_type	Considerations based on bioinformatics suggests that EctC belongs to the cupin superfamily.	RESULTS
87	91	iron	chemical	Most of these proteins contain catalytically important transition state metals such as iron, copper, zinc, manganese, cobalt, or nickel.	RESULTS
93	99	copper	chemical	Most of these proteins contain catalytically important transition state metals such as iron, copper, zinc, manganese, cobalt, or nickel.	RESULTS
101	105	zinc	chemical	Most of these proteins contain catalytically important transition state metals such as iron, copper, zinc, manganese, cobalt, or nickel.	RESULTS
107	116	manganese	chemical	Most of these proteins contain catalytically important transition state metals such as iron, copper, zinc, manganese, cobalt, or nickel.	RESULTS
118	124	cobalt	chemical	Most of these proteins contain catalytically important transition state metals such as iron, copper, zinc, manganese, cobalt, or nickel.	RESULTS
129	135	nickel	chemical	Most of these proteins contain catalytically important transition state metals such as iron, copper, zinc, manganese, cobalt, or nickel.	RESULTS
0	6	Cupins	protein_type	Cupins contain two conserved motifs: G(X)5HXH(X)3,4E(X)6G and G(X)5PXG(X)2H(X)3N (the letters in bold represent those residues that often coordinate the metal).	RESULTS
19	28	conserved	protein_state	Cupins contain two conserved motifs: G(X)5HXH(X)3,4E(X)6G and G(X)5PXG(X)2H(X)3N (the letters in bold represent those residues that often coordinate the metal).	RESULTS
37	57	G(X)5HXH(X)3,4E(X)6G	structure_element	Cupins contain two conserved motifs: G(X)5HXH(X)3,4E(X)6G and G(X)5PXG(X)2H(X)3N (the letters in bold represent those residues that often coordinate the metal).	RESULTS
62	80	G(X)5PXG(X)2H(X)3N	structure_element	Cupins contain two conserved motifs: G(X)5HXH(X)3,4E(X)6G and G(X)5PXG(X)2H(X)3N (the letters in bold represent those residues that often coordinate the metal).	RESULTS
153	158	metal	chemical	Cupins contain two conserved motifs: G(X)5HXH(X)3,4E(X)6G and G(X)5PXG(X)2H(X)3N (the letters in bold represent those residues that often coordinate the metal).	RESULTS
25	62	alignment of the amino acid sequences	experimental_method	Inspection of a previous alignment of the amino acid sequences of 440 EctC-type proteins revealed that the canonical metal-binding motif(s) of cupin-type proteins is not conserved among members of the extended ectoine synthase protein family.	RESULTS
70	88	EctC-type proteins	protein_type	Inspection of a previous alignment of the amino acid sequences of 440 EctC-type proteins revealed that the canonical metal-binding motif(s) of cupin-type proteins is not conserved among members of the extended ectoine synthase protein family.	RESULTS
117	136	metal-binding motif	structure_element	Inspection of a previous alignment of the amino acid sequences of 440 EctC-type proteins revealed that the canonical metal-binding motif(s) of cupin-type proteins is not conserved among members of the extended ectoine synthase protein family.	RESULTS
143	162	cupin-type proteins	protein_type	Inspection of a previous alignment of the amino acid sequences of 440 EctC-type proteins revealed that the canonical metal-binding motif(s) of cupin-type proteins is not conserved among members of the extended ectoine synthase protein family.	RESULTS
166	179	not conserved	protein_state	Inspection of a previous alignment of the amino acid sequences of 440 EctC-type proteins revealed that the canonical metal-binding motif(s) of cupin-type proteins is not conserved among members of the extended ectoine synthase protein family.	RESULTS
210	241	ectoine synthase protein family	protein_type	Inspection of a previous alignment of the amino acid sequences of 440 EctC-type proteins revealed that the canonical metal-binding motif(s) of cupin-type proteins is not conserved among members of the extended ectoine synthase protein family.	RESULTS
15	51	alignment of the amino acid sequence	experimental_method	An abbreviated alignment of the amino acid sequence of EctC-type proteins is shown in Fig 2.	RESULTS
55	73	EctC-type proteins	protein_type	An abbreviated alignment of the amino acid sequence of EctC-type proteins is shown in Fig 2.	RESULTS
12	21	alignment	experimental_method	Abbreviated alignment of EctC-type proteins.	FIG
25	43	EctC-type proteins	protein_type	Abbreviated alignment of EctC-type proteins.	FIG
40	58	EctC-type proteins	protein_type	The amino acid sequences of 20 selected EctC-type proteins are compared.	FIG
0	18	Strictly conserved	protein_state	Strictly conserved amino acid residues are shown in yellow.	FIG
22	24	Sa	species	Dots shown above the (Sa)EctC protein sequence indicate residues likely to be involved in iron-binding (red), ligand-binding (green) and stabilization of the loop-architecture (blue).	FIG
25	29	EctC	protein	Dots shown above the (Sa)EctC protein sequence indicate residues likely to be involved in iron-binding (red), ligand-binding (green) and stabilization of the loop-architecture (blue).	FIG
90	94	iron	chemical	Dots shown above the (Sa)EctC protein sequence indicate residues likely to be involved in iron-binding (red), ligand-binding (green) and stabilization of the loop-architecture (blue).	FIG
4	13	conserved	protein_state	The conserved residue Tyr-52 with so-far undefined functions is indicated by a green dot circled in red.	FIG
22	28	Tyr-52	residue_name_number	The conserved residue Tyr-52 with so-far undefined functions is indicated by a green dot circled in red.	FIG
31	40	α-helices	structure_element	Secondary structural elements (α-helices and β-sheets) found in the (Sa)EctC crystal structure are projected onto the amino acid sequences of EctC-type proteins.	FIG
45	53	β-sheets	structure_element	Secondary structural elements (α-helices and β-sheets) found in the (Sa)EctC crystal structure are projected onto the amino acid sequences of EctC-type proteins.	FIG
69	71	Sa	species	Secondary structural elements (α-helices and β-sheets) found in the (Sa)EctC crystal structure are projected onto the amino acid sequences of EctC-type proteins.	FIG
72	76	EctC	protein	Secondary structural elements (α-helices and β-sheets) found in the (Sa)EctC crystal structure are projected onto the amino acid sequences of EctC-type proteins.	FIG
77	94	crystal structure	evidence	Secondary structural elements (α-helices and β-sheets) found in the (Sa)EctC crystal structure are projected onto the amino acid sequences of EctC-type proteins.	FIG
142	160	EctC-type proteins	protein_type	Secondary structural elements (α-helices and β-sheets) found in the (Sa)EctC crystal structure are projected onto the amino acid sequences of EctC-type proteins.	FIG
40	59	metal-binding motif	structure_element	Since variations of the above-described metal-binding motif occur frequently, we experimentally investigated the presence and nature of the metal that might be contained in the (Sa)EctC protein by inductive-coupled plasma mass spectrometry (ICP-MS).	RESULTS
140	145	metal	chemical	Since variations of the above-described metal-binding motif occur frequently, we experimentally investigated the presence and nature of the metal that might be contained in the (Sa)EctC protein by inductive-coupled plasma mass spectrometry (ICP-MS).	RESULTS
178	180	Sa	species	Since variations of the above-described metal-binding motif occur frequently, we experimentally investigated the presence and nature of the metal that might be contained in the (Sa)EctC protein by inductive-coupled plasma mass spectrometry (ICP-MS).	RESULTS
181	185	EctC	protein	Since variations of the above-described metal-binding motif occur frequently, we experimentally investigated the presence and nature of the metal that might be contained in the (Sa)EctC protein by inductive-coupled plasma mass spectrometry (ICP-MS).	RESULTS
197	239	inductive-coupled plasma mass spectrometry	experimental_method	Since variations of the above-described metal-binding motif occur frequently, we experimentally investigated the presence and nature of the metal that might be contained in the (Sa)EctC protein by inductive-coupled plasma mass spectrometry (ICP-MS).	RESULTS
241	247	ICP-MS	experimental_method	Since variations of the above-described metal-binding motif occur frequently, we experimentally investigated the presence and nature of the metal that might be contained in the (Sa)EctC protein by inductive-coupled plasma mass spectrometry (ICP-MS).	RESULTS
39	41	Sa	species	For this analysis we used recombinant (Sa)EctC preparations from three independent protein overproduction and purification experiments.	RESULTS
42	46	EctC	protein	For this analysis we used recombinant (Sa)EctC preparations from three independent protein overproduction and purification experiments.	RESULTS
4	10	ICP-MS	experimental_method	The ICP-MS analyses yielded an iron content of 0.66 ± 0.06 mol iron per mol of protein and the used (Sa)EctC protein preparations also contained a minor amount of zinc (0.08 mol zinc per mol of protein).	RESULTS
31	35	iron	chemical	The ICP-MS analyses yielded an iron content of 0.66 ± 0.06 mol iron per mol of protein and the used (Sa)EctC protein preparations also contained a minor amount of zinc (0.08 mol zinc per mol of protein).	RESULTS
63	67	iron	chemical	The ICP-MS analyses yielded an iron content of 0.66 ± 0.06 mol iron per mol of protein and the used (Sa)EctC protein preparations also contained a minor amount of zinc (0.08 mol zinc per mol of protein).	RESULTS
101	103	Sa	species	The ICP-MS analyses yielded an iron content of 0.66 ± 0.06 mol iron per mol of protein and the used (Sa)EctC protein preparations also contained a minor amount of zinc (0.08 mol zinc per mol of protein).	RESULTS
104	108	EctC	protein	The ICP-MS analyses yielded an iron content of 0.66 ± 0.06 mol iron per mol of protein and the used (Sa)EctC protein preparations also contained a minor amount of zinc (0.08 mol zinc per mol of protein).	RESULTS
163	167	zinc	chemical	The ICP-MS analyses yielded an iron content of 0.66 ± 0.06 mol iron per mol of protein and the used (Sa)EctC protein preparations also contained a minor amount of zinc (0.08 mol zinc per mol of protein).	RESULTS
178	182	zinc	chemical	The ICP-MS analyses yielded an iron content of 0.66 ± 0.06 mol iron per mol of protein and the used (Sa)EctC protein preparations also contained a minor amount of zinc (0.08 mol zinc per mol of protein).	RESULTS
26	32	copper	chemical	All other assayed metals (copper and nickel) were only present in trace amounts (0.01 mol metal per mol of protein, respectively).	RESULTS
37	43	nickel	chemical	All other assayed metals (copper and nickel) were only present in trace amounts (0.01 mol metal per mol of protein, respectively).	RESULTS
90	95	metal	chemical	All other assayed metals (copper and nickel) were only present in trace amounts (0.01 mol metal per mol of protein, respectively).	RESULTS
16	20	iron	chemical	The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein.	RESULTS
31	33	Sa	species	The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein.	RESULTS
34	38	EctC	protein	The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein.	RESULTS
87	106	colorimetric method	experimental_method	The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein.	RESULTS
127	131	iron	chemical	The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein.	RESULTS
178	182	iron	chemical	The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein.	RESULTS
222	224	Sa	species	The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein.	RESULTS
225	229	EctC	protein	The presence of iron in these (Sa)EctC protein preparations was further confirmed by a colorimetric method that is based on an iron-complexing reagent; this procedure yielded an iron-content of 0.84 ± 0.05 mol per mol of (Sa)EctC protein.	RESULTS
12	18	ICP-MS	experimental_method	Hence, both ICP-MS and the colorimetric method clearly established that the recombinantly produced ectoine synthase from S. alaskensis is an iron-containing protein.	RESULTS
27	46	colorimetric method	experimental_method	Hence, both ICP-MS and the colorimetric method clearly established that the recombinantly produced ectoine synthase from S. alaskensis is an iron-containing protein.	RESULTS
99	115	ectoine synthase	protein_type	Hence, both ICP-MS and the colorimetric method clearly established that the recombinantly produced ectoine synthase from S. alaskensis is an iron-containing protein.	RESULTS
121	134	S. alaskensis	species	Hence, both ICP-MS and the colorimetric method clearly established that the recombinantly produced ectoine synthase from S. alaskensis is an iron-containing protein.	RESULTS
141	145	iron	chemical	Hence, both ICP-MS and the colorimetric method clearly established that the recombinantly produced ectoine synthase from S. alaskensis is an iron-containing protein.	RESULTS
58	62	iron	chemical	We note in this context, that the values obtained for the iron content of the (Sa)EctC proteins varied by approximately 10 to 20% between the two methods.	RESULTS
79	81	Sa	species	We note in this context, that the values obtained for the iron content of the (Sa)EctC proteins varied by approximately 10 to 20% between the two methods.	RESULTS
82	86	EctC	protein	We note in this context, that the values obtained for the iron content of the (Sa)EctC proteins varied by approximately 10 to 20% between the two methods.	RESULTS
85	103	colorimetric assay	experimental_method	The reason for this difference is not known, but indicates that the well established colorimetric assay probably overestimates the iron content of (Sa)EctC protein preparations to a certain degree.	RESULTS
131	135	iron	chemical	The reason for this difference is not known, but indicates that the well established colorimetric assay probably overestimates the iron content of (Sa)EctC protein preparations to a certain degree.	RESULTS
148	150	Sa	species	The reason for this difference is not known, but indicates that the well established colorimetric assay probably overestimates the iron content of (Sa)EctC protein preparations to a certain degree.	RESULTS
151	155	EctC	protein	The reason for this difference is not known, but indicates that the well established colorimetric assay probably overestimates the iron content of (Sa)EctC protein preparations to a certain degree.	RESULTS
2	7	metal	chemical	A metal cofactor is important for the catalytic activity of EctC	RESULTS
60	64	EctC	protein	A metal cofactor is important for the catalytic activity of EctC	RESULTS
4	8	iron	chemical	The iron detected in the (Sa)EctC protein preparations could serve a structural role, or most likely, could be critical for enzyme catalysis as is the case for many members of the cupin superfamily.	RESULTS
26	28	Sa	species	The iron detected in the (Sa)EctC protein preparations could serve a structural role, or most likely, could be critical for enzyme catalysis as is the case for many members of the cupin superfamily.	RESULTS
29	33	EctC	protein	The iron detected in the (Sa)EctC protein preparations could serve a structural role, or most likely, could be critical for enzyme catalysis as is the case for many members of the cupin superfamily.	RESULTS
180	197	cupin superfamily	protein_type	The iron detected in the (Sa)EctC protein preparations could serve a structural role, or most likely, could be critical for enzyme catalysis as is the case for many members of the cupin superfamily.	RESULTS
31	40	incubated	experimental_method	To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity.	RESULTS
46	48	Sa	species	To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity.	RESULTS
49	53	EctC	protein	To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity.	RESULTS
61	91	with increasing concentrations	experimental_method	To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity.	RESULTS
99	104	metal	chemical	To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity.	RESULTS
114	147	ethylene-diamine-tetraacetic-acid	chemical	To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity.	RESULTS
149	153	EDTA	chemical	To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity.	RESULTS
180	196	ectoine synthase	protein_type	To address these questions, we incubated the (Sa)EctC enzyme with increasing concentrations of the metal chelator ethylene-diamine-tetraacetic-acid (EDTA) and subsequently assayed ectoine synthase activity.	RESULTS
43	47	EDTA	chemical	The addition of very low concentrations of EDTA (0.05 mM) to the EctC enzyme already led to a noticeable inhibition of the ectoine synthase activity and the presence of 1 mM EDTA completely inhibited the enzyme (Fig 3a).	RESULTS
65	69	EctC	protein	The addition of very low concentrations of EDTA (0.05 mM) to the EctC enzyme already led to a noticeable inhibition of the ectoine synthase activity and the presence of 1 mM EDTA completely inhibited the enzyme (Fig 3a).	RESULTS
123	139	ectoine synthase	protein_type	The addition of very low concentrations of EDTA (0.05 mM) to the EctC enzyme already led to a noticeable inhibition of the ectoine synthase activity and the presence of 1 mM EDTA completely inhibited the enzyme (Fig 3a).	RESULTS
174	178	EDTA	chemical	The addition of very low concentrations of EDTA (0.05 mM) to the EctC enzyme already led to a noticeable inhibition of the ectoine synthase activity and the presence of 1 mM EDTA completely inhibited the enzyme (Fig 3a).	RESULTS
18	34	ectoine synthase	protein_type	Dependency of the ectoine synthase activity on metals.	FIG
18	22	iron	chemical	(a) Impact of the iron-chelator EDTA on the enzyme activity of the purified (Sa)EctC protein.	FIG
32	36	EDTA	chemical	(a) Impact of the iron-chelator EDTA on the enzyme activity of the purified (Sa)EctC protein.	FIG
77	79	Sa	species	(a) Impact of the iron-chelator EDTA on the enzyme activity of the purified (Sa)EctC protein.	FIG
80	84	EctC	protein	(a) Impact of the iron-chelator EDTA on the enzyme activity of the purified (Sa)EctC protein.	FIG
0	46	Metal depletion and reconstitution experiments	experimental_method	Metal depletion and reconstitution experiments with (b) stoichiometric and (c) excess amounts of metals.	FIG
5	7	Sa	species	The (Sa)EctC protein was present at a concentration of 10 μM. The level of enzyme activity given in (b) is benchmarked relative to that of ectoine synthase enzyme assays in which 1 mM FeCl2 was added.	FIG
8	12	EctC	protein	The (Sa)EctC protein was present at a concentration of 10 μM. The level of enzyme activity given in (b) is benchmarked relative to that of ectoine synthase enzyme assays in which 1 mM FeCl2 was added.	FIG
139	155	ectoine synthase	protein_type	The (Sa)EctC protein was present at a concentration of 10 μM. The level of enzyme activity given in (b) is benchmarked relative to that of ectoine synthase enzyme assays in which 1 mM FeCl2 was added.	FIG
156	169	enzyme assays	experimental_method	The (Sa)EctC protein was present at a concentration of 10 μM. The level of enzyme activity given in (b) is benchmarked relative to that of ectoine synthase enzyme assays in which 1 mM FeCl2 was added.	FIG
184	189	FeCl2	chemical	The (Sa)EctC protein was present at a concentration of 10 μM. The level of enzyme activity given in (b) is benchmarked relative to that of ectoine synthase enzyme assays in which 1 mM FeCl2 was added.	FIG
21	32	inactivated	protein_state	We then took such an inactivated enzyme preparation, removed the EDTA by dialysis, and added stoichiometric amounts (10 μM) of various metals to the (Sa)EctC enzyme.	RESULTS
65	69	EDTA	chemical	We then took such an inactivated enzyme preparation, removed the EDTA by dialysis, and added stoichiometric amounts (10 μM) of various metals to the (Sa)EctC enzyme.	RESULTS
73	81	dialysis	experimental_method	We then took such an inactivated enzyme preparation, removed the EDTA by dialysis, and added stoichiometric amounts (10 μM) of various metals to the (Sa)EctC enzyme.	RESULTS
150	152	Sa	species	We then took such an inactivated enzyme preparation, removed the EDTA by dialysis, and added stoichiometric amounts (10 μM) of various metals to the (Sa)EctC enzyme.	RESULTS
153	157	EctC	protein	We then took such an inactivated enzyme preparation, removed the EDTA by dialysis, and added stoichiometric amounts (10 μM) of various metals to the (Sa)EctC enzyme.	RESULTS
16	21	FeCl2	chemical	The addition of FeCl2 to the enzyme assay restored enzyme activity to about 38%, whereas the addition of ZnCl2 or CoCl2 rescued (Sa)EctC enzyme activity only to 5% and 3%, respectively.	RESULTS
29	41	enzyme assay	experimental_method	The addition of FeCl2 to the enzyme assay restored enzyme activity to about 38%, whereas the addition of ZnCl2 or CoCl2 rescued (Sa)EctC enzyme activity only to 5% and 3%, respectively.	RESULTS
105	110	ZnCl2	chemical	The addition of FeCl2 to the enzyme assay restored enzyme activity to about 38%, whereas the addition of ZnCl2 or CoCl2 rescued (Sa)EctC enzyme activity only to 5% and 3%, respectively.	RESULTS
114	119	CoCl2	chemical	The addition of FeCl2 to the enzyme assay restored enzyme activity to about 38%, whereas the addition of ZnCl2 or CoCl2 rescued (Sa)EctC enzyme activity only to 5% and 3%, respectively.	RESULTS
129	131	Sa	species	The addition of FeCl2 to the enzyme assay restored enzyme activity to about 38%, whereas the addition of ZnCl2 or CoCl2 rescued (Sa)EctC enzyme activity only to 5% and 3%, respectively.	RESULTS
132	136	EctC	protein	The addition of FeCl2 to the enzyme assay restored enzyme activity to about 38%, whereas the addition of ZnCl2 or CoCl2 rescued (Sa)EctC enzyme activity only to 5% and 3%, respectively.	RESULTS
35	39	Fe3+	chemical	All other tested metals, including Fe3+, were unable to restore activity (Fig 3b).	RESULTS
52	64	enzyme assay	experimental_method	When the concentration of the various metals in the enzyme assay was increased 100-fold, Fe2+ exhibited again the strongest stimulating effect on enzyme activity, and rescued enzyme activity to a degree similar to that exhibited by (Sa)EctC protein preparations that had not been inactivated through EDTA treatment (Fig 3c).	RESULTS
89	93	Fe2+	chemical	When the concentration of the various metals in the enzyme assay was increased 100-fold, Fe2+ exhibited again the strongest stimulating effect on enzyme activity, and rescued enzyme activity to a degree similar to that exhibited by (Sa)EctC protein preparations that had not been inactivated through EDTA treatment (Fig 3c).	RESULTS
233	235	Sa	species	When the concentration of the various metals in the enzyme assay was increased 100-fold, Fe2+ exhibited again the strongest stimulating effect on enzyme activity, and rescued enzyme activity to a degree similar to that exhibited by (Sa)EctC protein preparations that had not been inactivated through EDTA treatment (Fig 3c).	RESULTS
236	240	EctC	protein	When the concentration of the various metals in the enzyme assay was increased 100-fold, Fe2+ exhibited again the strongest stimulating effect on enzyme activity, and rescued enzyme activity to a degree similar to that exhibited by (Sa)EctC protein preparations that had not been inactivated through EDTA treatment (Fig 3c).	RESULTS
300	304	EDTA	chemical	When the concentration of the various metals in the enzyme assay was increased 100-fold, Fe2+ exhibited again the strongest stimulating effect on enzyme activity, and rescued enzyme activity to a degree similar to that exhibited by (Sa)EctC protein preparations that had not been inactivated through EDTA treatment (Fig 3c).	RESULTS
64	68	zinc	chemical	However, a large molar excess of other transition-state metals (zinc, cobalt, nickel, copper, and manganese) typically found in members of the cupin superfamily allowed the partial rescue of ectoine synthase activity as well (Fig 3c).	RESULTS
70	76	cobalt	chemical	However, a large molar excess of other transition-state metals (zinc, cobalt, nickel, copper, and manganese) typically found in members of the cupin superfamily allowed the partial rescue of ectoine synthase activity as well (Fig 3c).	RESULTS
78	84	nickel	chemical	However, a large molar excess of other transition-state metals (zinc, cobalt, nickel, copper, and manganese) typically found in members of the cupin superfamily allowed the partial rescue of ectoine synthase activity as well (Fig 3c).	RESULTS
86	92	copper	chemical	However, a large molar excess of other transition-state metals (zinc, cobalt, nickel, copper, and manganese) typically found in members of the cupin superfamily allowed the partial rescue of ectoine synthase activity as well (Fig 3c).	RESULTS
98	107	manganese	chemical	However, a large molar excess of other transition-state metals (zinc, cobalt, nickel, copper, and manganese) typically found in members of the cupin superfamily allowed the partial rescue of ectoine synthase activity as well (Fig 3c).	RESULTS
143	160	cupin superfamily	protein_type	However, a large molar excess of other transition-state metals (zinc, cobalt, nickel, copper, and manganese) typically found in members of the cupin superfamily allowed the partial rescue of ectoine synthase activity as well (Fig 3c).	RESULTS
191	207	ectoine synthase	protein_type	However, a large molar excess of other transition-state metals (zinc, cobalt, nickel, copper, and manganese) typically found in members of the cupin superfamily allowed the partial rescue of ectoine synthase activity as well (Fig 3c).	RESULTS
50	68	cupin-type enzymes	protein_type	This is in line with literature data showing that cupin-type enzymes are often promiscuous with respect to the use of the catalytically important metal.	RESULTS
146	151	metal	chemical	This is in line with literature data showing that cupin-type enzymes are often promiscuous with respect to the use of the catalytically important metal.	RESULTS
22	26	EctC	protein	Kinetic parameters of EctC for N-γ-ADABA and N-α-ADABA	RESULTS
31	40	N-γ-ADABA	chemical	Kinetic parameters of EctC for N-γ-ADABA and N-α-ADABA	RESULTS
45	54	N-α-ADABA	chemical	Kinetic parameters of EctC for N-γ-ADABA and N-α-ADABA	RESULTS
66	80	activity assay	experimental_method	Based on the data presented in S3 Fig, we formulated an optimized activity assay for the ectoine synthase of S. alaskensis and used it to determined the kinetic parameters for the (Sa)EctC enzyme for both its natural substrate N-γ-ADABA and the isomer N-α-ADABA.	RESULTS
89	105	ectoine synthase	protein_type	Based on the data presented in S3 Fig, we formulated an optimized activity assay for the ectoine synthase of S. alaskensis and used it to determined the kinetic parameters for the (Sa)EctC enzyme for both its natural substrate N-γ-ADABA and the isomer N-α-ADABA.	RESULTS
109	122	S. alaskensis	species	Based on the data presented in S3 Fig, we formulated an optimized activity assay for the ectoine synthase of S. alaskensis and used it to determined the kinetic parameters for the (Sa)EctC enzyme for both its natural substrate N-γ-ADABA and the isomer N-α-ADABA.	RESULTS
181	183	Sa	species	Based on the data presented in S3 Fig, we formulated an optimized activity assay for the ectoine synthase of S. alaskensis and used it to determined the kinetic parameters for the (Sa)EctC enzyme for both its natural substrate N-γ-ADABA and the isomer N-α-ADABA.	RESULTS
184	188	EctC	protein	Based on the data presented in S3 Fig, we formulated an optimized activity assay for the ectoine synthase of S. alaskensis and used it to determined the kinetic parameters for the (Sa)EctC enzyme for both its natural substrate N-γ-ADABA and the isomer N-α-ADABA.	RESULTS
227	236	N-γ-ADABA	chemical	Based on the data presented in S3 Fig, we formulated an optimized activity assay for the ectoine synthase of S. alaskensis and used it to determined the kinetic parameters for the (Sa)EctC enzyme for both its natural substrate N-γ-ADABA and the isomer N-α-ADABA.	RESULTS
252	261	N-α-ADABA	chemical	Based on the data presented in S3 Fig, we formulated an optimized activity assay for the ectoine synthase of S. alaskensis and used it to determined the kinetic parameters for the (Sa)EctC enzyme for both its natural substrate N-γ-ADABA and the isomer N-α-ADABA.	RESULTS
4	8	EctC	protein	The EctC-catalyzed ring-closure of N-γ-ADABA to form ectoine exhibited Michaelis-Menten-kinetics with an apparent Km of 4.9 ± 0.5 mM, a vmax of 25.0 ± 0.8 U/mg and a kcat of 7.2 s-1 (S4a Fig).	RESULTS
35	44	N-γ-ADABA	chemical	The EctC-catalyzed ring-closure of N-γ-ADABA to form ectoine exhibited Michaelis-Menten-kinetics with an apparent Km of 4.9 ± 0.5 mM, a vmax of 25.0 ± 0.8 U/mg and a kcat of 7.2 s-1 (S4a Fig).	RESULTS
53	60	ectoine	chemical	The EctC-catalyzed ring-closure of N-γ-ADABA to form ectoine exhibited Michaelis-Menten-kinetics with an apparent Km of 4.9 ± 0.5 mM, a vmax of 25.0 ± 0.8 U/mg and a kcat of 7.2 s-1 (S4a Fig).	RESULTS
71	96	Michaelis-Menten-kinetics	experimental_method	The EctC-catalyzed ring-closure of N-γ-ADABA to form ectoine exhibited Michaelis-Menten-kinetics with an apparent Km of 4.9 ± 0.5 mM, a vmax of 25.0 ± 0.8 U/mg and a kcat of 7.2 s-1 (S4a Fig).	RESULTS
114	116	Km	evidence	The EctC-catalyzed ring-closure of N-γ-ADABA to form ectoine exhibited Michaelis-Menten-kinetics with an apparent Km of 4.9 ± 0.5 mM, a vmax of 25.0 ± 0.8 U/mg and a kcat of 7.2 s-1 (S4a Fig).	RESULTS
136	140	vmax	evidence	The EctC-catalyzed ring-closure of N-γ-ADABA to form ectoine exhibited Michaelis-Menten-kinetics with an apparent Km of 4.9 ± 0.5 mM, a vmax of 25.0 ± 0.8 U/mg and a kcat of 7.2 s-1 (S4a Fig).	RESULTS
166	170	kcat	evidence	The EctC-catalyzed ring-closure of N-γ-ADABA to form ectoine exhibited Michaelis-Menten-kinetics with an apparent Km of 4.9 ± 0.5 mM, a vmax of 25.0 ± 0.8 U/mg and a kcat of 7.2 s-1 (S4a Fig).	RESULTS
34	43	N-α-ADABA	chemical	Given the chemical relatedness of N-α-ADABA to the natural substrate (N-γ-ADABA) of the ectoine synthase (S1a and S1b Fig), we wondered whether (Sa)EctC could also use N-α-ADABA to produce ectoine.	RESULTS
70	79	N-γ-ADABA	chemical	Given the chemical relatedness of N-α-ADABA to the natural substrate (N-γ-ADABA) of the ectoine synthase (S1a and S1b Fig), we wondered whether (Sa)EctC could also use N-α-ADABA to produce ectoine.	RESULTS
88	104	ectoine synthase	protein_type	Given the chemical relatedness of N-α-ADABA to the natural substrate (N-γ-ADABA) of the ectoine synthase (S1a and S1b Fig), we wondered whether (Sa)EctC could also use N-α-ADABA to produce ectoine.	RESULTS
145	147	Sa	species	Given the chemical relatedness of N-α-ADABA to the natural substrate (N-γ-ADABA) of the ectoine synthase (S1a and S1b Fig), we wondered whether (Sa)EctC could also use N-α-ADABA to produce ectoine.	RESULTS
148	152	EctC	protein	Given the chemical relatedness of N-α-ADABA to the natural substrate (N-γ-ADABA) of the ectoine synthase (S1a and S1b Fig), we wondered whether (Sa)EctC could also use N-α-ADABA to produce ectoine.	RESULTS
168	177	N-α-ADABA	chemical	Given the chemical relatedness of N-α-ADABA to the natural substrate (N-γ-ADABA) of the ectoine synthase (S1a and S1b Fig), we wondered whether (Sa)EctC could also use N-α-ADABA to produce ectoine.	RESULTS
189	196	ectoine	chemical	Given the chemical relatedness of N-α-ADABA to the natural substrate (N-γ-ADABA) of the ectoine synthase (S1a and S1b Fig), we wondered whether (Sa)EctC could also use N-α-ADABA to produce ectoine.	RESULTS
1	3	Sa	species	(Sa)EctC catalyzed this reaction with Michaelis-Menten-kinetics exhibiting an apparent Km of 25.4 ± 2.9 mM, a vmax of 24.6 ± 1.0 U/mg and a kcat 0.6 s-1 (S4b Fig).	RESULTS
4	8	EctC	protein	(Sa)EctC catalyzed this reaction with Michaelis-Menten-kinetics exhibiting an apparent Km of 25.4 ± 2.9 mM, a vmax of 24.6 ± 1.0 U/mg and a kcat 0.6 s-1 (S4b Fig).	RESULTS
38	63	Michaelis-Menten-kinetics	experimental_method	(Sa)EctC catalyzed this reaction with Michaelis-Menten-kinetics exhibiting an apparent Km of 25.4 ± 2.9 mM, a vmax of 24.6 ± 1.0 U/mg and a kcat 0.6 s-1 (S4b Fig).	RESULTS
87	89	Km	evidence	(Sa)EctC catalyzed this reaction with Michaelis-Menten-kinetics exhibiting an apparent Km of 25.4 ± 2.9 mM, a vmax of 24.6 ± 1.0 U/mg and a kcat 0.6 s-1 (S4b Fig).	RESULTS
110	114	vmax	evidence	(Sa)EctC catalyzed this reaction with Michaelis-Menten-kinetics exhibiting an apparent Km of 25.4 ± 2.9 mM, a vmax of 24.6 ± 1.0 U/mg and a kcat 0.6 s-1 (S4b Fig).	RESULTS
140	144	kcat	evidence	(Sa)EctC catalyzed this reaction with Michaelis-Menten-kinetics exhibiting an apparent Km of 25.4 ± 2.9 mM, a vmax of 24.6 ± 1.0 U/mg and a kcat 0.6 s-1 (S4b Fig).	RESULTS
7	16	N-α-ADABA	chemical	Hence, N-α-ADABA is a newly recognized substrate for ectoine synthase.	RESULTS
53	69	ectoine synthase	protein_type	Hence, N-α-ADABA is a newly recognized substrate for ectoine synthase.	RESULTS
18	26	affinity	evidence	However, both the affinity (Km) of the (Sa)EctC protein and its catalytic efficiency (kcat/Km) were strongly reduced in comparison with N-γ-ADABA.	RESULTS
28	30	Km	evidence	However, both the affinity (Km) of the (Sa)EctC protein and its catalytic efficiency (kcat/Km) were strongly reduced in comparison with N-γ-ADABA.	RESULTS
40	42	Sa	species	However, both the affinity (Km) of the (Sa)EctC protein and its catalytic efficiency (kcat/Km) were strongly reduced in comparison with N-γ-ADABA.	RESULTS
43	47	EctC	protein	However, both the affinity (Km) of the (Sa)EctC protein and its catalytic efficiency (kcat/Km) were strongly reduced in comparison with N-γ-ADABA.	RESULTS
64	84	catalytic efficiency	evidence	However, both the affinity (Km) of the (Sa)EctC protein and its catalytic efficiency (kcat/Km) were strongly reduced in comparison with N-γ-ADABA.	RESULTS
86	93	kcat/Km	evidence	However, both the affinity (Km) of the (Sa)EctC protein and its catalytic efficiency (kcat/Km) were strongly reduced in comparison with N-γ-ADABA.	RESULTS
136	145	N-γ-ADABA	chemical	However, both the affinity (Km) of the (Sa)EctC protein and its catalytic efficiency (kcat/Km) were strongly reduced in comparison with N-γ-ADABA.	RESULTS
4	6	Km	evidence	The Km dropped fife-fold from 4.9 ± 0.5 mM to 25.4 ± 2.9 mM, and the catalytic efficiency was reduced from 1.47 mM-1 s-1 to 0.02 mM-1 s-1, a 73-fold decrease.	RESULTS
69	89	catalytic efficiency	evidence	The Km dropped fife-fold from 4.9 ± 0.5 mM to 25.4 ± 2.9 mM, and the catalytic efficiency was reduced from 1.47 mM-1 s-1 to 0.02 mM-1 s-1, a 73-fold decrease.	RESULTS
5	14	N-γ-ADABA	chemical	Both N-γ-ADABA and N-α-ADABA are concomitantly formed during the enzymatic hydrolysis of the ectoine ring during catabolism.	RESULTS
19	28	N-α-ADABA	chemical	Both N-γ-ADABA and N-α-ADABA are concomitantly formed during the enzymatic hydrolysis of the ectoine ring during catabolism.	RESULTS
93	100	ectoine	chemical	Both N-γ-ADABA and N-α-ADABA are concomitantly formed during the enzymatic hydrolysis of the ectoine ring during catabolism.	RESULTS
17	26	N-α-ADABA	chemical	Our finding that N-α-ADABA is a substrate for ectoine synthase has bearings for an understanding of the physiology of those microorganisms that can both synthesize and catabolize ectoine.	RESULTS
46	62	ectoine synthase	protein_type	Our finding that N-α-ADABA is a substrate for ectoine synthase has bearings for an understanding of the physiology of those microorganisms that can both synthesize and catabolize ectoine.	RESULTS
124	138	microorganisms	taxonomy_domain	Our finding that N-α-ADABA is a substrate for ectoine synthase has bearings for an understanding of the physiology of those microorganisms that can both synthesize and catabolize ectoine.	RESULTS
179	186	ectoine	chemical	Our finding that N-α-ADABA is a substrate for ectoine synthase has bearings for an understanding of the physiology of those microorganisms that can both synthesize and catabolize ectoine.	RESULTS
24	38	microorganisms	taxonomy_domain	However, these types of microorganisms should still be able to largely avoid a futile cycle since the affinity of ectoine synthase for N-γ-ADABA and N-α-ADABA, and its catalytic efficiency for the two compounds, differs substantially (S4a and S4b Fig).	RESULTS
102	110	affinity	evidence	However, these types of microorganisms should still be able to largely avoid a futile cycle since the affinity of ectoine synthase for N-γ-ADABA and N-α-ADABA, and its catalytic efficiency for the two compounds, differs substantially (S4a and S4b Fig).	RESULTS
114	130	ectoine synthase	protein_type	However, these types of microorganisms should still be able to largely avoid a futile cycle since the affinity of ectoine synthase for N-γ-ADABA and N-α-ADABA, and its catalytic efficiency for the two compounds, differs substantially (S4a and S4b Fig).	RESULTS
135	144	N-γ-ADABA	chemical	However, these types of microorganisms should still be able to largely avoid a futile cycle since the affinity of ectoine synthase for N-γ-ADABA and N-α-ADABA, and its catalytic efficiency for the two compounds, differs substantially (S4a and S4b Fig).	RESULTS
149	158	N-α-ADABA	chemical	However, these types of microorganisms should still be able to largely avoid a futile cycle since the affinity of ectoine synthase for N-γ-ADABA and N-α-ADABA, and its catalytic efficiency for the two compounds, differs substantially (S4a and S4b Fig).	RESULTS
168	188	catalytic efficiency	evidence	However, these types of microorganisms should still be able to largely avoid a futile cycle since the affinity of ectoine synthase for N-γ-ADABA and N-α-ADABA, and its catalytic efficiency for the two compounds, differs substantially (S4a and S4b Fig).	RESULTS
0	15	Crystallization	experimental_method	Crystallization of the (Sa)EctC protein	RESULTS
24	26	Sa	species	Crystallization of the (Sa)EctC protein	RESULTS
27	31	EctC	protein	Crystallization of the (Sa)EctC protein	RESULTS
9	26	crystal structure	evidence	Since no crystal structure of ectoine synthase has been reported, we set out to crystallize the (Sa)EctC protein.	RESULTS
30	46	ectoine synthase	protein_type	Since no crystal structure of ectoine synthase has been reported, we set out to crystallize the (Sa)EctC protein.	RESULTS
80	91	crystallize	experimental_method	Since no crystal structure of ectoine synthase has been reported, we set out to crystallize the (Sa)EctC protein.	RESULTS
97	99	Sa	species	Since no crystal structure of ectoine synthase has been reported, we set out to crystallize the (Sa)EctC protein.	RESULTS
100	104	EctC	protein	Since no crystal structure of ectoine synthase has been reported, we set out to crystallize the (Sa)EctC protein.	RESULTS
19	27	crystals	evidence	Attempts to obtain crystals of (Sa)EctC in complex either with its substrate N-γ-ADABA or its reaction product ectoine were not successful.	RESULTS
32	34	Sa	species	Attempts to obtain crystals of (Sa)EctC in complex either with its substrate N-γ-ADABA or its reaction product ectoine were not successful.	RESULTS
35	39	EctC	protein	Attempts to obtain crystals of (Sa)EctC in complex either with its substrate N-γ-ADABA or its reaction product ectoine were not successful.	RESULTS
40	50	in complex	protein_state	Attempts to obtain crystals of (Sa)EctC in complex either with its substrate N-γ-ADABA or its reaction product ectoine were not successful.	RESULTS
77	86	N-γ-ADABA	chemical	Attempts to obtain crystals of (Sa)EctC in complex either with its substrate N-γ-ADABA or its reaction product ectoine were not successful.	RESULTS
111	118	ectoine	chemical	Attempts to obtain crystals of (Sa)EctC in complex either with its substrate N-γ-ADABA or its reaction product ectoine were not successful.	RESULTS
13	26	crystal forms	evidence	However, two crystal forms of the (Sa)EctC protein in the absence of the substrate were obtained.	RESULTS
35	37	Sa	species	However, two crystal forms of the (Sa)EctC protein in the absence of the substrate were obtained.	RESULTS
38	42	EctC	protein	However, two crystal forms of the (Sa)EctC protein in the absence of the substrate were obtained.	RESULTS
58	68	absence of	protein_state	However, two crystal forms of the (Sa)EctC protein in the absence of the substrate were obtained.	RESULTS
22	39	crystal structure	evidence	Attempts to solve the crystal structure of the (Sa)EctC protein by molecular replacement has previously failed.	RESULTS
48	50	Sa	species	Attempts to solve the crystal structure of the (Sa)EctC protein by molecular replacement has previously failed.	RESULTS
51	55	EctC	protein	Attempts to solve the crystal structure of the (Sa)EctC protein by molecular replacement has previously failed.	RESULTS
67	88	molecular replacement	experimental_method	Attempts to solve the crystal structure of the (Sa)EctC protein by molecular replacement has previously failed.	RESULTS
32	40	crystals	evidence	However, we were able to obtain crystals of form B that were derivatized with mercury and these diffracted up to 2.8 Å (S1 Table).	RESULTS
78	85	mercury	chemical	However, we were able to obtain crystals of form B that were derivatized with mercury and these diffracted up to 2.8 Å (S1 Table).	RESULTS
43	59	structural model	evidence	This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table).	RESULTS
68	70	Sa	species	This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table).	RESULTS
71	75	EctC	protein	This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table).	RESULTS
130	151	molecular replacement	experimental_method	This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table).	RESULTS
274	282	monomers	oligomeric_state	This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table).	RESULTS
287	289	Sa	species	This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table).	RESULTS
290	294	EctC	protein	This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table).	RESULTS
319	336	crystal structure	evidence	This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table).	RESULTS
360	366	Rcryst	evidence	This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table).	RESULTS
383	388	Rfree	evidence	This dataset was used to derive an initial structural model of the (Sa)EctC protein, which in turn was employed as a template for molecular replacement to phase the native dataset (2.0 Å) of crystal form B. After several rounds of manual model building and refinement, four monomers of (Sa)EctC were identified and the crystal structure was refined to a final Rcryst of 21.1% and an Rfree of 24.8% (S1 Table).	RESULTS
11	18	monomer	oligomeric_state	Finally, a monomer of this structure was used as a template for molecular replacement to phase the high-resolution (1.2 Å) dataset of crystal form A, which was subsequently refined to a final Rcryst of 12.4% and an Rfree of 14.9% (S1 Table).	RESULTS
27	36	structure	evidence	Finally, a monomer of this structure was used as a template for molecular replacement to phase the high-resolution (1.2 Å) dataset of crystal form A, which was subsequently refined to a final Rcryst of 12.4% and an Rfree of 14.9% (S1 Table).	RESULTS
64	85	molecular replacement	experimental_method	Finally, a monomer of this structure was used as a template for molecular replacement to phase the high-resolution (1.2 Å) dataset of crystal form A, which was subsequently refined to a final Rcryst of 12.4% and an Rfree of 14.9% (S1 Table).	RESULTS
192	198	Rcryst	evidence	Finally, a monomer of this structure was used as a template for molecular replacement to phase the high-resolution (1.2 Å) dataset of crystal form A, which was subsequently refined to a final Rcryst of 12.4% and an Rfree of 14.9% (S1 Table).	RESULTS
215	220	Rfree	evidence	Finally, a monomer of this structure was used as a template for molecular replacement to phase the high-resolution (1.2 Å) dataset of crystal form A, which was subsequently refined to a final Rcryst of 12.4% and an Rfree of 14.9% (S1 Table).	RESULTS
21	23	Sa	species	Overall fold of the (Sa)EctC protein	RESULTS
24	28	EctC	protein	Overall fold of the (Sa)EctC protein	RESULTS
8	12	EctC	protein	The two EctC structures that we determined revealed that the ectoine synthase belongs to the cupin superfamily with respect to its overall fold (Fig 4a–4c).	RESULTS
13	23	structures	evidence	The two EctC structures that we determined revealed that the ectoine synthase belongs to the cupin superfamily with respect to its overall fold (Fig 4a–4c).	RESULTS
61	77	ectoine synthase	protein_type	The two EctC structures that we determined revealed that the ectoine synthase belongs to the cupin superfamily with respect to its overall fold (Fig 4a–4c).	RESULTS
93	110	cupin superfamily	protein_type	The two EctC structures that we determined revealed that the ectoine synthase belongs to the cupin superfamily with respect to its overall fold (Fig 4a–4c).	RESULTS
52	67	137 amino acids	residue_range	However, they represent two different states of the 137 amino acids comprising (Sa)EctC protein (Fig 2).	RESULTS
80	82	Sa	species	However, they represent two different states of the 137 amino acids comprising (Sa)EctC protein (Fig 2).	RESULTS
83	87	EctC	protein	However, they represent two different states of the 137 amino acids comprising (Sa)EctC protein (Fig 2).	RESULTS
17	26	structure	evidence	First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein.	RESULTS
69	71	Sa	species	First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein.	RESULTS
72	76	EctC	protein	First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein.	RESULTS
109	125	Met-1 to Glu-115	residue_range	First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein.	RESULTS
137	142	lacks	protein_state	First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein.	RESULTS
143	157	22 amino acids	residue_range	First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein.	RESULTS
165	181	carboxy-terminus	structure_element	First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein.	RESULTS
200	202	Sa	species	First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein.	RESULTS
203	207	EctC	protein	First, the 1.2 Å structure reveals the spatial configuration of the (Sa)EctC protein ranging from amino acid Met-1 to Glu-115; hence, it lacks 22 amino acids at the carboxy-terminus of the authentic (Sa)EctC protein.	RESULTS
5	14	structure	evidence	This structure adopts an open conformation with respect to the typical fold of cupin barrels and is therefore termed in the following the “open” (Sa)EctC structure (Fig 4b).	RESULTS
25	29	open	protein_state	This structure adopts an open conformation with respect to the typical fold of cupin barrels and is therefore termed in the following the “open” (Sa)EctC structure (Fig 4b).	RESULTS
79	92	cupin barrels	structure_element	This structure adopts an open conformation with respect to the typical fold of cupin barrels and is therefore termed in the following the “open” (Sa)EctC structure (Fig 4b).	RESULTS
139	143	open	protein_state	This structure adopts an open conformation with respect to the typical fold of cupin barrels and is therefore termed in the following the “open” (Sa)EctC structure (Fig 4b).	RESULTS
146	148	Sa	species	This structure adopts an open conformation with respect to the typical fold of cupin barrels and is therefore termed in the following the “open” (Sa)EctC structure (Fig 4b).	RESULTS
149	153	EctC	protein	This structure adopts an open conformation with respect to the typical fold of cupin barrels and is therefore termed in the following the “open” (Sa)EctC structure (Fig 4b).	RESULTS
154	163	structure	evidence	This structure adopts an open conformation with respect to the typical fold of cupin barrels and is therefore termed in the following the “open” (Sa)EctC structure (Fig 4b).	RESULTS
8	17	structure	evidence	In this structure no metal co-factor was identified.	RESULTS
21	26	metal	chemical	In this structure no metal co-factor was identified.	RESULTS
11	28	crystal structure	evidence	The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation.	RESULTS
37	39	Sa	species	The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation.	RESULTS
40	44	EctC	protein	The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation.	RESULTS
57	63	solved	experimental_method	The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation.	RESULTS
165	173	protomer	oligomeric_state	The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation.	RESULTS
174	175	A	structure_element	The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation.	RESULTS
197	213	Met-1 to Gly-121	residue_range	The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation.	RESULTS
227	233	closed	protein_state	The second crystal structure of the (Sa)EctC protein was solved at a resolution of 2.0 Å and contained four molecules of the protein in the asymmetric unit of which protomer A comprised amino acid Met-1 to Gly-121 and adopts a closed conformation.	RESULTS
16	21	lacks	protein_state	Hence, it still lacks 16 amino acid residues of the carboxy-terminus of the authentic 137 amino acids comprising (Sa)EctC protein (Fig 2).	RESULTS
22	35	16 amino acid	residue_range	Hence, it still lacks 16 amino acid residues of the carboxy-terminus of the authentic 137 amino acids comprising (Sa)EctC protein (Fig 2).	RESULTS
52	68	carboxy-terminus	structure_element	Hence, it still lacks 16 amino acid residues of the carboxy-terminus of the authentic 137 amino acids comprising (Sa)EctC protein (Fig 2).	RESULTS
86	101	137 amino acids	residue_range	Hence, it still lacks 16 amino acid residues of the carboxy-terminus of the authentic 137 amino acids comprising (Sa)EctC protein (Fig 2).	RESULTS
114	116	Sa	species	Hence, it still lacks 16 amino acid residues of the carboxy-terminus of the authentic 137 amino acids comprising (Sa)EctC protein (Fig 2).	RESULTS
117	121	EctC	protein	Hence, it still lacks 16 amino acid residues of the carboxy-terminus of the authentic 137 amino acids comprising (Sa)EctC protein (Fig 2).	RESULTS
38	55	crystal structure	evidence	We therefore cannot exclude that this crystal structure does not represent the fully closed state of the ectoine synthase; consequently, we tentatively termed it the “semi-closed” (Sa)EctC structure.	RESULTS
79	91	fully closed	protein_state	We therefore cannot exclude that this crystal structure does not represent the fully closed state of the ectoine synthase; consequently, we tentatively termed it the “semi-closed” (Sa)EctC structure.	RESULTS
105	121	ectoine synthase	protein_type	We therefore cannot exclude that this crystal structure does not represent the fully closed state of the ectoine synthase; consequently, we tentatively termed it the “semi-closed” (Sa)EctC structure.	RESULTS
167	178	semi-closed	protein_state	We therefore cannot exclude that this crystal structure does not represent the fully closed state of the ectoine synthase; consequently, we tentatively termed it the “semi-closed” (Sa)EctC structure.	RESULTS
181	183	Sa	species	We therefore cannot exclude that this crystal structure does not represent the fully closed state of the ectoine synthase; consequently, we tentatively termed it the “semi-closed” (Sa)EctC structure.	RESULTS
184	188	EctC	protein	We therefore cannot exclude that this crystal structure does not represent the fully closed state of the ectoine synthase; consequently, we tentatively termed it the “semi-closed” (Sa)EctC structure.	RESULTS
189	198	structure	evidence	We therefore cannot exclude that this crystal structure does not represent the fully closed state of the ectoine synthase; consequently, we tentatively termed it the “semi-closed” (Sa)EctC structure.	RESULTS
31	39	monomers	oligomeric_state	Interestingly, the three other monomers present in the asymmetric unit all range from Met-1 to Glu-115 and adopt a conformation similar to the “open” EctC structure.	RESULTS
86	102	Met-1 to Glu-115	residue_range	Interestingly, the three other monomers present in the asymmetric unit all range from Met-1 to Glu-115 and adopt a conformation similar to the “open” EctC structure.	RESULTS
144	148	open	protein_state	Interestingly, the three other monomers present in the asymmetric unit all range from Met-1 to Glu-115 and adopt a conformation similar to the “open” EctC structure.	RESULTS
150	154	EctC	protein	Interestingly, the three other monomers present in the asymmetric unit all range from Met-1 to Glu-115 and adopt a conformation similar to the “open” EctC structure.	RESULTS
155	164	structure	evidence	Interestingly, the three other monomers present in the asymmetric unit all range from Met-1 to Glu-115 and adopt a conformation similar to the “open” EctC structure.	RESULTS
8	17	structure	evidence	Overall structure of the “open” and “semi-closed” crystal structures of (Sa)EctC.	FIG
26	30	open	protein_state	Overall structure of the “open” and “semi-closed” crystal structures of (Sa)EctC.	FIG
37	48	semi-closed	protein_state	Overall structure of the “open” and “semi-closed” crystal structures of (Sa)EctC.	FIG
50	68	crystal structures	evidence	Overall structure of the “open” and “semi-closed” crystal structures of (Sa)EctC.	FIG
73	75	Sa	species	Overall structure of the “open” and “semi-closed” crystal structures of (Sa)EctC.	FIG
76	80	EctC	protein	Overall structure of the “open” and “semi-closed” crystal structures of (Sa)EctC.	FIG
16	25	structure	evidence	(a) The overall structure of the “semi-closed” (Sa)EctC resolved at 2.0 Å is depicted in green in a cartoon (upper panel) and surface (lower panel) representation.	FIG
34	45	semi-closed	protein_state	(a) The overall structure of the “semi-closed” (Sa)EctC resolved at 2.0 Å is depicted in green in a cartoon (upper panel) and surface (lower panel) representation.	FIG
48	50	Sa	species	(a) The overall structure of the “semi-closed” (Sa)EctC resolved at 2.0 Å is depicted in green in a cartoon (upper panel) and surface (lower panel) representation.	FIG
51	55	EctC	protein	(a) The overall structure of the “semi-closed” (Sa)EctC resolved at 2.0 Å is depicted in green in a cartoon (upper panel) and surface (lower panel) representation.	FIG
4	13	β-strands	structure_element	The β-strands are numbered β1-β11 and the helices α-I to α-II.	FIG
27	33	β1-β11	structure_element	The β-strands are numbered β1-β11 and the helices α-I to α-II.	FIG
42	49	helices	structure_element	The β-strands are numbered β1-β11 and the helices α-I to α-II.	FIG
50	61	α-I to α-II	structure_element	The β-strands are numbered β1-β11 and the helices α-I to α-II.	FIG
16	25	structure	evidence	(b) The overall structure of the “open” (Sa)EctC was resolved at 1.2 Å and is depicted in yellow in a cartoon (upper panel) and surface (lower panel) representation.	FIG
34	38	open	protein_state	(b) The overall structure of the “open” (Sa)EctC was resolved at 1.2 Å and is depicted in yellow in a cartoon (upper panel) and surface (lower panel) representation.	FIG
41	43	Sa	species	(b) The overall structure of the “open” (Sa)EctC was resolved at 1.2 Å and is depicted in yellow in a cartoon (upper panel) and surface (lower panel) representation.	FIG
44	48	EctC	protein	(b) The overall structure of the “open” (Sa)EctC was resolved at 1.2 Å and is depicted in yellow in a cartoon (upper panel) and surface (lower panel) representation.	FIG
20	31	active site	site	The entrance to the active site of the ectoine synthase is marked.	FIG
39	55	ectoine synthase	protein_type	The entrance to the active site of the ectoine synthase is marked.	FIG
4	11	Overlay	experimental_method	(c) Overlay of the “semi-closed” and “open” (Sa)EctC structures.	FIG
20	31	semi-closed	protein_state	(c) Overlay of the “semi-closed” and “open” (Sa)EctC structures.	FIG
38	42	open	protein_state	(c) Overlay of the “semi-closed” and “open” (Sa)EctC structures.	FIG
45	47	Sa	species	(c) Overlay of the “semi-closed” and “open” (Sa)EctC structures.	FIG
48	52	EctC	protein	(c) Overlay of the “semi-closed” and “open” (Sa)EctC structures.	FIG
53	63	structures	evidence	(c) Overlay of the “semi-closed” and “open” (Sa)EctC structures.	FIG
12	21	structure	evidence	The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure.	RESULTS
26	28	Sa	species	The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure.	RESULTS
29	33	EctC	protein	The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure.	RESULTS
64	72	crystals	evidence	The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure.	RESULTS
88	104	carboxy-terminus	structure_element	The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure.	RESULTS
148	160	cupin barrel	structure_element	The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure.	RESULTS
186	193	monomer	oligomeric_state	The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure.	RESULTS
194	195	A	structure_element	The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure.	RESULTS
204	215	semi-closed	protein_state	The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure.	RESULTS
217	226	structure	evidence	The overall structure of (Sa)EctC is basically the same in both crystals except for the carboxy-terminus, which covers the entry of one side of the cupin barrel from the surroundings in monomer A in the “semi-closed” structure.	RESULTS
36	62	root mean square deviation	evidence	This is reflected by the calculated root mean square deviation (RMSD) of the Cα atoms that was about 0.56 Å (over 117 residues) when the four “open” monomers were compared with each other.	RESULTS
64	68	RMSD	evidence	This is reflected by the calculated root mean square deviation (RMSD) of the Cα atoms that was about 0.56 Å (over 117 residues) when the four “open” monomers were compared with each other.	RESULTS
143	147	open	protein_state	This is reflected by the calculated root mean square deviation (RMSD) of the Cα atoms that was about 0.56 Å (over 117 residues) when the four “open” monomers were compared with each other.	RESULTS
149	157	monomers	oligomeric_state	This is reflected by the calculated root mean square deviation (RMSD) of the Cα atoms that was about 0.56 Å (over 117 residues) when the four “open” monomers were compared with each other.	RESULTS
14	25	semi-closed	protein_state	However, the “semi-closed” monomer has a slightly higher RMSD of 1.4 Å (over 117 residues) when compared with the “open” 2.0 Å structure.	RESULTS
27	34	monomer	oligomeric_state	However, the “semi-closed” monomer has a slightly higher RMSD of 1.4 Å (over 117 residues) when compared with the “open” 2.0 Å structure.	RESULTS
57	61	RMSD	evidence	However, the “semi-closed” monomer has a slightly higher RMSD of 1.4 Å (over 117 residues) when compared with the “open” 2.0 Å structure.	RESULTS
115	119	open	protein_state	However, the “semi-closed” monomer has a slightly higher RMSD of 1.4 Å (over 117 residues) when compared with the “open” 2.0 Å structure.	RESULTS
127	136	structure	evidence	However, the “semi-closed” monomer has a slightly higher RMSD of 1.4 Å (over 117 residues) when compared with the “open” 2.0 Å structure.	RESULTS
52	61	structure	evidence	Therefore, we describe in the following the overall structure for the “semi-closed” form of the (Sa)EctC protein and subsequently highlight the structural differences between the “open” and “semi-closed” forms in more detail.	RESULTS
71	82	semi-closed	protein_state	Therefore, we describe in the following the overall structure for the “semi-closed” form of the (Sa)EctC protein and subsequently highlight the structural differences between the “open” and “semi-closed” forms in more detail.	RESULTS
97	99	Sa	species	Therefore, we describe in the following the overall structure for the “semi-closed” form of the (Sa)EctC protein and subsequently highlight the structural differences between the “open” and “semi-closed” forms in more detail.	RESULTS
100	104	EctC	protein	Therefore, we describe in the following the overall structure for the “semi-closed” form of the (Sa)EctC protein and subsequently highlight the structural differences between the “open” and “semi-closed” forms in more detail.	RESULTS
180	184	open	protein_state	Therefore, we describe in the following the overall structure for the “semi-closed” form of the (Sa)EctC protein and subsequently highlight the structural differences between the “open” and “semi-closed” forms in more detail.	RESULTS
191	202	semi-closed	protein_state	Therefore, we describe in the following the overall structure for the “semi-closed” form of the (Sa)EctC protein and subsequently highlight the structural differences between the “open” and “semi-closed” forms in more detail.	RESULTS
4	13	structure	evidence	The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a).	RESULTS
22	33	semi-closed	protein_state	The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a).	RESULTS
36	38	Sa	species	The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a).	RESULTS
39	43	EctC	protein	The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a).	RESULTS
67	76	β-strands	structure_element	The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a).	RESULTS
78	84	β1-β11	structure_element	The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a).	RESULTS
94	103	α-helices	structure_element	The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a).	RESULTS
105	108	α-I	structure_element	The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a).	RESULTS
113	117	α-II	structure_element	The structure of the “semi-closed” (Sa)EctC protein consists of 11 β-strands (β1-β11) and two α-helices (α-I and α-II) (Fig 4a).	RESULTS
4	13	β-strands	structure_element	The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively.	RESULTS
23	45	anti-parallel β-sheets	structure_element	The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively.	RESULTS
47	49	β2	structure_element	The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively.	RESULTS
50	52	β3	structure_element	The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively.	RESULTS
54	56	β4	structure_element	The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively.	RESULTS
58	61	β11	structure_element	The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively.	RESULTS
63	65	β6	structure_element	The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively.	RESULTS
71	73	β9	structure_element	The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively.	RESULTS
89	111	three-stranded β-sheet	structure_element	The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively.	RESULTS
113	115	β7	structure_element	The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively.	RESULTS
117	119	β8	structure_element	The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively.	RESULTS
125	128	β10	structure_element	The β-strands form two anti-parallel β-sheets: β2 β3, β4, β11, β6, and β9, and a smaller three-stranded β-sheet (β7, β8, and β10), respectively.	RESULTS
10	18	β-sheets	structure_element	These two β-sheets pack against each other, forming a cup-shaped β-sandwich with a topology characteristic for the cupin-fold.	RESULTS
54	75	cup-shaped β-sandwich	structure_element	These two β-sheets pack against each other, forming a cup-shaped β-sandwich with a topology characteristic for the cupin-fold.	RESULTS
115	125	cupin-fold	structure_element	These two β-sheets pack against each other, forming a cup-shaped β-sandwich with a topology characteristic for the cupin-fold.	RESULTS
8	10	Sa	species	Hence, (Sa)EctC adopts an overall bowl shape in which one side is opened towards the solvent (Fig 4a to 4c).	RESULTS
11	15	EctC	protein	Hence, (Sa)EctC adopts an overall bowl shape in which one side is opened towards the solvent (Fig 4a to 4c).	RESULTS
8	19	semi-closed	protein_state	In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a).	RESULTS
21	30	structure	evidence	In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a).	RESULTS
41	62	carboxy-terminal tail	structure_element	In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a).	RESULTS
81	97	electron density	evidence	In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a).	RESULTS
114	125	small helix	structure_element	In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a).	RESULTS
127	131	α-II	structure_element	In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a).	RESULTS
149	160	active site	site	In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a).	RESULTS
169	171	Sa	species	In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a).	RESULTS
172	176	EctC	protein	In the “semi-closed” structure, a longer carboxy-terminal tail is visible in the electron density, folding into a small helix (α-II) that closes the active site of the (Sa)EctC protein (Fig 4a).	RESULTS
22	32	α-II helix	structure_element	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
77	89	unstructured	protein_state	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
90	94	loop	structure_element	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
116	120	open	protein_state	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
122	131	structure	evidence	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
144	146	β4	structure_element	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
151	153	β6	structure_element	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
189	195	stable	protein_state	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
196	204	β-strand	structure_element	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
205	207	β5	structure_element	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
228	239	semi-closed	protein_state	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
254	256	Sa	species	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
257	261	EctC	protein	The formation of this α-II helix induces a reorientation and shift of a long unstructured loop (as observed in the “open” structure) connecting β4 and β6, resulting in the formation of the stable β-strand β5 as observed in the “semi-closed”state of the (Sa)EctC protein (Fig 4a).	RESULTS
0	30	Structural comparison analyses	experimental_method	Structural comparison analyses using the DALI server revealed that (Sa)EctC adopts a fold similar to other members of the cupin superfamily.	RESULTS
41	52	DALI server	experimental_method	Structural comparison analyses using the DALI server revealed that (Sa)EctC adopts a fold similar to other members of the cupin superfamily.	RESULTS
68	70	Sa	species	Structural comparison analyses using the DALI server revealed that (Sa)EctC adopts a fold similar to other members of the cupin superfamily.	RESULTS
71	75	EctC	protein	Structural comparison analyses using the DALI server revealed that (Sa)EctC adopts a fold similar to other members of the cupin superfamily.	RESULTS
122	139	cupin superfamily	protein_type	Structural comparison analyses using the DALI server revealed that (Sa)EctC adopts a fold similar to other members of the cupin superfamily.	RESULTS
57	96	Cupin 2 conserved barrel domain protein	protein	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
98	109	YP_751781.1	protein	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
116	140	Shewanella frigidimarina	species	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
175	182	Z-score	evidence	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
198	202	RMSD	evidence	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
332	341	structure	evidence	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
369	395	manganese-containing cupin	protein	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
397	403	TM1459	protein	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
410	429	Thermotoga maritima	species	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
464	471	Z-score	evidence	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
487	491	RMSD	evidence	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
524	531	cyclase	protein_type	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
532	536	RemF	protein	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
542	571	Streptomyces resistomycificus	species	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
605	612	Z-score	evidence	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
628	632	RMSD	evidence	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
669	692	auxin-binding protein 1	protein	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
698	706	Zea mays	species	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
742	749	Z-score	evidence	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
765	769	RMSD	evidence	The highest structural similarities are observed for the Cupin 2 conserved barrel domain protein (YP_751781.1) from Shewanella frigidimarina (PDB accession code: 2PFW) with a Z-score of 13.1 and an RMSD of 2.2 Å over 104 Cα-atoms (structural data for this protein have been deposited in the PDB but no publication connected to this structure is currently available), a manganese-containing cupin (TM1459) from Thermotoga maritima (PDB accession code: 1VJ2) with a Z-score of 12.8 and an RMSD of 2.0 Å over 103 Cα-atoms, the cyclase RemF from Streptomyces resistomycificus (PDB accession code: 3HT1 with a Z-score of 11.9 and an RMSD of 1.9 Å over 102 Cα-atoms), and an auxin-binding protein 1 from Zea mays (PDB accession code: 1LR5) with an Z-score of 11.8 and an RMSD of 2.8 Å over 104 Cα-atoms).	RESULTS
18	22	EctC	protein	Our data classify EctC, in addition to the polyketide cyclase RemF, as the second known cupin-related enzyme that catalyze a cyclocondensation reaction.	RESULTS
43	61	polyketide cyclase	protein_type	Our data classify EctC, in addition to the polyketide cyclase RemF, as the second known cupin-related enzyme that catalyze a cyclocondensation reaction.	RESULTS
62	66	RemF	protein	Our data classify EctC, in addition to the polyketide cyclase RemF, as the second known cupin-related enzyme that catalyze a cyclocondensation reaction.	RESULTS
88	101	cupin-related	protein_type	Our data classify EctC, in addition to the polyketide cyclase RemF, as the second known cupin-related enzyme that catalyze a cyclocondensation reaction.	RESULTS
8	12	RemF	protein	Next to RemF and the aldos-2-ulose dehydratase/isomerase, the ectoine synthase is only the third characterized dehydratase within the cupin superfamily.	RESULTS
21	46	aldos-2-ulose dehydratase	protein_type	Next to RemF and the aldos-2-ulose dehydratase/isomerase, the ectoine synthase is only the third characterized dehydratase within the cupin superfamily.	RESULTS
47	56	isomerase	protein_type	Next to RemF and the aldos-2-ulose dehydratase/isomerase, the ectoine synthase is only the third characterized dehydratase within the cupin superfamily.	RESULTS
62	78	ectoine synthase	protein_type	Next to RemF and the aldos-2-ulose dehydratase/isomerase, the ectoine synthase is only the third characterized dehydratase within the cupin superfamily.	RESULTS
111	122	dehydratase	protein_type	Next to RemF and the aldos-2-ulose dehydratase/isomerase, the ectoine synthase is only the third characterized dehydratase within the cupin superfamily.	RESULTS
134	151	cupin superfamily	protein_type	Next to RemF and the aldos-2-ulose dehydratase/isomerase, the ectoine synthase is only the third characterized dehydratase within the cupin superfamily.	RESULTS
16	20	EctC	protein	Analysis of the EctC dimer interface as observed in the (Sa)EctC crystal structure	RESULTS
21	36	dimer interface	site	Analysis of the EctC dimer interface as observed in the (Sa)EctC crystal structure	RESULTS
57	59	Sa	species	Analysis of the EctC dimer interface as observed in the (Sa)EctC crystal structure	RESULTS
60	64	EctC	protein	Analysis of the EctC dimer interface as observed in the (Sa)EctC crystal structure	RESULTS
65	82	crystal structure	evidence	Analysis of the EctC dimer interface as observed in the (Sa)EctC crystal structure	RESULTS
9	12	SEC	experimental_method	Both the SEC analysis and the HPLC-MALS experiments (S2b Fig) have shown that the ectoine synthase from S. alaskensis is a dimer in solution.	RESULTS
30	39	HPLC-MALS	experimental_method	Both the SEC analysis and the HPLC-MALS experiments (S2b Fig) have shown that the ectoine synthase from S. alaskensis is a dimer in solution.	RESULTS
82	98	ectoine synthase	protein_type	Both the SEC analysis and the HPLC-MALS experiments (S2b Fig) have shown that the ectoine synthase from S. alaskensis is a dimer in solution.	RESULTS
104	117	S. alaskensis	species	Both the SEC analysis and the HPLC-MALS experiments (S2b Fig) have shown that the ectoine synthase from S. alaskensis is a dimer in solution.	RESULTS
123	128	dimer	oligomeric_state	Both the SEC analysis and the HPLC-MALS experiments (S2b Fig) have shown that the ectoine synthase from S. alaskensis is a dimer in solution.	RESULTS
4	21	crystal structure	evidence	The crystal structure of this protein reflects this quaternary arrangement.	RESULTS
8	19	semi-closed	protein_state	In the “semi-closed” crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit.	RESULTS
21	38	crystal structure	evidence	In the “semi-closed” crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit.	RESULTS
41	43	Sa	species	In the “semi-closed” crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit.	RESULTS
44	48	EctC	protein	In the “semi-closed” crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit.	RESULTS
53	65	crystallized	experimental_method	In the “semi-closed” crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit.	RESULTS
71	76	dimer	oligomeric_state	In the “semi-closed” crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit.	RESULTS
80	86	dimers	oligomeric_state	In the “semi-closed” crystal structure, (Sa)EctC has crystallized as a dimer of dimers within the asymmetric unit.	RESULTS
5	10	dimer	oligomeric_state	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
46	54	monomers	oligomeric_state	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
69	81	head-to-tail	protein_state	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
152	174	antiparallel β-strands	structure_element	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
176	184	β-strand	structure_element	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
185	187	β1	structure_element	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
198	205	1MIVRN5	structure_element	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
212	219	monomer	oligomeric_state	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
220	221	A	structure_element	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
226	234	β-strand	structure_element	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
235	237	β8	structure_element	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
243	250	monomer	oligomeric_state	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
251	252	B	structure_element	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
263	273	82GVMYAL87	structure_element	This dimer (Fig 5a and 5b) is composed of two monomers arranged in a head-to-tail orientation and is stabilized via strong interactions mediated by two antiparallel β-strands, β-strand β1 (sequence 1MIVRN5) from monomer A and β-strand β8 from monomer B (sequence 82GVMYAL87) (Fig 5c).	RESULTS
38	47	β-strands	structure_element	The strong interactions between these β-strands rely primarily on backbone contacts.	RESULTS
47	71	hydrophobic interactions	bond_interaction	In addition to these interactions, some weaker hydrophobic interactions are also observed between the two monomers in some loops connecting the β-strands.	RESULTS
106	114	monomers	oligomeric_state	In addition to these interactions, some weaker hydrophobic interactions are also observed between the two monomers in some loops connecting the β-strands.	RESULTS
123	128	loops	structure_element	In addition to these interactions, some weaker hydrophobic interactions are also observed between the two monomers in some loops connecting the β-strands.	RESULTS
144	153	β-strands	structure_element	In addition to these interactions, some weaker hydrophobic interactions are also observed between the two monomers in some loops connecting the β-strands.	RESULTS
19	27	PDBePISA	experimental_method	As calculated with PDBePISA, the surface area buried upon dimer formation is 1462 Å2, which is 20.5% of the total accessible surface of a monomer of this protein.	RESULTS
58	63	dimer	oligomeric_state	As calculated with PDBePISA, the surface area buried upon dimer formation is 1462 Å2, which is 20.5% of the total accessible surface of a monomer of this protein.	RESULTS
138	145	monomer	oligomeric_state	As calculated with PDBePISA, the surface area buried upon dimer formation is 1462 Å2, which is 20.5% of the total accessible surface of a monomer of this protein.	RESULTS
55	61	dimers	oligomeric_state	Both values fall within the range for known functional dimers.	RESULTS
0	17	Crystal structure	evidence	Crystal structure of (Sa)EctC.	FIG
22	24	Sa	species	Crystal structure of (Sa)EctC.	FIG
25	29	EctC	protein	Crystal structure of (Sa)EctC.	FIG
20	25	dimer	oligomeric_state	(a) Top-view of the dimer of the (Sa)EctC protein.	FIG
34	36	Sa	species	(a) Top-view of the dimer of the (Sa)EctC protein.	FIG
37	41	EctC	protein	(a) Top-view of the dimer of the (Sa)EctC protein.	FIG
20	25	water	chemical	The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer.	FIG
92	100	monomers	oligomeric_state	The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer.	FIG
142	144	Sa	species	The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer.	FIG
145	149	EctC	protein	The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer.	FIG
150	155	dimer	oligomeric_state	The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer.	FIG
186	201	dimer interface	site	The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer.	FIG
216	225	β-strands	structure_element	The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer.	FIG
234	241	monomer	oligomeric_state	The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (Sa)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer.	FIG
35	50	dimer interface	site	(c) Close-up representation of the dimer interface mediated by beta-strand β1 and β6.	FIG
63	74	beta-strand	structure_element	(c) Close-up representation of the dimer interface mediated by beta-strand β1 and β6.	FIG
75	77	β1	structure_element	(c) Close-up representation of the dimer interface mediated by beta-strand β1 and β6.	FIG
82	84	β6	structure_element	(c) Close-up representation of the dimer interface mediated by beta-strand β1 and β6.	FIG
8	12	open	protein_state	In the “open” (Sa)EctC structure, one monomer is present in the asymmetric unit.	RESULTS
15	17	Sa	species	In the “open” (Sa)EctC structure, one monomer is present in the asymmetric unit.	RESULTS
18	22	EctC	protein	In the “open” (Sa)EctC structure, one monomer is present in the asymmetric unit.	RESULTS
23	32	structure	evidence	In the “open” (Sa)EctC structure, one monomer is present in the asymmetric unit.	RESULTS
38	45	monomer	oligomeric_state	In the “open” (Sa)EctC structure, one monomer is present in the asymmetric unit.	RESULTS
35	42	packing	experimental_method	We therefore inspected the crystal packing and analyzed the monomer-monomer interactions with symmetry related molecules to elucidate whether a physiologically relevant dimer could be deduced from this crystal form as well.	RESULTS
60	67	monomer	oligomeric_state	We therefore inspected the crystal packing and analyzed the monomer-monomer interactions with symmetry related molecules to elucidate whether a physiologically relevant dimer could be deduced from this crystal form as well.	RESULTS
68	75	monomer	oligomeric_state	We therefore inspected the crystal packing and analyzed the monomer-monomer interactions with symmetry related molecules to elucidate whether a physiologically relevant dimer could be deduced from this crystal form as well.	RESULTS
169	174	dimer	oligomeric_state	We therefore inspected the crystal packing and analyzed the monomer-monomer interactions with symmetry related molecules to elucidate whether a physiologically relevant dimer could be deduced from this crystal form as well.	RESULTS
202	214	crystal form	evidence	We therefore inspected the crystal packing and analyzed the monomer-monomer interactions with symmetry related molecules to elucidate whether a physiologically relevant dimer could be deduced from this crystal form as well.	RESULTS
18	23	dimer	oligomeric_state	Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets.	RESULTS
68	79	semi-closed	protein_state	Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets.	RESULTS
82	84	Sa	species	Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets.	RESULTS
85	89	EctC	protein	Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets.	RESULTS
90	99	structure	evidence	Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets.	RESULTS
126	133	monomer	oligomeric_state	Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets.	RESULTS
134	141	monomer	oligomeric_state	Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets.	RESULTS
175	183	β-sheets	structure_element	Indeed, a similar dimer configuration to the one described for the “semi-closed” (Sa)EctC structure is observed with the same monomer-monomer interactions mediated by the two β-sheets.	RESULTS
109	117	monomers	oligomeric_state	The crystallographic two-fold axis present within the crystal symmetry is located exactly in between the two monomers, resulting in a monomer within the asymmetric unit.	RESULTS
134	141	monomer	oligomeric_state	The crystallographic two-fold axis present within the crystal symmetry is located exactly in between the two monomers, resulting in a monomer within the asymmetric unit.	RESULTS
16	21	dimer	oligomeric_state	Hence, the same dimer observed in the “semi-closed” structure of (Sa)EctC can also be observed in the “open” structure.	RESULTS
39	50	semi-closed	protein_state	Hence, the same dimer observed in the “semi-closed” structure of (Sa)EctC can also be observed in the “open” structure.	RESULTS
52	61	structure	evidence	Hence, the same dimer observed in the “semi-closed” structure of (Sa)EctC can also be observed in the “open” structure.	RESULTS
66	68	Sa	species	Hence, the same dimer observed in the “semi-closed” structure of (Sa)EctC can also be observed in the “open” structure.	RESULTS
69	73	EctC	protein	Hence, the same dimer observed in the “semi-closed” structure of (Sa)EctC can also be observed in the “open” structure.	RESULTS
103	107	open	protein_state	Hence, the same dimer observed in the “semi-closed” structure of (Sa)EctC can also be observed in the “open” structure.	RESULTS
109	118	structure	evidence	Hence, the same dimer observed in the “semi-closed” structure of (Sa)EctC can also be observed in the “open” structure.	RESULTS
62	73	DALI search	experimental_method	Interestingly, the proteins identified by the above-described DALI search not only have folds similar to EctC, but are also functional dimers that adopt similar monomer-monomer interactions within the dimer assembly as deduced from the inspection of the corresponding PDB files (2PFW, 3HT1, 1VJ2, 1LR5).	RESULTS
105	109	EctC	protein	Interestingly, the proteins identified by the above-described DALI search not only have folds similar to EctC, but are also functional dimers that adopt similar monomer-monomer interactions within the dimer assembly as deduced from the inspection of the corresponding PDB files (2PFW, 3HT1, 1VJ2, 1LR5).	RESULTS
135	141	dimers	oligomeric_state	Interestingly, the proteins identified by the above-described DALI search not only have folds similar to EctC, but are also functional dimers that adopt similar monomer-monomer interactions within the dimer assembly as deduced from the inspection of the corresponding PDB files (2PFW, 3HT1, 1VJ2, 1LR5).	RESULTS
161	168	monomer	oligomeric_state	Interestingly, the proteins identified by the above-described DALI search not only have folds similar to EctC, but are also functional dimers that adopt similar monomer-monomer interactions within the dimer assembly as deduced from the inspection of the corresponding PDB files (2PFW, 3HT1, 1VJ2, 1LR5).	RESULTS
169	176	monomer	oligomeric_state	Interestingly, the proteins identified by the above-described DALI search not only have folds similar to EctC, but are also functional dimers that adopt similar monomer-monomer interactions within the dimer assembly as deduced from the inspection of the corresponding PDB files (2PFW, 3HT1, 1VJ2, 1LR5).	RESULTS
201	206	dimer	oligomeric_state	Interestingly, the proteins identified by the above-described DALI search not only have folds similar to EctC, but are also functional dimers that adopt similar monomer-monomer interactions within the dimer assembly as deduced from the inspection of the corresponding PDB files (2PFW, 3HT1, 1VJ2, 1LR5).	RESULTS
33	41	flexible	protein_state	Structural rearrangements of the flexible (Sa)EctC carboxy-terminus	RESULTS
43	45	Sa	species	Structural rearrangements of the flexible (Sa)EctC carboxy-terminus	RESULTS
46	50	EctC	protein	Structural rearrangements of the flexible (Sa)EctC carboxy-terminus	RESULTS
51	67	carboxy-terminus	structure_element	Structural rearrangements of the flexible (Sa)EctC carboxy-terminus	RESULTS
54	70	ectoine synthase	protein_type	The cupin core represents the structural framework of ectoine synthase (Figs 4 and 5).	RESULTS
32	50	crystal structures	evidence	The major difference in the two crystal structures of the (Sa)EctC protein reported here is the orientation of the carboxy-terminus.	RESULTS
59	61	Sa	species	The major difference in the two crystal structures of the (Sa)EctC protein reported here is the orientation of the carboxy-terminus.	RESULTS
62	66	EctC	protein	The major difference in the two crystal structures of the (Sa)EctC protein reported here is the orientation of the carboxy-terminus.	RESULTS
115	131	carboxy-terminus	structure_element	The major difference in the two crystal structures of the (Sa)EctC protein reported here is the orientation of the carboxy-terminus.	RESULTS
32	55	carboxy-terminal region	structure_element	Some amino acids located in the carboxy-terminal region of the 137 amino acids comprising (Sa)EctC protein are highly conserved (Fig 2) within the extended EctC protein family.	RESULTS
63	78	137 amino acids	residue_range	Some amino acids located in the carboxy-terminal region of the 137 amino acids comprising (Sa)EctC protein are highly conserved (Fig 2) within the extended EctC protein family.	RESULTS
91	93	Sa	species	Some amino acids located in the carboxy-terminal region of the 137 amino acids comprising (Sa)EctC protein are highly conserved (Fig 2) within the extended EctC protein family.	RESULTS
94	98	EctC	protein	Some amino acids located in the carboxy-terminal region of the 137 amino acids comprising (Sa)EctC protein are highly conserved (Fig 2) within the extended EctC protein family.	RESULTS
111	127	highly conserved	protein_state	Some amino acids located in the carboxy-terminal region of the 137 amino acids comprising (Sa)EctC protein are highly conserved (Fig 2) within the extended EctC protein family.	RESULTS
147	155	extended	protein_state	Some amino acids located in the carboxy-terminal region of the 137 amino acids comprising (Sa)EctC protein are highly conserved (Fig 2) within the extended EctC protein family.	RESULTS
156	168	EctC protein	protein_type	Some amino acids located in the carboxy-terminal region of the 137 amino acids comprising (Sa)EctC protein are highly conserved (Fig 2) within the extended EctC protein family.	RESULTS
14	22	β-strand	structure_element	At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein.	RESULTS
23	26	β11	structure_element	At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein.	RESULTS
44	53	conserved	protein_state	At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein.	RESULTS
54	61	proline	residue_name	At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein.	RESULTS
72	79	Pro-109	residue_name_number	At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein.	RESULTS
84	91	Pro-110	residue_name_number	At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein.	RESULTS
163	165	Sa	species	At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein.	RESULTS
166	170	EctC	protein	At the end of β-strand β11, two consecutive conserved proline residues (Pro-109 and Pro-110) are present that are responsible for a turn in the main chain of the (Sa)EctC protein.	RESULTS
8	19	semi-closed	protein_state	In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121.	RESULTS
22	24	Sa	species	In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121.	RESULTS
25	29	EctC	protein	In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121.	RESULTS
30	39	structure	evidence	In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121.	RESULTS
53	69	electron density	evidence	In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121.	RESULTS
77	93	carboxy-terminus	structure_element	In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121.	RESULTS
109	130	7 amino acid residues	residue_range	In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121.	RESULTS
152	159	Gly-121	residue_name_number	In the “semi-closed” (Sa)EctC structure, the visible electron density of the carboxy-terminus is extended by 7 amino acid residues and ends at position Gly-121.	RESULTS
41	52	small helix	structure_element	These additional amino acids fold into a small helix, which seals the open cavity of the cupin-fold of the (Sa)EctC protein (Fig 4a).	RESULTS
70	74	open	protein_state	These additional amino acids fold into a small helix, which seals the open cavity of the cupin-fold of the (Sa)EctC protein (Fig 4a).	RESULTS
75	81	cavity	site	These additional amino acids fold into a small helix, which seals the open cavity of the cupin-fold of the (Sa)EctC protein (Fig 4a).	RESULTS
89	99	cupin-fold	structure_element	These additional amino acids fold into a small helix, which seals the open cavity of the cupin-fold of the (Sa)EctC protein (Fig 4a).	RESULTS
108	110	Sa	species	These additional amino acids fold into a small helix, which seals the open cavity of the cupin-fold of the (Sa)EctC protein (Fig 4a).	RESULTS
111	115	EctC	protein	These additional amino acids fold into a small helix, which seals the open cavity of the cupin-fold of the (Sa)EctC protein (Fig 4a).	RESULTS
18	23	helix	structure_element	Furthermore, this helix is stabilized via interactions with the loop region between β-strands β4 and β6, thereby inducing a structural rearrangement.	RESULTS
64	75	loop region	structure_element	Furthermore, this helix is stabilized via interactions with the loop region between β-strands β4 and β6, thereby inducing a structural rearrangement.	RESULTS
84	93	β-strands	structure_element	Furthermore, this helix is stabilized via interactions with the loop region between β-strands β4 and β6, thereby inducing a structural rearrangement.	RESULTS
94	96	β4	structure_element	Furthermore, this helix is stabilized via interactions with the loop region between β-strands β4 and β6, thereby inducing a structural rearrangement.	RESULTS
101	103	β6	structure_element	Furthermore, this helix is stabilized via interactions with the loop region between β-strands β4 and β6, thereby inducing a structural rearrangement.	RESULTS
30	38	β-strand	structure_element	This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure.	RESULTS
39	41	β5	structure_element	This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure.	RESULTS
73	95	small C-terminal helix	structure_element	This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure.	RESULTS
99	105	absent	protein_state	This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure.	RESULTS
126	130	open	protein_state	This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure.	RESULTS
133	135	Sa	species	This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure.	RESULTS
136	140	EctC	protein	This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure.	RESULTS
141	150	structure	evidence	This induces the formation of β-strand β5, which is not present when the small C-terminal helix is absent as observed in the “open” (Sa)EctC structure.	RESULTS
30	38	β-strand	structure_element	As a result, the newly formed β-strand β5 is reoriented and moved by 2.4 Å within the “semi-closed” (Sa)EctC structure (Fig 4a to 4c).	RESULTS
39	41	β5	structure_element	As a result, the newly formed β-strand β5 is reoriented and moved by 2.4 Å within the “semi-closed” (Sa)EctC structure (Fig 4a to 4c).	RESULTS
87	98	semi-closed	protein_state	As a result, the newly formed β-strand β5 is reoriented and moved by 2.4 Å within the “semi-closed” (Sa)EctC structure (Fig 4a to 4c).	RESULTS
101	103	Sa	species	As a result, the newly formed β-strand β5 is reoriented and moved by 2.4 Å within the “semi-closed” (Sa)EctC structure (Fig 4a to 4c).	RESULTS
104	108	EctC	protein	As a result, the newly formed β-strand β5 is reoriented and moved by 2.4 Å within the “semi-closed” (Sa)EctC structure (Fig 4a to 4c).	RESULTS
109	118	structure	evidence	As a result, the newly formed β-strand β5 is reoriented and moved by 2.4 Å within the “semi-closed” (Sa)EctC structure (Fig 4a to 4c).	RESULTS
28	36	β-strand	structure_element	It is worth mentioning that β-strand β5 is located next to His-93, which in all likelihood involved in metal binding (see below).	RESULTS
37	39	β5	structure_element	It is worth mentioning that β-strand β5 is located next to His-93, which in all likelihood involved in metal binding (see below).	RESULTS
59	65	His-93	residue_name_number	It is worth mentioning that β-strand β5 is located next to His-93, which in all likelihood involved in metal binding (see below).	RESULTS
103	108	metal	chemical	It is worth mentioning that β-strand β5 is located next to His-93, which in all likelihood involved in metal binding (see below).	RESULTS
21	24	His	residue_name	The position of this His residue is slightly shifted in both (Sa)EctC structures, likely the result of the formation of β-strand β5.	RESULTS
62	64	Sa	species	The position of this His residue is slightly shifted in both (Sa)EctC structures, likely the result of the formation of β-strand β5.	RESULTS
65	69	EctC	protein	The position of this His residue is slightly shifted in both (Sa)EctC structures, likely the result of the formation of β-strand β5.	RESULTS
70	80	structures	evidence	The position of this His residue is slightly shifted in both (Sa)EctC structures, likely the result of the formation of β-strand β5.	RESULTS
120	128	β-strand	structure_element	The position of this His residue is slightly shifted in both (Sa)EctC structures, likely the result of the formation of β-strand β5.	RESULTS
129	131	β5	structure_element	The position of this His residue is slightly shifted in both (Sa)EctC structures, likely the result of the formation of β-strand β5.	RESULTS
29	39	cupin fold	structure_element	Therefore the sealing of the cupin fold, as described above, seem to have an indirect influence on the architecture of the postulated iron-binding site.	RESULTS
134	151	iron-binding site	site	Therefore the sealing of the cupin fold, as described above, seem to have an indirect influence on the architecture of the postulated iron-binding site.	RESULTS
16	23	Pro-109	residue_name_number	The consecutive Pro-109 and Pro-110 residues found at the end of β-strand β11are highly conserved in EctC-type proteins (Fig 2).	RESULTS
28	35	Pro-110	residue_name_number	The consecutive Pro-109 and Pro-110 residues found at the end of β-strand β11are highly conserved in EctC-type proteins (Fig 2).	RESULTS
65	73	β-strand	structure_element	The consecutive Pro-109 and Pro-110 residues found at the end of β-strand β11are highly conserved in EctC-type proteins (Fig 2).	RESULTS
74	77	β11	structure_element	The consecutive Pro-109 and Pro-110 residues found at the end of β-strand β11are highly conserved in EctC-type proteins (Fig 2).	RESULTS
81	97	highly conserved	protein_state	The consecutive Pro-109 and Pro-110 residues found at the end of β-strand β11are highly conserved in EctC-type proteins (Fig 2).	RESULTS
101	119	EctC-type proteins	protein_type	The consecutive Pro-109 and Pro-110 residues found at the end of β-strand β11are highly conserved in EctC-type proteins (Fig 2).	RESULTS
69	85	carboxy-terminus	structure_element	They are responsible for redirecting the main chain of the remaining carboxy-terminus (27 amino acid residues) of (Sa)EctC to close the cupin fold.	RESULTS
87	109	27 amino acid residues	residue_range	They are responsible for redirecting the main chain of the remaining carboxy-terminus (27 amino acid residues) of (Sa)EctC to close the cupin fold.	RESULTS
115	117	Sa	species	They are responsible for redirecting the main chain of the remaining carboxy-terminus (27 amino acid residues) of (Sa)EctC to close the cupin fold.	RESULTS
118	122	EctC	protein	They are responsible for redirecting the main chain of the remaining carboxy-terminus (27 amino acid residues) of (Sa)EctC to close the cupin fold.	RESULTS
136	146	cupin fold	structure_element	They are responsible for redirecting the main chain of the remaining carboxy-terminus (27 amino acid residues) of (Sa)EctC to close the cupin fold.	RESULTS
8	19	semi-closed	protein_state	In the “semi-closed” structure this results in a complete closure of the entry of the cupin barrel (Fig 4a to 4c).	RESULTS
21	30	structure	evidence	In the “semi-closed” structure this results in a complete closure of the entry of the cupin barrel (Fig 4a to 4c).	RESULTS
86	98	cupin barrel	structure_element	In the “semi-closed” structure this results in a complete closure of the entry of the cupin barrel (Fig 4a to 4c).	RESULTS
8	12	open	protein_state	In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus.	RESULTS
15	17	Sa	species	In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus.	RESULTS
18	22	EctC	protein	In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus.	RESULTS
23	32	structure	evidence	In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus.	RESULTS
39	46	proline	residue_name	In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus.	RESULTS
75	91	electron density	evidence	In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus.	RESULTS
124	131	Pro-110	residue_name_number	In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus.	RESULTS
137	153	electron density	evidence	In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus.	RESULTS
213	229	carboxy-terminus	structure_element	In the “open” (Sa)EctC structure, both proline residues are visible in the electron density; however, almost directly after Pro-110, the electron density is drastically diminished caused by the flexibility of the carboxy-terminus.	RESULTS
39	46	Pro-109	residue_name_number	A search for partners interacting with Pro-109 revealed that it interacts via its backbone oxygen with the side chain of His-55 as visible in both the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
121	127	His-55	residue_name_number	A search for partners interacting with Pro-109 revealed that it interacts via its backbone oxygen with the side chain of His-55 as visible in both the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
152	156	open	protein_state	A search for partners interacting with Pro-109 revealed that it interacts via its backbone oxygen with the side chain of His-55 as visible in both the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
163	174	semi-closed	protein_state	A search for partners interacting with Pro-109 revealed that it interacts via its backbone oxygen with the side chain of His-55 as visible in both the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
177	179	Sa	species	A search for partners interacting with Pro-109 revealed that it interacts via its backbone oxygen with the side chain of His-55 as visible in both the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
180	184	EctC	protein	A search for partners interacting with Pro-109 revealed that it interacts via its backbone oxygen with the side chain of His-55 as visible in both the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
185	195	structures	evidence	A search for partners interacting with Pro-109 revealed that it interacts via its backbone oxygen with the side chain of His-55 as visible in both the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
4	11	Pro-109	residue_name_number	The Pro-109/His-55 interaction ensures the stable orientation of both proline residues at the end of β-strand β11.	RESULTS
12	18	His-55	residue_name_number	The Pro-109/His-55 interaction ensures the stable orientation of both proline residues at the end of β-strand β11.	RESULTS
43	49	stable	protein_state	The Pro-109/His-55 interaction ensures the stable orientation of both proline residues at the end of β-strand β11.	RESULTS
70	77	proline	residue_name	The Pro-109/His-55 interaction ensures the stable orientation of both proline residues at the end of β-strand β11.	RESULTS
101	109	β-strand	structure_element	The Pro-109/His-55 interaction ensures the stable orientation of both proline residues at the end of β-strand β11.	RESULTS
110	113	β11	structure_element	The Pro-109/His-55 interaction ensures the stable orientation of both proline residues at the end of β-strand β11.	RESULTS
12	19	proline	residue_name	Since these proline residues are followed by the carboxy-terminal region of the (Sa)EctC protein, the interaction of His-55 with Pro-109 will likely play a substantial role in spatially orienting this very flexible part of the protein.	RESULTS
49	72	carboxy-terminal region	structure_element	Since these proline residues are followed by the carboxy-terminal region of the (Sa)EctC protein, the interaction of His-55 with Pro-109 will likely play a substantial role in spatially orienting this very flexible part of the protein.	RESULTS
81	83	Sa	species	Since these proline residues are followed by the carboxy-terminal region of the (Sa)EctC protein, the interaction of His-55 with Pro-109 will likely play a substantial role in spatially orienting this very flexible part of the protein.	RESULTS
84	88	EctC	protein	Since these proline residues are followed by the carboxy-terminal region of the (Sa)EctC protein, the interaction of His-55 with Pro-109 will likely play a substantial role in spatially orienting this very flexible part of the protein.	RESULTS
117	123	His-55	residue_name_number	Since these proline residues are followed by the carboxy-terminal region of the (Sa)EctC protein, the interaction of His-55 with Pro-109 will likely play a substantial role in spatially orienting this very flexible part of the protein.	RESULTS
129	136	Pro-109	residue_name_number	Since these proline residues are followed by the carboxy-terminal region of the (Sa)EctC protein, the interaction of His-55 with Pro-109 will likely play a substantial role in spatially orienting this very flexible part of the protein.	RESULTS
40	47	Pro-109	residue_name_number	In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further.	RESULTS
52	58	His-55	residue_name_number	In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further.	RESULTS
64	87	carboxy-terminal region	structure_element	In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further.	RESULTS
92	94	Sa	species	In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further.	RESULTS
95	99	EctC	protein	In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further.	RESULTS
142	149	Glu-115	residue_name_number	In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further.	RESULTS
155	161	His-55	residue_name_number	In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further.	RESULTS
204	215	small helix	structure_element	In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further.	RESULTS
223	239	carboxy-terminus	structure_element	In addition to the interactions between Pro-109 and His-55, the carboxy-terminal region of (Sa)EctC is held in position via an interaction of Glu-115 with His-55, which stabilizes the conformation of the small helix in the carboxy-terminus further.	RESULTS
24	31	Glu-115	residue_name_number	The interaction between Glu-115 and His-55 is only visible in the “semi-closed” structure where the partially extended carboxy-terminus is resolved in the electron density.	RESULTS
36	42	His-55	residue_name_number	The interaction between Glu-115 and His-55 is only visible in the “semi-closed” structure where the partially extended carboxy-terminus is resolved in the electron density.	RESULTS
67	78	semi-closed	protein_state	The interaction between Glu-115 and His-55 is only visible in the “semi-closed” structure where the partially extended carboxy-terminus is resolved in the electron density.	RESULTS
80	89	structure	evidence	The interaction between Glu-115 and His-55 is only visible in the “semi-closed” structure where the partially extended carboxy-terminus is resolved in the electron density.	RESULTS
100	118	partially extended	protein_state	The interaction between Glu-115 and His-55 is only visible in the “semi-closed” structure where the partially extended carboxy-terminus is resolved in the electron density.	RESULTS
119	135	carboxy-terminus	structure_element	The interaction between Glu-115 and His-55 is only visible in the “semi-closed” structure where the partially extended carboxy-terminus is resolved in the electron density.	RESULTS
155	171	electron density	evidence	The interaction between Glu-115 and His-55 is only visible in the “semi-closed” structure where the partially extended carboxy-terminus is resolved in the electron density.	RESULTS
8	12	open	protein_state	In the “open” structure of the (Sa)EctC protein, this interaction does not occur since Glu-115 is rotated outwards (Fig 6a and 6b).	RESULTS
14	23	structure	evidence	In the “open” structure of the (Sa)EctC protein, this interaction does not occur since Glu-115 is rotated outwards (Fig 6a and 6b).	RESULTS
32	34	Sa	species	In the “open” structure of the (Sa)EctC protein, this interaction does not occur since Glu-115 is rotated outwards (Fig 6a and 6b).	RESULTS
35	39	EctC	protein	In the “open” structure of the (Sa)EctC protein, this interaction does not occur since Glu-115 is rotated outwards (Fig 6a and 6b).	RESULTS
87	94	Glu-115	residue_name_number	In the “open” structure of the (Sa)EctC protein, this interaction does not occur since Glu-115 is rotated outwards (Fig 6a and 6b).	RESULTS
105	121	carboxy-terminus	structure_element	Hence, one might speculate that this missing interaction might be responsible for the flexibility of the carboxy-terminus in the “open” (Sa)EctC structure and consequently results in less well defined electron density in this region.	RESULTS
130	134	open	protein_state	Hence, one might speculate that this missing interaction might be responsible for the flexibility of the carboxy-terminus in the “open” (Sa)EctC structure and consequently results in less well defined electron density in this region.	RESULTS
137	139	Sa	species	Hence, one might speculate that this missing interaction might be responsible for the flexibility of the carboxy-terminus in the “open” (Sa)EctC structure and consequently results in less well defined electron density in this region.	RESULTS
140	144	EctC	protein	Hence, one might speculate that this missing interaction might be responsible for the flexibility of the carboxy-terminus in the “open” (Sa)EctC structure and consequently results in less well defined electron density in this region.	RESULTS
145	154	structure	evidence	Hence, one might speculate that this missing interaction might be responsible for the flexibility of the carboxy-terminus in the “open” (Sa)EctC structure and consequently results in less well defined electron density in this region.	RESULTS
201	217	electron density	evidence	Hence, one might speculate that this missing interaction might be responsible for the flexibility of the carboxy-terminus in the “open” (Sa)EctC structure and consequently results in less well defined electron density in this region.	RESULTS
29	47	metal-binding site	site	Architecture of the presumed metal-binding site of the (Sa)EctC protein and its flexible carboxy-terminus.	FIG
56	58	Sa	species	Architecture of the presumed metal-binding site of the (Sa)EctC protein and its flexible carboxy-terminus.	FIG
59	63	EctC	protein	Architecture of the presumed metal-binding site of the (Sa)EctC protein and its flexible carboxy-terminus.	FIG
80	88	flexible	protein_state	Architecture of the presumed metal-binding site of the (Sa)EctC protein and its flexible carboxy-terminus.	FIG
89	105	carboxy-terminus	structure_element	Architecture of the presumed metal-binding site of the (Sa)EctC protein and its flexible carboxy-terminus.	FIG
18	23	water	chemical	(a) The described water molecule (depicted as orange sphere) is bound via interactions with the side chains of Glu-57, Tyr-85, and His-93.	FIG
111	117	Glu-57	residue_name_number	(a) The described water molecule (depicted as orange sphere) is bound via interactions with the side chains of Glu-57, Tyr-85, and His-93.	FIG
119	125	Tyr-85	residue_name_number	(a) The described water molecule (depicted as orange sphere) is bound via interactions with the side chains of Glu-57, Tyr-85, and His-93.	FIG
131	137	His-93	residue_name_number	(a) The described water molecule (depicted as orange sphere) is bound via interactions with the side chains of Glu-57, Tyr-85, and His-93.	FIG
30	35	water	chemical	The position occupied by this water molecule represents probably the position of the Fe2+ cofactor in the active side of the ectoine synthase.	FIG
85	89	Fe2+	chemical	The position occupied by this water molecule represents probably the position of the Fe2+ cofactor in the active side of the ectoine synthase.	FIG
106	117	active side	site	The position occupied by this water molecule represents probably the position of the Fe2+ cofactor in the active side of the ectoine synthase.	FIG
125	141	ectoine synthase	protein_type	The position occupied by this water molecule represents probably the position of the Fe2+ cofactor in the active side of the ectoine synthase.	FIG
0	6	His-55	residue_name_number	His-55 interacts with the double proline motif (Pro-109 and Pro-110).	FIG
26	46	double proline motif	structure_element	His-55 interacts with the double proline motif (Pro-109 and Pro-110).	FIG
48	55	Pro-109	residue_name_number	His-55 interacts with the double proline motif (Pro-109 and Pro-110).	FIG
60	67	Pro-110	residue_name_number	His-55 interacts with the double proline motif (Pro-109 and Pro-110).	FIG
67	74	Glu-115	residue_name_number	It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure.	FIG
101	109	flexible	protein_state	It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure.	FIG
110	126	carboxy-terminus	structure_element	It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure.	FIG
151	153	Sa	species	It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure.	FIG
154	158	EctC	protein	It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure.	FIG
183	194	semi-closed	protein_state	It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure.	FIG
197	199	Sa	species	It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure.	FIG
200	204	EctC	protein	It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure.	FIG
205	214	structure	evidence	It is further stabilized via an interaction with the side chain of Glu-115 which is localized in the flexible carboxy-terminus (colored in orange) of (Sa)EctC that is visible in the “semi-closed” (Sa)EctC structure.	FIG
7	14	overlay	experimental_method	(b) An overlay of the “open” (colored in light blue) and the “semi-closed” (colored in green) structure of the (Sa)EctC protein.	FIG
23	27	open	protein_state	(b) An overlay of the “open” (colored in light blue) and the “semi-closed” (colored in green) structure of the (Sa)EctC protein.	FIG
62	73	semi-closed	protein_state	(b) An overlay of the “open” (colored in light blue) and the “semi-closed” (colored in green) structure of the (Sa)EctC protein.	FIG
94	103	structure	evidence	(b) An overlay of the “open” (colored in light blue) and the “semi-closed” (colored in green) structure of the (Sa)EctC protein.	FIG
112	114	Sa	species	(b) An overlay of the “open” (colored in light blue) and the “semi-closed” (colored in green) structure of the (Sa)EctC protein.	FIG
115	119	EctC	protein	(b) An overlay of the “open” (colored in light blue) and the “semi-closed” (colored in green) structure of the (Sa)EctC protein.	FIG
13	30	iron binding site	site	The putative iron binding site of (Sa)EctC	RESULTS
35	37	Sa	species	The putative iron binding site of (Sa)EctC	RESULTS
38	42	EctC	protein	The putative iron binding site of (Sa)EctC	RESULTS
8	19	semi-closed	protein_state	In the “semi-closed” structure of (Sa)EctC, each of the four monomers in the asymmetric unit contains a relative strong electron density positioned within the cupin barrel.	RESULTS
21	30	structure	evidence	In the “semi-closed” structure of (Sa)EctC, each of the four monomers in the asymmetric unit contains a relative strong electron density positioned within the cupin barrel.	RESULTS
35	37	Sa	species	In the “semi-closed” structure of (Sa)EctC, each of the four monomers in the asymmetric unit contains a relative strong electron density positioned within the cupin barrel.	RESULTS
38	42	EctC	protein	In the “semi-closed” structure of (Sa)EctC, each of the four monomers in the asymmetric unit contains a relative strong electron density positioned within the cupin barrel.	RESULTS
61	69	monomers	oligomeric_state	In the “semi-closed” structure of (Sa)EctC, each of the four monomers in the asymmetric unit contains a relative strong electron density positioned within the cupin barrel.	RESULTS
120	136	electron density	evidence	In the “semi-closed” structure of (Sa)EctC, each of the four monomers in the asymmetric unit contains a relative strong electron density positioned within the cupin barrel.	RESULTS
159	171	cupin barrel	structure_element	In the “semi-closed” structure of (Sa)EctC, each of the four monomers in the asymmetric unit contains a relative strong electron density positioned within the cupin barrel.	RESULTS
7	9	Sa	species	Since (Sa)EctC is a metal containing protein (Fig 3), we tried to fit either Fe2+, or Zn2+ ions into this density and also refined occupancy.	RESULTS
10	14	EctC	protein	Since (Sa)EctC is a metal containing protein (Fig 3), we tried to fit either Fe2+, or Zn2+ ions into this density and also refined occupancy.	RESULTS
20	25	metal	chemical	Since (Sa)EctC is a metal containing protein (Fig 3), we tried to fit either Fe2+, or Zn2+ ions into this density and also refined occupancy.	RESULTS
77	81	Fe2+	chemical	Since (Sa)EctC is a metal containing protein (Fig 3), we tried to fit either Fe2+, or Zn2+ ions into this density and also refined occupancy.	RESULTS
86	90	Zn2+	chemical	Since (Sa)EctC is a metal containing protein (Fig 3), we tried to fit either Fe2+, or Zn2+ ions into this density and also refined occupancy.	RESULTS
106	113	density	evidence	Since (Sa)EctC is a metal containing protein (Fig 3), we tried to fit either Fe2+, or Zn2+ ions into this density and also refined occupancy.	RESULTS
123	140	refined occupancy	experimental_method	Since (Sa)EctC is a metal containing protein (Fig 3), we tried to fit either Fe2+, or Zn2+ ions into this density and also refined occupancy.	RESULTS
23	27	Fe2+	chemical	Only the refinement of Fe2+ resulted in a visibly improved electron density, however with a low degree of occupancy.	RESULTS
59	75	electron density	evidence	Only the refinement of Fe2+ resulted in a visibly improved electron density, however with a low degree of occupancy.	RESULTS
14	18	iron	chemical	This possible iron molecule is bound via interactions with Glu-57, Tyr-85 and His-93 (Fig 6a and 6b).	RESULTS
59	65	Glu-57	residue_name_number	This possible iron molecule is bound via interactions with Glu-57, Tyr-85 and His-93 (Fig 6a and 6b).	RESULTS
67	73	Tyr-85	residue_name_number	This possible iron molecule is bound via interactions with Glu-57, Tyr-85 and His-93 (Fig 6a and 6b).	RESULTS
78	84	His-93	residue_name_number	This possible iron molecule is bound via interactions with Glu-57, Tyr-85 and His-93 (Fig 6a and 6b).	RESULTS
74	78	iron	chemical	The distance between the side chains of these residues and the (putative) iron co-factor is 3.1 Å for Glu-57, 2.9 Å for Tyr-85, and 2.9 Å for His-93, respectively.	RESULTS
102	108	Glu-57	residue_name_number	The distance between the side chains of these residues and the (putative) iron co-factor is 3.1 Å for Glu-57, 2.9 Å for Tyr-85, and 2.9 Å for His-93, respectively.	RESULTS
120	126	Tyr-85	residue_name_number	The distance between the side chains of these residues and the (putative) iron co-factor is 3.1 Å for Glu-57, 2.9 Å for Tyr-85, and 2.9 Å for His-93, respectively.	RESULTS
142	148	His-93	residue_name_number	The distance between the side chains of these residues and the (putative) iron co-factor is 3.1 Å for Glu-57, 2.9 Å for Tyr-85, and 2.9 Å for His-93, respectively.	RESULTS
51	69	iron binding sites	site	These distances are to long when compared to other iron binding sites, a fact that might be caused by the absence of the proper substrate in the (Sa)EctC crystal structure.	RESULTS
106	116	absence of	protein_state	These distances are to long when compared to other iron binding sites, a fact that might be caused by the absence of the proper substrate in the (Sa)EctC crystal structure.	RESULTS
146	148	Sa	species	These distances are to long when compared to other iron binding sites, a fact that might be caused by the absence of the proper substrate in the (Sa)EctC crystal structure.	RESULTS
149	153	EctC	protein	These distances are to long when compared to other iron binding sites, a fact that might be caused by the absence of the proper substrate in the (Sa)EctC crystal structure.	RESULTS
154	171	crystal structure	evidence	These distances are to long when compared to other iron binding sites, a fact that might be caused by the absence of the proper substrate in the (Sa)EctC crystal structure.	RESULTS
71	75	iron	chemical	Since both the refinement and the distance did not clearly identify an iron molecule, we decided to conservatively place a water molecule at this position.	RESULTS
123	128	water	chemical	Since both the refinement and the distance did not clearly identify an iron molecule, we decided to conservatively place a water molecule at this position.	RESULTS
21	26	water	chemical	The position of this water molecule is described in more detail below and is highlighted in Figs 5a and 5b and 6a and 6b as a sphere.	RESULTS
55	60	water	chemical	Interestingly, all three amino acids coordinating this water molecule are strictly conserved within an alignment of 440 members of the EctC protein family (for an abbreviated alignment of EctC-type proteins see Fig 2).	RESULTS
74	92	strictly conserved	protein_state	Interestingly, all three amino acids coordinating this water molecule are strictly conserved within an alignment of 440 members of the EctC protein family (for an abbreviated alignment of EctC-type proteins see Fig 2).	RESULTS
103	112	alignment	experimental_method	Interestingly, all three amino acids coordinating this water molecule are strictly conserved within an alignment of 440 members of the EctC protein family (for an abbreviated alignment of EctC-type proteins see Fig 2).	RESULTS
135	147	EctC protein	protein_type	Interestingly, all three amino acids coordinating this water molecule are strictly conserved within an alignment of 440 members of the EctC protein family (for an abbreviated alignment of EctC-type proteins see Fig 2).	RESULTS
188	206	EctC-type proteins	protein_type	Interestingly, all three amino acids coordinating this water molecule are strictly conserved within an alignment of 440 members of the EctC protein family (for an abbreviated alignment of EctC-type proteins see Fig 2).	RESULTS
8	12	open	protein_state	In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure.	RESULTS
14	23	structure	evidence	In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure.	RESULTS
32	34	Sa	species	In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure.	RESULTS
35	39	EctC	protein	In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure.	RESULTS
49	65	electron density	evidence	In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure.	RESULTS
99	103	iron	chemical	In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure.	RESULTS
126	137	semi-closed	protein_state	In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure.	RESULTS
139	148	structure	evidence	In the “open” structure of the (Sa)EctC protein, electron density is visible where the presumptive iron is positioned in the “semi-closed” structure.	RESULTS
14	30	electron density	evidence	However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure.	RESULTS
51	56	water	chemical	However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure.	RESULTS
80	84	iron	chemical	However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure.	RESULTS
94	99	water	chemical	However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure.	RESULTS
190	194	open	protein_state	However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure.	RESULTS
197	199	Sa	species	However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure.	RESULTS
200	204	EctC	protein	However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure.	RESULTS
205	214	structure	evidence	However, this electron density fits perfectly to a water molecule and not to an iron, and the water molecule was clearly visible after the refinement at this high resolution (1.2 Å) of the “open” (Sa)EctC structure.	RESULTS
5	20	superimposition	experimental_method	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
30	32	Sa	species	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
33	37	EctC	protein	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
38	56	crystal structures	evidence	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
128	134	Glu-57	residue_name_number	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
136	142	Tyr-85	residue_name_number	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
148	154	His-93	residue_name_number	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
178	182	iron	chemical	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
191	202	semi-closed	protein_state	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
204	213	structure	evidence	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
276	285	iron-free	protein_state	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
288	292	open	protein_state	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
294	303	structure	evidence	In a superimposition of both (Sa)EctC crystal structures, the spatial arrangements of the side chains of the three amino acids (Glu-57, Tyr-85, and His-93) likely to contact the iron in the “semi-closed” structure match nicely with those of the corresponding residues of the “iron-free” “open” structure (Fig 6b).	RESULTS
5	11	His-93	residue_name_number	Only His-93 is slightly rotated inwards in the “semi-closed” structure, most likely due to formation of β-strand β5 as described above.	RESULTS
48	59	semi-closed	protein_state	Only His-93 is slightly rotated inwards in the “semi-closed” structure, most likely due to formation of β-strand β5 as described above.	RESULTS
61	70	structure	evidence	Only His-93 is slightly rotated inwards in the “semi-closed” structure, most likely due to formation of β-strand β5 as described above.	RESULTS
104	112	β-strand	structure_element	Only His-93 is slightly rotated inwards in the “semi-closed” structure, most likely due to formation of β-strand β5 as described above.	RESULTS
113	115	β5	structure_element	Only His-93 is slightly rotated inwards in the “semi-closed” structure, most likely due to formation of β-strand β5 as described above.	RESULTS
85	102	iron-binding site	site	Taken together, this observations indicate, that the architecture of the presumptive iron-binding site is pre-set for the binding of the catalytically important metal by the ectoine synthase.	RESULTS
161	166	metal	chemical	Taken together, this observations indicate, that the architecture of the presumptive iron-binding site is pre-set for the binding of the catalytically important metal by the ectoine synthase.	RESULTS
174	190	ectoine synthase	protein_type	Taken together, this observations indicate, that the architecture of the presumptive iron-binding site is pre-set for the binding of the catalytically important metal by the ectoine synthase.	RESULTS
66	72	Tyr-52	residue_name_number	Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
87	91	loop	structure_element	Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
109	117	β-strand	structure_element	Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
118	120	β5	structure_element	Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
130	134	open	protein_state	Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
141	152	semi-closed	protein_state	Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
155	157	Sa	species	Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
158	162	EctC	protein	Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
163	173	structures	evidence	Of note is the different spatial arrangement of the side-chain of Tyr-52 (located in a loop after the end of β-strand β5) in the “open” and “semi-closed” (Sa)EctC structures.	RESULTS
8	19	semi-closed	protein_state	In the “semi-closed” structure, the hydroxyl-group of the side-chain of Tyr-52 points towards the iron (Fig 6a and 6b), but the corresponding distance (3.9 Å) makes it highly unlikely that Tyr-52 is directly involved in metal binding.	RESULTS
21	30	structure	evidence	In the “semi-closed” structure, the hydroxyl-group of the side-chain of Tyr-52 points towards the iron (Fig 6a and 6b), but the corresponding distance (3.9 Å) makes it highly unlikely that Tyr-52 is directly involved in metal binding.	RESULTS
72	78	Tyr-52	residue_name_number	In the “semi-closed” structure, the hydroxyl-group of the side-chain of Tyr-52 points towards the iron (Fig 6a and 6b), but the corresponding distance (3.9 Å) makes it highly unlikely that Tyr-52 is directly involved in metal binding.	RESULTS
98	102	iron	chemical	In the “semi-closed” structure, the hydroxyl-group of the side-chain of Tyr-52 points towards the iron (Fig 6a and 6b), but the corresponding distance (3.9 Å) makes it highly unlikely that Tyr-52 is directly involved in metal binding.	RESULTS
189	195	Tyr-52	residue_name_number	In the “semi-closed” structure, the hydroxyl-group of the side-chain of Tyr-52 points towards the iron (Fig 6a and 6b), but the corresponding distance (3.9 Å) makes it highly unlikely that Tyr-52 is directly involved in metal binding.	RESULTS
220	225	metal	chemical	In the “semi-closed” structure, the hydroxyl-group of the side-chain of Tyr-52 points towards the iron (Fig 6a and 6b), but the corresponding distance (3.9 Å) makes it highly unlikely that Tyr-52 is directly involved in metal binding.	RESULTS
18	30	substitution	experimental_method	Nevertheless, its substitution by an Ala residue causes a strong decrease in iron-content and enzyme activity of the mutant protein (Table 1).	RESULTS
37	40	Ala	residue_name	Nevertheless, its substitution by an Ala residue causes a strong decrease in iron-content and enzyme activity of the mutant protein (Table 1).	RESULTS
77	81	iron	chemical	Nevertheless, its substitution by an Ala residue causes a strong decrease in iron-content and enzyme activity of the mutant protein (Table 1).	RESULTS
117	123	mutant	protein_state	Nevertheless, its substitution by an Ala residue causes a strong decrease in iron-content and enzyme activity of the mutant protein (Table 1).	RESULTS
28	35	overlay	experimental_method	It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).	RESULTS
44	48	open	protein_state	It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).	RESULTS
55	66	semi-closed	protein_state	It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).	RESULTS
69	71	Sa	species	It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).	RESULTS
72	76	EctC	protein	It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).	RESULTS
77	95	crystal structures	evidence	It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).	RESULTS
119	125	Tyr-52	residue_name_number	It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).	RESULTS
176	180	iron	chemical	It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).	RESULTS
253	258	metal	chemical	It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).	RESULTS
269	275	Glu-57	residue_name_number	It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).	RESULTS
277	283	Tyr-85	residue_name_number	It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).	RESULTS
289	295	His-93	residue_name_number	It becomes apparent from an overlay of the “open” and “semi-closed” (Sa)EctC crystal structures that the side-chain of Tyr-52 rotates away from the position of the presumptive iron, whereas the side-chains of those residues that probably contacting the metal directly [Glu-57, Tyr-85, and His-93], remain in place (Fig 6a and 6b).	RESULTS
6	12	Tyr-52	residue_name_number	Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility.	RESULTS
16	34	strictly conserved	protein_state	Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility.	RESULTS
41	50	alignment	experimental_method	Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility.	RESULTS
58	76	EctC-type proteins	protein_type	Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility.	RESULTS
160	176	ectoine synthase	protein_type	Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility.	RESULTS
190	200	absence of	protein_state	Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility.	RESULTS
201	210	N-γ-ADABA	chemical	Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility.	RESULTS
219	221	Sa	species	Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility.	RESULTS
222	226	EctC	protein	Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility.	RESULTS
227	245	crystal structures	evidence	Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility.	RESULTS
276	282	Tyr-52	residue_name_number	Since Tyr-52 is strictly conserved in an alignment of 440 EctC-type proteins (Fig 2), we speculate that it might be involved in contacting the substrate of the ectoine synthase and that the absence of N-γ-ADABA in our (Sa)EctC crystal structures might endow the side chain of Tyr-52 with extra spatial flexibility.	RESULTS
32	49	iron binding site	site	To further analyze the putative iron binding site (Fig 6a), we performed structure-guided site-directed mutagenesis and assessed the resulting (Sa)EctC variants for their iron content and studied their enzyme activity.	RESULTS
73	115	structure-guided site-directed mutagenesis	experimental_method	To further analyze the putative iron binding site (Fig 6a), we performed structure-guided site-directed mutagenesis and assessed the resulting (Sa)EctC variants for their iron content and studied their enzyme activity.	RESULTS
144	146	Sa	species	To further analyze the putative iron binding site (Fig 6a), we performed structure-guided site-directed mutagenesis and assessed the resulting (Sa)EctC variants for their iron content and studied their enzyme activity.	RESULTS
147	151	EctC	protein	To further analyze the putative iron binding site (Fig 6a), we performed structure-guided site-directed mutagenesis and assessed the resulting (Sa)EctC variants for their iron content and studied their enzyme activity.	RESULTS
171	175	iron	chemical	To further analyze the putative iron binding site (Fig 6a), we performed structure-guided site-directed mutagenesis and assessed the resulting (Sa)EctC variants for their iron content and studied their enzyme activity.	RESULTS
27	33	Glu-57	residue_name_number	When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1).	RESULTS
35	41	Tyr-85	residue_name_number	When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1).	RESULTS
43	49	His-93	residue_name_number	When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1).	RESULTS
72	96	mono-nuclear iron center	site	When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1).	RESULTS
105	107	Sa	species	When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1).	RESULTS
108	112	EctC	protein	When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1).	RESULTS
113	130	crystal structure	evidence	When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1).	RESULTS
149	157	replaced	experimental_method	When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1).	RESULTS
164	167	Ala	residue_name	When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1).	RESULTS
213	217	iron	chemical	When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1).	RESULTS
233	239	mutant	protein_state	When those three residues (Glu-57, Tyr-85, His-93) that likely form the mono-nuclear iron center in the (Sa)EctC crystal structure were individually replaced by an Ala residue, both the catalytic activity and the iron content of the mutant proteins was strongly reduced (Table 1).	RESULTS
28	54	iron-coordinating residues	site	For some of the presumptive iron-coordinating residues, additional site-directed mutagenesis experiments were carried out.	RESULTS
67	92	site-directed mutagenesis	experimental_method	For some of the presumptive iron-coordinating residues, additional site-directed mutagenesis experiments were carried out.	RESULTS
67	73	Glu-57	residue_name_number	To verify the importance of the negative charge in the position of Glu-57, we created an Asp variant.	RESULTS
89	92	Asp	residue_name	To verify the importance of the negative charge in the position of Glu-57, we created an Asp variant.	RESULTS
93	100	variant	protein_state	To verify the importance of the negative charge in the position of Glu-57, we created an Asp variant.	RESULTS
5	11	mutant	protein_state	This mutant protein rescued the enzyme activity and iron content of the Ala substitution substantially (Table 1).	RESULTS
52	56	iron	chemical	This mutant protein rescued the enzyme activity and iron content of the Ala substitution substantially (Table 1).	RESULTS
72	75	Ala	residue_name	This mutant protein rescued the enzyme activity and iron content of the Ala substitution substantially (Table 1).	RESULTS
76	88	substitution	experimental_method	This mutant protein rescued the enzyme activity and iron content of the Ala substitution substantially (Table 1).	RESULTS
8	16	replaced	experimental_method	We also replaced Tyr-85 with either a Phe or a Trp residue and both mutant proteins largely lost their catalytic activity and iron content (Table 1) despite the fact that these substitutions were conservative.	RESULTS
17	23	Tyr-85	residue_name_number	We also replaced Tyr-85 with either a Phe or a Trp residue and both mutant proteins largely lost their catalytic activity and iron content (Table 1) despite the fact that these substitutions were conservative.	RESULTS
38	41	Phe	residue_name	We also replaced Tyr-85 with either a Phe or a Trp residue and both mutant proteins largely lost their catalytic activity and iron content (Table 1) despite the fact that these substitutions were conservative.	RESULTS
47	50	Trp	residue_name	We also replaced Tyr-85 with either a Phe or a Trp residue and both mutant proteins largely lost their catalytic activity and iron content (Table 1) despite the fact that these substitutions were conservative.	RESULTS
68	74	mutant	protein_state	We also replaced Tyr-85 with either a Phe or a Trp residue and both mutant proteins largely lost their catalytic activity and iron content (Table 1) despite the fact that these substitutions were conservative.	RESULTS
126	130	iron	chemical	We also replaced Tyr-85 with either a Phe or a Trp residue and both mutant proteins largely lost their catalytic activity and iron content (Table 1) despite the fact that these substitutions were conservative.	RESULTS
64	70	Tyr-85	residue_name_number	Collectively, these data suggest that the hydroxyl group of the Tyr-85 side chain is needed for the binding of the iron (Fig 6a).	RESULTS
115	119	iron	chemical	Collectively, these data suggest that the hydroxyl group of the Tyr-85 side chain is needed for the binding of the iron (Fig 6a).	RESULTS
8	16	replaced	experimental_method	We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1).	RESULTS
33	53	iron-binding residue	site	We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1).	RESULTS
54	60	His-93	residue_name_number	We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1).	RESULTS
67	70	Asn	residue_name	We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1).	RESULTS
92	94	Sa	species	We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1).	RESULTS
95	99	EctC	protein	We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1).	RESULTS
161	165	iron	chemical	We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1).	RESULTS
210	219	wild-type	protein_state	We also replaced the presumptive iron-binding residue His-93 by an Asn residue, yielding a (Sa)EctC protein variant that possessed an enzyme activity of 23% and iron content of only 14% relative to that of the wild-type protein (Table 1).	RESULTS
68	94	iron-coordinating residues	site	Collectively, the data addressing the functionality of the putative iron-coordinating residues (Glu-57, Tyr-85, His-93) buttress our notion that the Fe2+ present in the (Sa)EctC protein is of catalytic importance.	RESULTS
96	102	Glu-57	residue_name_number	Collectively, the data addressing the functionality of the putative iron-coordinating residues (Glu-57, Tyr-85, His-93) buttress our notion that the Fe2+ present in the (Sa)EctC protein is of catalytic importance.	RESULTS
104	110	Tyr-85	residue_name_number	Collectively, the data addressing the functionality of the putative iron-coordinating residues (Glu-57, Tyr-85, His-93) buttress our notion that the Fe2+ present in the (Sa)EctC protein is of catalytic importance.	RESULTS
112	118	His-93	residue_name_number	Collectively, the data addressing the functionality of the putative iron-coordinating residues (Glu-57, Tyr-85, His-93) buttress our notion that the Fe2+ present in the (Sa)EctC protein is of catalytic importance.	RESULTS
149	153	Fe2+	chemical	Collectively, the data addressing the functionality of the putative iron-coordinating residues (Glu-57, Tyr-85, His-93) buttress our notion that the Fe2+ present in the (Sa)EctC protein is of catalytic importance.	RESULTS
170	172	Sa	species	Collectively, the data addressing the functionality of the putative iron-coordinating residues (Glu-57, Tyr-85, His-93) buttress our notion that the Fe2+ present in the (Sa)EctC protein is of catalytic importance.	RESULTS
173	177	EctC	protein	Collectively, the data addressing the functionality of the putative iron-coordinating residues (Glu-57, Tyr-85, His-93) buttress our notion that the Fe2+ present in the (Sa)EctC protein is of catalytic importance.	RESULTS
38	40	Sa	species	A chemically undefined ligand in the (Sa)EctC structure provides clues for the binding of the N-γ-ADABA substrate	RESULTS
41	45	EctC	protein	A chemically undefined ligand in the (Sa)EctC structure provides clues for the binding of the N-γ-ADABA substrate	RESULTS
46	55	structure	evidence	A chemically undefined ligand in the (Sa)EctC structure provides clues for the binding of the N-γ-ADABA substrate	RESULTS
94	103	N-γ-ADABA	chemical	A chemically undefined ligand in the (Sa)EctC structure provides clues for the binding of the N-γ-ADABA substrate	RESULTS
47	65	co-crystallization	experimental_method	Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1).	RESULTS
69	88	soaking experiments	experimental_method	Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1).	RESULTS
120	122	Sa	species	Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1).	RESULTS
123	127	EctC	protein	Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1).	RESULTS
128	146	crystal structures	evidence	Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1).	RESULTS
183	192	N-γ-ADABA	chemical	Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1).	RESULTS
197	204	ectoine	chemical	Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1).	RESULTS
230	246	ectoine synthase	protein_type	Despite considerable efforts, either by trying co-crystallization or soaking experiments, we were not able to obtain a (Sa)EctC crystal structures that contained either the substrate N-γ-ADABA, or ectoine, the reaction product of ectoine synthase (Fig 1).	RESULTS
17	28	semi-closed	protein_state	However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement.	RESULTS
31	33	Sa	species	However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement.	RESULTS
34	38	EctC	protein	However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement.	RESULTS
39	48	structure	evidence	However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement.	RESULTS
59	80	carboxy-terminal loop	structure_element	However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement.	RESULTS
119	135	electron density	evidence	However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement.	RESULTS
174	185	active site	site	However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement.	RESULTS
227	254	crystallographic refinement	experimental_method	However, in the “semi-closed” (Sa)EctC structure where the carboxy-terminal loop is largely resolved, a long stretched electron density feature was detected in the predicted active site of the enzyme; it remained visible after crystallographic refinement.	RESULTS
44	48	open	protein_state	This is in contrast to the high-resolution “open” structure of the (Sa)EctC protein where no additional electron density was observed after refinement.	RESULTS
50	59	structure	evidence	This is in contrast to the high-resolution “open” structure of the (Sa)EctC protein where no additional electron density was observed after refinement.	RESULTS
68	70	Sa	species	This is in contrast to the high-resolution “open” structure of the (Sa)EctC protein where no additional electron density was observed after refinement.	RESULTS
71	75	EctC	protein	This is in contrast to the high-resolution “open” structure of the (Sa)EctC protein where no additional electron density was observed after refinement.	RESULTS
104	120	electron density	evidence	This is in contrast to the high-resolution “open” structure of the (Sa)EctC protein where no additional electron density was observed after refinement.	RESULTS
57	69	purification	experimental_method	We tried to fit all compounds used in the buffers during purification and crystallization into the observed electron density, but none matched.	RESULTS
74	89	crystallization	experimental_method	We tried to fit all compounds used in the buffers during purification and crystallization into the observed electron density, but none matched.	RESULTS
108	124	electron density	evidence	We tried to fit all compounds used in the buffers during purification and crystallization into the observed electron density, but none matched.	RESULTS
91	93	Sa	species	This observation indicates that the chemically undefined ligand was either trapped by the (Sa)EctC protein during its heterologous production in E. coli or during crystallization.	RESULTS
94	98	EctC	protein	This observation indicates that the chemically undefined ligand was either trapped by the (Sa)EctC protein during its heterologous production in E. coli or during crystallization.	RESULTS
145	152	E. coli	species	This observation indicates that the chemically undefined ligand was either trapped by the (Sa)EctC protein during its heterologous production in E. coli or during crystallization.	RESULTS
163	178	crystallization	experimental_method	This observation indicates that the chemically undefined ligand was either trapped by the (Sa)EctC protein during its heterologous production in E. coli or during crystallization.	RESULTS
14	17	PEG	chemical	Since we used PEG molecules in the crystallization conditions, the observed density might stem from an ordered part of a PEG molecule, or low molecular weight PEG species that might have been present in the PEG preparation used in our experiments.	RESULTS
76	83	density	evidence	Since we used PEG molecules in the crystallization conditions, the observed density might stem from an ordered part of a PEG molecule, or low molecular weight PEG species that might have been present in the PEG preparation used in our experiments.	RESULTS
121	124	PEG	chemical	Since we used PEG molecules in the crystallization conditions, the observed density might stem from an ordered part of a PEG molecule, or low molecular weight PEG species that might have been present in the PEG preparation used in our experiments.	RESULTS
159	162	PEG	chemical	Since we used PEG molecules in the crystallization conditions, the observed density might stem from an ordered part of a PEG molecule, or low molecular weight PEG species that might have been present in the PEG preparation used in our experiments.	RESULTS
207	210	PEG	chemical	Since we used PEG molecules in the crystallization conditions, the observed density might stem from an ordered part of a PEG molecule, or low molecular weight PEG species that might have been present in the PEG preparation used in our experiments.	RESULTS
38	62	electron density feature	evidence	Estimating from the dimensions of the electron density feature, we modeled the chemically undefined compound trapped by the (Sa)EctC protein as a hexane-1,6-diol molecule (PDB identifier: HEZ) to best fit the observed electron density.	RESULTS
125	127	Sa	species	Estimating from the dimensions of the electron density feature, we modeled the chemically undefined compound trapped by the (Sa)EctC protein as a hexane-1,6-diol molecule (PDB identifier: HEZ) to best fit the observed electron density.	RESULTS
128	132	EctC	protein	Estimating from the dimensions of the electron density feature, we modeled the chemically undefined compound trapped by the (Sa)EctC protein as a hexane-1,6-diol molecule (PDB identifier: HEZ) to best fit the observed electron density.	RESULTS
146	161	hexane-1,6-diol	chemical	Estimating from the dimensions of the electron density feature, we modeled the chemically undefined compound trapped by the (Sa)EctC protein as a hexane-1,6-diol molecule (PDB identifier: HEZ) to best fit the observed electron density.	RESULTS
218	234	electron density	evidence	Estimating from the dimensions of the electron density feature, we modeled the chemically undefined compound trapped by the (Sa)EctC protein as a hexane-1,6-diol molecule (PDB identifier: HEZ) to best fit the observed electron density.	RESULTS
39	54	hexane-1,6-diol	chemical	However, to the best of our knowledge, hexane-1,6-diol is not part of the E. coli metabolome.	RESULTS
74	81	E. coli	species	However, to the best of our knowledge, hexane-1,6-diol is not part of the E. coli metabolome.	RESULTS
179	183	EctC	protein	Despite these notable limitations, we considered the serendipitously trapped compound as a mock ligand that might provide useful insights into the spatial positioning of the true EctC substrate and those residues that coordinate it within the ectoine synthase active site.	RESULTS
243	259	ectoine synthase	protein_type	Despite these notable limitations, we considered the serendipitously trapped compound as a mock ligand that might provide useful insights into the spatial positioning of the true EctC substrate and those residues that coordinate it within the ectoine synthase active site.	RESULTS
260	271	active site	site	Despite these notable limitations, we considered the serendipitously trapped compound as a mock ligand that might provide useful insights into the spatial positioning of the true EctC substrate and those residues that coordinate it within the ectoine synthase active site.	RESULTS
18	27	N-γ-ADABA	chemical	We note that both N-γ-ADABA and hexane-1,6-diol are both C6-compounds and display similar length (Fig 7a).	RESULTS
32	47	hexane-1,6-diol	chemical	We note that both N-γ-ADABA and hexane-1,6-diol are both C6-compounds and display similar length (Fig 7a).	RESULTS
49	60	active site	site	A chemically undefined ligand is captured in the active site of the “semi-closed” (Sa)EctC crystal structure.	FIG
69	80	semi-closed	protein_state	A chemically undefined ligand is captured in the active site of the “semi-closed” (Sa)EctC crystal structure.	FIG
83	85	Sa	species	A chemically undefined ligand is captured in the active site of the “semi-closed” (Sa)EctC crystal structure.	FIG
86	90	EctC	protein	A chemically undefined ligand is captured in the active site of the “semi-closed” (Sa)EctC crystal structure.	FIG
91	108	crystal structure	evidence	A chemically undefined ligand is captured in the active site of the “semi-closed” (Sa)EctC crystal structure.	FIG
17	33	electron density	evidence	(a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds.	FIG
41	52	active site	site	(a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds.	FIG
61	72	semi-closed	protein_state	(a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds.	FIG
74	83	structure	evidence	(a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds.	FIG
88	90	Sa	species	(a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds.	FIG
91	95	EctC	protein	(a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds.	FIG
112	127	hexane-1,6-diol	chemical	(a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds.	FIG
159	175	electron density	evidence	(a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds.	FIG
183	192	N-γ-ADABA	chemical	(a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds.	FIG
210	226	ectoine synthase	protein_type	(a) The observed electron density in the active site of the “semi-closed” structure of (Sa)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the N-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds.	FIG
19	31	binding site	site	(b) The presumable binding site of the iron co-factor and of the modeled hexane-1,6-diol molecule is depicted.	FIG
39	43	iron	chemical	(b) The presumable binding site of the iron co-factor and of the modeled hexane-1,6-diol molecule is depicted.	FIG
73	88	hexane-1,6-diol	chemical	(b) The presumable binding site of the iron co-factor and of the modeled hexane-1,6-diol molecule is depicted.	FIG
39	43	iron	chemical	The amino acid side chains involved in iron-ligand binding are colored in blue and those involved in the binding of the chemically undefined ligand are colored in green using a ball and stick representation.	FIG
4	12	flexible	protein_state	The flexible carboxy-terminal loop of (Sa)EctC is highlighted in orange.	FIG
13	34	carboxy-terminal loop	structure_element	The flexible carboxy-terminal loop of (Sa)EctC is highlighted in orange.	FIG
39	41	Sa	species	The flexible carboxy-terminal loop of (Sa)EctC is highlighted in orange.	FIG
42	46	EctC	protein	The flexible carboxy-terminal loop of (Sa)EctC is highlighted in orange.	FIG
4	20	electron density	evidence	The electron density was calculated as an omit map and contoured at 1.0 σ.	FIG
42	50	omit map	evidence	The electron density was calculated as an omit map and contoured at 1.0 σ.	FIG
3	10	refined	experimental_method	We refined the (Sa)EctC structure with the trapped compound, and by doing so, the refinement parameters (especially R- and Rfree-factor) dropped by 1.5%.	RESULTS
16	18	Sa	species	We refined the (Sa)EctC structure with the trapped compound, and by doing so, the refinement parameters (especially R- and Rfree-factor) dropped by 1.5%.	RESULTS
19	23	EctC	protein	We refined the (Sa)EctC structure with the trapped compound, and by doing so, the refinement parameters (especially R- and Rfree-factor) dropped by 1.5%.	RESULTS
24	33	structure	evidence	We refined the (Sa)EctC structure with the trapped compound, and by doing so, the refinement parameters (especially R- and Rfree-factor) dropped by 1.5%.	RESULTS
116	135	R- and Rfree-factor	evidence	We refined the (Sa)EctC structure with the trapped compound, and by doing so, the refinement parameters (especially R- and Rfree-factor) dropped by 1.5%.	RESULTS
22	30	omit map	evidence	We also calculated an omit map and the electron density reappeared (Fig 7b).	RESULTS
39	55	electron density	evidence	We also calculated an omit map and the electron density reappeared (Fig 7b).	RESULTS
61	63	Sa	species	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
64	68	EctC	protein	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
98	103	bound	protein_state	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
126	132	Trp-21	residue_name_number	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
137	143	Ser-23	residue_name_number	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
147	154	β-sheet	structure_element	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
155	157	β3	structure_element	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
159	165	Thr-40	residue_name_number	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
177	184	β-sheet	structure_element	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
185	187	β4	structure_element	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
193	200	Cys-105	residue_name_number	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
205	212	Phe-107	residue_name_number	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
237	244	β-sheet	structure_element	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
245	248	β11	structure_element	When analyzing the interactions of this compound within the (Sa)EctC protein, we found that it is bound via interactions with Trp-21 and Ser-23 of β-sheet β3, Thr-40 located in β-sheet β4, and Cys-105 and Phe-107, which are both part of β-sheet β11.	RESULTS
38	54	highly conserved	protein_state	Remarkably, all of these residues are highly conserved throughout the extended EctC protein family (Fig 2).	RESULTS
79	91	EctC protein	protein_type	Remarkably, all of these residues are highly conserved throughout the extended EctC protein family (Fig 2).	RESULTS
0	42	Structure-guided site-directed mutagenesis	experimental_method	Structure-guided site-directed mutagenesis of the catalytic core of the ectoine synthase	RESULTS
50	64	catalytic core	site	Structure-guided site-directed mutagenesis of the catalytic core of the ectoine synthase	RESULTS
72	88	ectoine synthase	protein_type	Structure-guided site-directed mutagenesis of the catalytic core of the ectoine synthase	RESULTS
14	51	alignment of the amino acid sequences	experimental_method	In a previous alignment of the amino acid sequences of 440 EctC-type proteins, 13 amino acids were identified as strictly conserved residues.	RESULTS
59	77	EctC-type proteins	protein_type	In a previous alignment of the amino acid sequences of 440 EctC-type proteins, 13 amino acids were identified as strictly conserved residues.	RESULTS
113	131	strictly conserved	protein_state	In a previous alignment of the amino acid sequences of 440 EctC-type proteins, 13 amino acids were identified as strictly conserved residues.	RESULTS
32	38	Thr-40	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
40	46	Tyr-52	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
48	54	His-55	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
56	62	Glu-57	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
64	70	Gly-64	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
72	78	Tyr-85	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
80	86	Leu-87	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
88	94	His-93	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
96	103	Phe-107	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
105	112	Pro-109	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
114	121	Gly-113	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
123	130	Glu-115	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
136	143	His-117	residue_name_number	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
152	154	Sa	species	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
155	159	EctC	protein	These correspond to amino acids Thr-40, Tyr-52, His-55, Glu-57, Gly-64, Tyr-85- Leu-87, His-93, Phe-107, Pro-109, Gly-113, Glu-115, and His-117 in the (Sa)EctC protein (Fig 2).	RESULTS
20	26	Gly-64	residue_name_number	Amino acid residues Gly-64, Pro-109, and Gly-113 likely fulfill structural roles since they are positioned either at the end or at the beginning of β-strands and α-helices.	RESULTS
28	35	Pro-109	residue_name_number	Amino acid residues Gly-64, Pro-109, and Gly-113 likely fulfill structural roles since they are positioned either at the end or at the beginning of β-strands and α-helices.	RESULTS
41	48	Gly-113	residue_name_number	Amino acid residues Gly-64, Pro-109, and Gly-113 likely fulfill structural roles since they are positioned either at the end or at the beginning of β-strands and α-helices.	RESULTS
148	157	β-strands	structure_element	Amino acid residues Gly-64, Pro-109, and Gly-113 likely fulfill structural roles since they are positioned either at the end or at the beginning of β-strands and α-helices.	RESULTS
162	171	α-helices	structure_element	Amino acid residues Gly-64, Pro-109, and Gly-113 likely fulfill structural roles since they are positioned either at the end or at the beginning of β-strands and α-helices.	RESULTS
147	155	flexible	protein_state	We considered the remaining ten residues as important either for ligand binding, for catalysis, or for the structurally correct orientation of the flexible carboxy-terminus of the (Sa)EctC protein.	RESULTS
156	172	carboxy-terminus	structure_element	We considered the remaining ten residues as important either for ligand binding, for catalysis, or for the structurally correct orientation of the flexible carboxy-terminus of the (Sa)EctC protein.	RESULTS
181	183	Sa	species	We considered the remaining ten residues as important either for ligand binding, for catalysis, or for the structurally correct orientation of the flexible carboxy-terminus of the (Sa)EctC protein.	RESULTS
184	188	EctC	protein	We considered the remaining ten residues as important either for ligand binding, for catalysis, or for the structurally correct orientation of the flexible carboxy-terminus of the (Sa)EctC protein.	RESULTS
39	45	Glu-57	residue_name_number	As described above, the side chains of Glu-57, Tyr-85, and His-93 are probably involved in iron binding (Table 1 and Fig 6a).	RESULTS
47	53	Tyr-85	residue_name_number	As described above, the side chains of Glu-57, Tyr-85, and His-93 are probably involved in iron binding (Table 1 and Fig 6a).	RESULTS
59	65	His-93	residue_name_number	As described above, the side chains of Glu-57, Tyr-85, and His-93 are probably involved in iron binding (Table 1 and Fig 6a).	RESULTS
91	95	iron	chemical	As described above, the side chains of Glu-57, Tyr-85, and His-93 are probably involved in iron binding (Table 1 and Fig 6a).	RESULTS
16	18	Sa	species	In view of the (Sa)EctC structure with the serendipitously trapped compound (Fig 7b), we probed the functional importance of the seven residues that contact this ligand by structure-guided site-directed mutagenesis (Table 1).	RESULTS
19	23	EctC	protein	In view of the (Sa)EctC structure with the serendipitously trapped compound (Fig 7b), we probed the functional importance of the seven residues that contact this ligand by structure-guided site-directed mutagenesis (Table 1).	RESULTS
24	33	structure	evidence	In view of the (Sa)EctC structure with the serendipitously trapped compound (Fig 7b), we probed the functional importance of the seven residues that contact this ligand by structure-guided site-directed mutagenesis (Table 1).	RESULTS
172	214	structure-guided site-directed mutagenesis	experimental_method	In view of the (Sa)EctC structure with the serendipitously trapped compound (Fig 7b), we probed the functional importance of the seven residues that contact this ligand by structure-guided site-directed mutagenesis (Table 1).	RESULTS
14	20	mutant	protein_state	Each of these mutant (Sa)EctC proteins was overproduced in E. coli and purified by affinity chromatography; they all yielded pure and stable protein preparations.	RESULTS
22	24	Sa	species	Each of these mutant (Sa)EctC proteins was overproduced in E. coli and purified by affinity chromatography; they all yielded pure and stable protein preparations.	RESULTS
25	29	EctC	protein	Each of these mutant (Sa)EctC proteins was overproduced in E. coli and purified by affinity chromatography; they all yielded pure and stable protein preparations.	RESULTS
59	66	E. coli	species	Each of these mutant (Sa)EctC proteins was overproduced in E. coli and purified by affinity chromatography; they all yielded pure and stable protein preparations.	RESULTS
83	106	affinity chromatography	experimental_method	Each of these mutant (Sa)EctC proteins was overproduced in E. coli and purified by affinity chromatography; they all yielded pure and stable protein preparations.	RESULTS
36	38	Sa	species	We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min.	RESULTS
39	43	EctC	protein	We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min.	RESULTS
58	88	single time-point enzyme assay	experimental_method	We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min.	RESULTS
125	134	wild-type	protein_state	We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min.	RESULTS
136	138	Sa	species	We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min.	RESULTS
139	143	EctC	protein	We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min.	RESULTS
199	208	N-γ-ADABA	chemical	We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min.	RESULTS
230	237	ectoine	chemical	We benchmarked the activity of the (Sa)EctC variants in a single time-point enzyme assay under conditions where 10 μM of the wild-type (Sa)EctC protein converted almost completely the supplied 10 mM N-γ-ADABA substrate to 9.33 mM ectoine within a time frame of 20 min.	RESULTS
31	35	iron	chemical	In addition, we determined the iron content of each of the mutant (Sa)EctC protein by a colorimetric assay (Table 1).	RESULTS
59	65	mutant	protein_state	In addition, we determined the iron content of each of the mutant (Sa)EctC protein by a colorimetric assay (Table 1).	RESULTS
67	69	Sa	species	In addition, we determined the iron content of each of the mutant (Sa)EctC protein by a colorimetric assay (Table 1).	RESULTS
70	74	EctC	protein	In addition, we determined the iron content of each of the mutant (Sa)EctC protein by a colorimetric assay (Table 1).	RESULTS
88	106	colorimetric assay	experimental_method	In addition, we determined the iron content of each of the mutant (Sa)EctC protein by a colorimetric assay (Table 1).	RESULTS
23	47	evolutionarily conserved	protein_state	The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b).	RESULTS
48	54	Trp-21	residue_name_number	The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b).	RESULTS
56	62	Ser-23	residue_name_number	The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b).	RESULTS
64	70	Thr-40	residue_name_number	The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b).	RESULTS
72	79	Cys-105	residue_name_number	The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b).	RESULTS
85	92	Phe-107	residue_name_number	The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b).	RESULTS
186	197	semi-closed	protein_state	The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b).	RESULTS
200	202	Sa	species	The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b).	RESULTS
203	207	EctC	protein	The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b).	RESULTS
208	217	structure	evidence	The side chains of the evolutionarily conserved Trp-21, Ser-23, Thr-40, Cys-105, and Phe-107 residues (Fig 2) make contacts with the chemically undefined ligand that we observed in the “semi-closed” (Sa)EctC structure (Fig 7b).	RESULTS
3	11	replaced	experimental_method	We replaced each of these residues with an Ala residue and found that none of them had an influence on the iron content of the mutant proteins.	RESULTS
43	46	Ala	residue_name	We replaced each of these residues with an Ala residue and found that none of them had an influence on the iron content of the mutant proteins.	RESULTS
107	111	iron	chemical	We replaced each of these residues with an Ala residue and found that none of them had an influence on the iron content of the mutant proteins.	RESULTS
127	133	mutant	protein_state	We replaced each of these residues with an Ala residue and found that none of them had an influence on the iron content of the mutant proteins.	RESULTS
0	6	Thr-40	residue_name_number	Thr-40 is positioned on β-strand β5 and its side chain protrudes into the lumen of the cupin barrel formed by the (Sa)EctC protein (Fig 7b).	RESULTS
24	32	β-strand	structure_element	Thr-40 is positioned on β-strand β5 and its side chain protrudes into the lumen of the cupin barrel formed by the (Sa)EctC protein (Fig 7b).	RESULTS
33	35	β5	structure_element	Thr-40 is positioned on β-strand β5 and its side chain protrudes into the lumen of the cupin barrel formed by the (Sa)EctC protein (Fig 7b).	RESULTS
87	99	cupin barrel	structure_element	Thr-40 is positioned on β-strand β5 and its side chain protrudes into the lumen of the cupin barrel formed by the (Sa)EctC protein (Fig 7b).	RESULTS
115	117	Sa	species	Thr-40 is positioned on β-strand β5 and its side chain protrudes into the lumen of the cupin barrel formed by the (Sa)EctC protein (Fig 7b).	RESULTS
118	122	EctC	protein	Thr-40 is positioned on β-strand β5 and its side chain protrudes into the lumen of the cupin barrel formed by the (Sa)EctC protein (Fig 7b).	RESULTS
8	16	replaced	experimental_method	We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.	RESULTS
17	24	Phe-107	residue_name_number	We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.	RESULTS
40	43	Tyr	residue_name	We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.	RESULTS
50	53	Trp	residue_name	We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.	RESULTS
67	78	Phe-107/Tyr	mutant	We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.	RESULTS
79	91	substitution	experimental_method	We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.	RESULTS
107	116	wild-type	protein_state	We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.	RESULTS
158	162	iron	chemical	We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.	RESULTS
180	191	Phe-107/Trp	mutant	We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.	RESULTS
192	204	substitution	experimental_method	We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.	RESULTS
248	252	iron	chemical	We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.	RESULTS
277	286	wild-type	protein_state	We also replaced Phe-107 with either an Tyr or an Trp residue: the Phe-107/Tyr substitution possessed near wild-type enzyme activity (about 95%) and the full iron content, but the Phe-107/Trp substitution possessed only 12% enzyme activity and 72% iron content compared to the wild-type protein.	RESULTS
24	30	mutant	protein_state	The properties of these mutant proteins indicate that the aromatic side chain at position 107 of (Sa)EctC is of importance but that a substitution with a bulky aromatic side chain is strongly detrimental to enzyme activity and concomitantly moderately impairs iron binding.	RESULTS
90	93	107	residue_number	The properties of these mutant proteins indicate that the aromatic side chain at position 107 of (Sa)EctC is of importance but that a substitution with a bulky aromatic side chain is strongly detrimental to enzyme activity and concomitantly moderately impairs iron binding.	RESULTS
98	100	Sa	species	The properties of these mutant proteins indicate that the aromatic side chain at position 107 of (Sa)EctC is of importance but that a substitution with a bulky aromatic side chain is strongly detrimental to enzyme activity and concomitantly moderately impairs iron binding.	RESULTS
101	105	EctC	protein	The properties of these mutant proteins indicate that the aromatic side chain at position 107 of (Sa)EctC is of importance but that a substitution with a bulky aromatic side chain is strongly detrimental to enzyme activity and concomitantly moderately impairs iron binding.	RESULTS
134	146	substitution	experimental_method	The properties of these mutant proteins indicate that the aromatic side chain at position 107 of (Sa)EctC is of importance but that a substitution with a bulky aromatic side chain is strongly detrimental to enzyme activity and concomitantly moderately impairs iron binding.	RESULTS
260	264	iron	chemical	The properties of these mutant proteins indicate that the aromatic side chain at position 107 of (Sa)EctC is of importance but that a substitution with a bulky aromatic side chain is strongly detrimental to enzyme activity and concomitantly moderately impairs iron binding.	RESULTS
0	11	Replacement	experimental_method	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
24	27	Cys	residue_name	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
40	42	Sa	species	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
43	47	EctC	protein	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
49	56	Cys-105	residue_name_number	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
70	73	Ser	residue_name	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
130	143	EctC proteins	protein_type	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
197	199	Sa	species	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
200	204	EctC	protein	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
205	212	variant	protein_state	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
222	231	wild-type	protein_state	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
248	252	iron	chemical	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
284	293	wild-type	protein_state	Replacement of the only Cys residue in (Sa)EctC (Cys-105; Fig 2) by a Ser residue, a configuration that is naturally found in two EctC proteins among 440 inspected amino acid sequences, yielded a (Sa)EctC variant with 84% wild-type activity and an iron content similar to that of the wild-type protein.	RESULTS
13	24	Cys-105/Ala	mutant	However, the Cys-105/Ala variant was practically catalytically inactive while largely maintaining its iron content (Table 1).	RESULTS
25	32	variant	protein_state	However, the Cys-105/Ala variant was practically catalytically inactive while largely maintaining its iron content (Table 1).	RESULTS
49	71	catalytically inactive	protein_state	However, the Cys-105/Ala variant was practically catalytically inactive while largely maintaining its iron content (Table 1).	RESULTS
102	106	iron	chemical	However, the Cys-105/Ala variant was practically catalytically inactive while largely maintaining its iron content (Table 1).	RESULTS
25	28	Cys	residue_name	Since the side-chains of Cys residues are chemically reactive and often participate in enzyme catalysis, Cys-105 (or Ser-105) might serve such a role for ectoine synthase.	RESULTS
105	112	Cys-105	residue_name_number	Since the side-chains of Cys residues are chemically reactive and often participate in enzyme catalysis, Cys-105 (or Ser-105) might serve such a role for ectoine synthase.	RESULTS
117	124	Ser-105	residue_name_number	Since the side-chains of Cys residues are chemically reactive and often participate in enzyme catalysis, Cys-105 (or Ser-105) might serve such a role for ectoine synthase.	RESULTS
154	170	ectoine synthase	protein_type	Since the side-chains of Cys residues are chemically reactive and often participate in enzyme catalysis, Cys-105 (or Ser-105) might serve such a role for ectoine synthase.	RESULTS
16	40	amino acid substitutions	experimental_method	We observed two amino acid substitutions that simultaneously strongly affected enzyme activity and iron content; these were the Tyr-52/Ala and the His-55/Ala (Sa)EctC protein variants (Table 1).	RESULTS
99	103	iron	chemical	We observed two amino acid substitutions that simultaneously strongly affected enzyme activity and iron content; these were the Tyr-52/Ala and the His-55/Ala (Sa)EctC protein variants (Table 1).	RESULTS
128	138	Tyr-52/Ala	mutant	We observed two amino acid substitutions that simultaneously strongly affected enzyme activity and iron content; these were the Tyr-52/Ala and the His-55/Ala (Sa)EctC protein variants (Table 1).	RESULTS
147	157	His-55/Ala	mutant	We observed two amino acid substitutions that simultaneously strongly affected enzyme activity and iron content; these were the Tyr-52/Ala and the His-55/Ala (Sa)EctC protein variants (Table 1).	RESULTS
159	161	Sa	species	We observed two amino acid substitutions that simultaneously strongly affected enzyme activity and iron content; these were the Tyr-52/Ala and the His-55/Ala (Sa)EctC protein variants (Table 1).	RESULTS
162	166	EctC	protein	We observed two amino acid substitutions that simultaneously strongly affected enzyme activity and iron content; these were the Tyr-52/Ala and the His-55/Ala (Sa)EctC protein variants (Table 1).	RESULTS
14	16	Sa	species	Based on the (Sa)EctC crystal structures that we present here, we can currently not firmly understand why the replacement of Tyr-52 by Ala impairs enzyme function and iron content so drastically (Table 1).	RESULTS
17	21	EctC	protein	Based on the (Sa)EctC crystal structures that we present here, we can currently not firmly understand why the replacement of Tyr-52 by Ala impairs enzyme function and iron content so drastically (Table 1).	RESULTS
22	40	crystal structures	evidence	Based on the (Sa)EctC crystal structures that we present here, we can currently not firmly understand why the replacement of Tyr-52 by Ala impairs enzyme function and iron content so drastically (Table 1).	RESULTS
110	121	replacement	experimental_method	Based on the (Sa)EctC crystal structures that we present here, we can currently not firmly understand why the replacement of Tyr-52 by Ala impairs enzyme function and iron content so drastically (Table 1).	RESULTS
125	131	Tyr-52	residue_name_number	Based on the (Sa)EctC crystal structures that we present here, we can currently not firmly understand why the replacement of Tyr-52 by Ala impairs enzyme function and iron content so drastically (Table 1).	RESULTS
135	138	Ala	residue_name	Based on the (Sa)EctC crystal structures that we present here, we can currently not firmly understand why the replacement of Tyr-52 by Ala impairs enzyme function and iron content so drastically (Table 1).	RESULTS
167	171	iron	chemical	Based on the (Sa)EctC crystal structures that we present here, we can currently not firmly understand why the replacement of Tyr-52 by Ala impairs enzyme function and iron content so drastically (Table 1).	RESULTS
26	36	His-55/Ala	mutant	This is different for the His-55/Ala substitution.	RESULTS
4	27	carboxy-terminal region	structure_element	The carboxy-terminal region of the (Sa)EctC protein is held in its position via an interaction of Glu-115 with His-55, where His-55 in turn interacts with Pro-110 (Fig 6a and 6b).	RESULTS
36	38	Sa	species	The carboxy-terminal region of the (Sa)EctC protein is held in its position via an interaction of Glu-115 with His-55, where His-55 in turn interacts with Pro-110 (Fig 6a and 6b).	RESULTS
39	43	EctC	protein	The carboxy-terminal region of the (Sa)EctC protein is held in its position via an interaction of Glu-115 with His-55, where His-55 in turn interacts with Pro-110 (Fig 6a and 6b).	RESULTS
98	105	Glu-115	residue_name_number	The carboxy-terminal region of the (Sa)EctC protein is held in its position via an interaction of Glu-115 with His-55, where His-55 in turn interacts with Pro-110 (Fig 6a and 6b).	RESULTS
111	117	His-55	residue_name_number	The carboxy-terminal region of the (Sa)EctC protein is held in its position via an interaction of Glu-115 with His-55, where His-55 in turn interacts with Pro-110 (Fig 6a and 6b).	RESULTS
125	131	His-55	residue_name_number	The carboxy-terminal region of the (Sa)EctC protein is held in its position via an interaction of Glu-115 with His-55, where His-55 in turn interacts with Pro-110 (Fig 6a and 6b).	RESULTS
155	162	Pro-110	residue_name_number	The carboxy-terminal region of the (Sa)EctC protein is held in its position via an interaction of Glu-115 with His-55, where His-55 in turn interacts with Pro-110 (Fig 6a and 6b).	RESULTS
26	57	evolutionarily highly conserved	protein_state	Each of these residues is evolutionarily highly conserved.	RESULTS
15	27	substitution	experimental_method	The individual substitution of either Glu-115 or His-55 by an Ala residue is predicted to disrupt this interactive network and therefore should affect enzyme activity.	RESULTS
38	45	Glu-115	residue_name_number	The individual substitution of either Glu-115 or His-55 by an Ala residue is predicted to disrupt this interactive network and therefore should affect enzyme activity.	RESULTS
49	55	His-55	residue_name_number	The individual substitution of either Glu-115 or His-55 by an Ala residue is predicted to disrupt this interactive network and therefore should affect enzyme activity.	RESULTS
62	65	Ala	residue_name	The individual substitution of either Glu-115 or His-55 by an Ala residue is predicted to disrupt this interactive network and therefore should affect enzyme activity.	RESULTS
103	122	interactive network	site	The individual substitution of either Glu-115 or His-55 by an Ala residue is predicted to disrupt this interactive network and therefore should affect enzyme activity.	RESULTS
12	23	Glu-115/Ala	mutant	Indeed, the Glu-115/Ala and the His-55/Ala substitutions possessed only 21% and 16% activity of the wild-type protein, respectively (Table 1).	RESULTS
32	42	His-55/Ala	mutant	Indeed, the Glu-115/Ala and the His-55/Ala substitutions possessed only 21% and 16% activity of the wild-type protein, respectively (Table 1).	RESULTS
100	109	wild-type	protein_state	Indeed, the Glu-115/Ala and the His-55/Ala substitutions possessed only 21% and 16% activity of the wild-type protein, respectively (Table 1).	RESULTS
4	15	Glu-115/Ala	mutant	The Glu-115/Ala mutant possessed wild-type levels of iron, whereas the iron content of the His-55/Ala substitutions dropped to 15% of the wild-type level (Table 1).	RESULTS
16	22	mutant	protein_state	The Glu-115/Ala mutant possessed wild-type levels of iron, whereas the iron content of the His-55/Ala substitutions dropped to 15% of the wild-type level (Table 1).	RESULTS
33	42	wild-type	protein_state	The Glu-115/Ala mutant possessed wild-type levels of iron, whereas the iron content of the His-55/Ala substitutions dropped to 15% of the wild-type level (Table 1).	RESULTS
53	57	iron	chemical	The Glu-115/Ala mutant possessed wild-type levels of iron, whereas the iron content of the His-55/Ala substitutions dropped to 15% of the wild-type level (Table 1).	RESULTS
71	75	iron	chemical	The Glu-115/Ala mutant possessed wild-type levels of iron, whereas the iron content of the His-55/Ala substitutions dropped to 15% of the wild-type level (Table 1).	RESULTS
91	101	His-55/Ala	mutant	The Glu-115/Ala mutant possessed wild-type levels of iron, whereas the iron content of the His-55/Ala substitutions dropped to 15% of the wild-type level (Table 1).	RESULTS
138	147	wild-type	protein_state	The Glu-115/Ala mutant possessed wild-type levels of iron, whereas the iron content of the His-55/Ala substitutions dropped to 15% of the wild-type level (Table 1).	RESULTS
8	16	replaced	experimental_method	We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity.	RESULTS
17	24	Glu-115	residue_name_number	We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity.	RESULTS
60	63	Asp	residue_name	We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity.	RESULTS
72	74	Sa	species	We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity.	RESULTS
75	79	EctC	protein	We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity.	RESULTS
98	107	wild-type	protein_state	We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity.	RESULTS
118	122	iron	chemical	We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity.	RESULTS
150	159	wild-type	protein_state	We also replaced Glu-115 with a negatively charged residue (Asp); this (Sa)EctC variant possessed wild-type levels of iron and still exhibited 77% of wild-type enzyme activity.	RESULTS
69	85	carboxy-terminus	structure_element	Collectively, these data suggest that the correct positioning of the carboxy-terminus of the (Sa)EctC protein is of structural and functional importance for the activity of the ectoine synthase.	RESULTS
94	96	Sa	species	Collectively, these data suggest that the correct positioning of the carboxy-terminus of the (Sa)EctC protein is of structural and functional importance for the activity of the ectoine synthase.	RESULTS
97	101	EctC	protein	Collectively, these data suggest that the correct positioning of the carboxy-terminus of the (Sa)EctC protein is of structural and functional importance for the activity of the ectoine synthase.	RESULTS
177	193	ectoine synthase	protein_type	Collectively, these data suggest that the correct positioning of the carboxy-terminus of the (Sa)EctC protein is of structural and functional importance for the activity of the ectoine synthase.	RESULTS
9	15	Leu-87	residue_name_number	Residues Leu-87 and Asp-91 are highly conserved in the ectoine synthase protein family.	RESULTS
20	26	Asp-91	residue_name_number	Residues Leu-87 and Asp-91 are highly conserved in the ectoine synthase protein family.	RESULTS
31	47	highly conserved	protein_state	Residues Leu-87 and Asp-91 are highly conserved in the ectoine synthase protein family.	RESULTS
55	71	ectoine synthase	protein_type	Residues Leu-87 and Asp-91 are highly conserved in the ectoine synthase protein family.	RESULTS
4	15	replacement	experimental_method	The replacement of Leu-87 by Ala led to a substantial drop in enzyme activity (Table 1).	RESULTS
19	25	Leu-87	residue_name_number	The replacement of Leu-87 by Ala led to a substantial drop in enzyme activity (Table 1).	RESULTS
29	32	Ala	residue_name	The replacement of Leu-87 by Ala led to a substantial drop in enzyme activity (Table 1).	RESULTS
16	27	replacement	experimental_method	Conversely, the replacement of Asp-91 by Ala and Glu, resulted in (Sa)EctC protein variants with 80% and 98% enzyme activity, respectively (Table 1).	RESULTS
31	37	Asp-91	residue_name_number	Conversely, the replacement of Asp-91 by Ala and Glu, resulted in (Sa)EctC protein variants with 80% and 98% enzyme activity, respectively (Table 1).	RESULTS
41	44	Ala	residue_name	Conversely, the replacement of Asp-91 by Ala and Glu, resulted in (Sa)EctC protein variants with 80% and 98% enzyme activity, respectively (Table 1).	RESULTS
49	52	Glu	residue_name	Conversely, the replacement of Asp-91 by Ala and Glu, resulted in (Sa)EctC protein variants with 80% and 98% enzyme activity, respectively (Table 1).	RESULTS
67	69	Sa	species	Conversely, the replacement of Asp-91 by Ala and Glu, resulted in (Sa)EctC protein variants with 80% and 98% enzyme activity, respectively (Table 1).	RESULTS
70	74	EctC	protein	Conversely, the replacement of Asp-91 by Ala and Glu, resulted in (Sa)EctC protein variants with 80% and 98% enzyme activity, respectively (Table 1).	RESULTS
56	62	Asp-91	residue_name_number	We currently cannot comment on possible functional role Asp-91.	RESULTS
9	15	Leu-87	residue_name_number	However, Leu-87 is positioned at the end of one of the β-sheets that form the dimer interface (Fig 5c) and it might therefore possess a structural role.	RESULTS
55	63	β-sheets	structure_element	However, Leu-87 is positioned at the end of one of the β-sheets that form the dimer interface (Fig 5c) and it might therefore possess a structural role.	RESULTS
78	93	dimer interface	site	However, Leu-87 is positioned at the end of one of the β-sheets that form the dimer interface (Fig 5c) and it might therefore possess a structural role.	RESULTS
24	30	Tyr-85	residue_name_number	It is also located near Tyr-85, one of the residues that probably coordinate the iron molecule with in the (Sa)EctC active site (Fig 6a) and therefore might exert indirect effects.	RESULTS
81	85	iron	chemical	It is also located near Tyr-85, one of the residues that probably coordinate the iron molecule with in the (Sa)EctC active site (Fig 6a) and therefore might exert indirect effects.	RESULTS
108	110	Sa	species	It is also located near Tyr-85, one of the residues that probably coordinate the iron molecule with in the (Sa)EctC active site (Fig 6a) and therefore might exert indirect effects.	RESULTS
111	115	EctC	protein	It is also located near Tyr-85, one of the residues that probably coordinate the iron molecule with in the (Sa)EctC active site (Fig 6a) and therefore might exert indirect effects.	RESULTS
116	127	active site	site	It is also located near Tyr-85, one of the residues that probably coordinate the iron molecule with in the (Sa)EctC active site (Fig 6a) and therefore might exert indirect effects.	RESULTS
0	7	His-117	residue_name_number	His-117 is a strictly conserved residue and its substitution by an Ala residue results in a drop of enzyme activity (down to 44%) and an iron content of 83% (Table 1).	RESULTS
13	31	strictly conserved	protein_state	His-117 is a strictly conserved residue and its substitution by an Ala residue results in a drop of enzyme activity (down to 44%) and an iron content of 83% (Table 1).	RESULTS
48	60	substitution	experimental_method	His-117 is a strictly conserved residue and its substitution by an Ala residue results in a drop of enzyme activity (down to 44%) and an iron content of 83% (Table 1).	RESULTS
67	70	Ala	residue_name	His-117 is a strictly conserved residue and its substitution by an Ala residue results in a drop of enzyme activity (down to 44%) and an iron content of 83% (Table 1).	RESULTS
137	141	iron	chemical	His-117 is a strictly conserved residue and its substitution by an Ala residue results in a drop of enzyme activity (down to 44%) and an iron content of 83% (Table 1).	RESULTS
13	20	His-117	residue_name_number	We note that His-117 is located close to the chemically undefined ligand in the (Sa)EctC structure (Fig 7b) and might thus play a role in contacting the natural substrate of the ectoine synthase.	RESULTS
81	83	Sa	species	We note that His-117 is located close to the chemically undefined ligand in the (Sa)EctC structure (Fig 7b) and might thus play a role in contacting the natural substrate of the ectoine synthase.	RESULTS
84	88	EctC	protein	We note that His-117 is located close to the chemically undefined ligand in the (Sa)EctC structure (Fig 7b) and might thus play a role in contacting the natural substrate of the ectoine synthase.	RESULTS
89	98	structure	evidence	We note that His-117 is located close to the chemically undefined ligand in the (Sa)EctC structure (Fig 7b) and might thus play a role in contacting the natural substrate of the ectoine synthase.	RESULTS
178	194	ectoine synthase	protein_type	We note that His-117 is located close to the chemically undefined ligand in the (Sa)EctC structure (Fig 7b) and might thus play a role in contacting the natural substrate of the ectoine synthase.	RESULTS
31	54	mutagenesis experiments	experimental_method	As an internal control for our mutagenesis experiments, we also substituted Thr-41 and His-51, two residues that are not evolutionarily conserved in EctC-type proteins with Ala residues.	RESULTS
64	75	substituted	experimental_method	As an internal control for our mutagenesis experiments, we also substituted Thr-41 and His-51, two residues that are not evolutionarily conserved in EctC-type proteins with Ala residues.	RESULTS
76	82	Thr-41	residue_name_number	As an internal control for our mutagenesis experiments, we also substituted Thr-41 and His-51, two residues that are not evolutionarily conserved in EctC-type proteins with Ala residues.	RESULTS
87	93	His-51	residue_name_number	As an internal control for our mutagenesis experiments, we also substituted Thr-41 and His-51, two residues that are not evolutionarily conserved in EctC-type proteins with Ala residues.	RESULTS
117	145	not evolutionarily conserved	protein_state	As an internal control for our mutagenesis experiments, we also substituted Thr-41 and His-51, two residues that are not evolutionarily conserved in EctC-type proteins with Ala residues.	RESULTS
149	167	EctC-type proteins	protein_type	As an internal control for our mutagenesis experiments, we also substituted Thr-41 and His-51, two residues that are not evolutionarily conserved in EctC-type proteins with Ala residues.	RESULTS
173	176	Ala	residue_name	As an internal control for our mutagenesis experiments, we also substituted Thr-41 and His-51, two residues that are not evolutionarily conserved in EctC-type proteins with Ala residues.	RESULTS
6	8	Sa	species	Both (Sa)EctC protein variants exhibited wild-type level enzyme activities and possessed a iron content matching that of the wild-type (Table 1).	RESULTS
9	13	EctC	protein	Both (Sa)EctC protein variants exhibited wild-type level enzyme activities and possessed a iron content matching that of the wild-type (Table 1).	RESULTS
41	50	wild-type	protein_state	Both (Sa)EctC protein variants exhibited wild-type level enzyme activities and possessed a iron content matching that of the wild-type (Table 1).	RESULTS
91	95	iron	chemical	Both (Sa)EctC protein variants exhibited wild-type level enzyme activities and possessed a iron content matching that of the wild-type (Table 1).	RESULTS
125	134	wild-type	protein_state	Both (Sa)EctC protein variants exhibited wild-type level enzyme activities and possessed a iron content matching that of the wild-type (Table 1).	RESULTS
64	66	Sa	species	This illustrates that not every amino acid substitution in the (Sa)EctC protein leads to an indiscriminate impairment of enzyme function and iron content.	RESULTS
67	71	EctC	protein	This illustrates that not every amino acid substitution in the (Sa)EctC protein leads to an indiscriminate impairment of enzyme function and iron content.	RESULTS
141	145	iron	chemical	This illustrates that not every amino acid substitution in the (Sa)EctC protein leads to an indiscriminate impairment of enzyme function and iron content.	RESULTS
4	25	crystallographic data	evidence	The crystallographic data presented here firmly identify ectoine synthase (EctC), an enzyme critical for the production of the microbial cytoprotectant and chemical chaperone ectoine, as a new member of the cupin superfamily.	DISCUSS
57	73	ectoine synthase	protein_type	The crystallographic data presented here firmly identify ectoine synthase (EctC), an enzyme critical for the production of the microbial cytoprotectant and chemical chaperone ectoine, as a new member of the cupin superfamily.	DISCUSS
75	79	EctC	protein	The crystallographic data presented here firmly identify ectoine synthase (EctC), an enzyme critical for the production of the microbial cytoprotectant and chemical chaperone ectoine, as a new member of the cupin superfamily.	DISCUSS
127	136	microbial	taxonomy_domain	The crystallographic data presented here firmly identify ectoine synthase (EctC), an enzyme critical for the production of the microbial cytoprotectant and chemical chaperone ectoine, as a new member of the cupin superfamily.	DISCUSS
175	182	ectoine	chemical	The crystallographic data presented here firmly identify ectoine synthase (EctC), an enzyme critical for the production of the microbial cytoprotectant and chemical chaperone ectoine, as a new member of the cupin superfamily.	DISCUSS
207	224	cupin superfamily	protein_type	The crystallographic data presented here firmly identify ectoine synthase (EctC), an enzyme critical for the production of the microbial cytoprotectant and chemical chaperone ectoine, as a new member of the cupin superfamily.	DISCUSS
40	42	Sa	species	The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins.	DISCUSS
43	47	EctC	protein	The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins.	DISCUSS
83	92	β-strands	structure_element	The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins.	DISCUSS
94	100	β1-β11	structure_element	The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins.	DISCUSS
110	119	α-helices	structure_element	The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins.	DISCUSS
121	124	α-I	structure_element	The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins.	DISCUSS
129	133	α-II	structure_element	The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins.	DISCUSS
195	213	crystal structures	evidence	The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins.	DISCUSS
217	223	cupins	protein_type	The overall fold and bowl shape of the (Sa)EctC protein (Figs 4 and 5) with its 11 β-strands (β1-β11) and two α-helices (α-I and α-II) closely adheres to the design principles typically found in crystal structures of cupins.	DISCUSS
19	35	ectoine synthase	protein_type	In addition to the ectoine synthase, the polyketide cyclase RemF is the only other currently known cupin-related enzyme that catalyze a cyclocondensation reaction although the substrates of EctC and RemF are rather different.	DISCUSS
41	59	polyketide cyclase	protein_type	In addition to the ectoine synthase, the polyketide cyclase RemF is the only other currently known cupin-related enzyme that catalyze a cyclocondensation reaction although the substrates of EctC and RemF are rather different.	DISCUSS
60	64	RemF	protein	In addition to the ectoine synthase, the polyketide cyclase RemF is the only other currently known cupin-related enzyme that catalyze a cyclocondensation reaction although the substrates of EctC and RemF are rather different.	DISCUSS
99	112	cupin-related	protein_type	In addition to the ectoine synthase, the polyketide cyclase RemF is the only other currently known cupin-related enzyme that catalyze a cyclocondensation reaction although the substrates of EctC and RemF are rather different.	DISCUSS
190	194	EctC	protein	In addition to the ectoine synthase, the polyketide cyclase RemF is the only other currently known cupin-related enzyme that catalyze a cyclocondensation reaction although the substrates of EctC and RemF are rather different.	DISCUSS
199	203	RemF	protein	In addition to the ectoine synthase, the polyketide cyclase RemF is the only other currently known cupin-related enzyme that catalyze a cyclocondensation reaction although the substrates of EctC and RemF are rather different.	DISCUSS
50	54	EctC	protein	As a consequence of the structural relatedness of EctC and RemF and the type of chemical reaction these two enzymes catalyze, is now understandable why bona fide EctC-type proteins are frequently (mis)-annotated in microbial genome sequences as “RemF-like” proteins.	DISCUSS
59	63	RemF	protein	As a consequence of the structural relatedness of EctC and RemF and the type of chemical reaction these two enzymes catalyze, is now understandable why bona fide EctC-type proteins are frequently (mis)-annotated in microbial genome sequences as “RemF-like” proteins.	DISCUSS
162	180	EctC-type proteins	protein_type	As a consequence of the structural relatedness of EctC and RemF and the type of chemical reaction these two enzymes catalyze, is now understandable why bona fide EctC-type proteins are frequently (mis)-annotated in microbial genome sequences as “RemF-like” proteins.	DISCUSS
215	224	microbial	taxonomy_domain	As a consequence of the structural relatedness of EctC and RemF and the type of chemical reaction these two enzymes catalyze, is now understandable why bona fide EctC-type proteins are frequently (mis)-annotated in microbial genome sequences as “RemF-like” proteins.	DISCUSS
246	255	RemF-like	protein_type	As a consequence of the structural relatedness of EctC and RemF and the type of chemical reaction these two enzymes catalyze, is now understandable why bona fide EctC-type proteins are frequently (mis)-annotated in microbial genome sequences as “RemF-like” proteins.	DISCUSS
4	8	pro-	taxonomy_domain	The pro- and eukaryotic members of the cupin superfamily perform a variety of both enzymatic and non-enzymatic functions that are built upon a common structural scaffold.	DISCUSS
13	23	eukaryotic	taxonomy_domain	The pro- and eukaryotic members of the cupin superfamily perform a variety of both enzymatic and non-enzymatic functions that are built upon a common structural scaffold.	DISCUSS
39	56	cupin superfamily	protein_type	The pro- and eukaryotic members of the cupin superfamily perform a variety of both enzymatic and non-enzymatic functions that are built upon a common structural scaffold.	DISCUSS
5	11	cupins	protein_type	Most cupins contain transition state metals that can promote different types of chemical reactions.	DISCUSS
16	38	cupin-related proteins	protein_type	Except for some cupin-related proteins that seem to function as metallo-chaperones, the bound metal is typically an essential part of the active sites.	DISCUSS
64	82	metallo-chaperones	protein_type	Except for some cupin-related proteins that seem to function as metallo-chaperones, the bound metal is typically an essential part of the active sites.	DISCUSS
88	93	bound	protein_state	Except for some cupin-related proteins that seem to function as metallo-chaperones, the bound metal is typically an essential part of the active sites.	DISCUSS
94	99	metal	chemical	Except for some cupin-related proteins that seem to function as metallo-chaperones, the bound metal is typically an essential part of the active sites.	DISCUSS
138	150	active sites	site	Except for some cupin-related proteins that seem to function as metallo-chaperones, the bound metal is typically an essential part of the active sites.	DISCUSS
43	59	ectoine synthase	protein_type	We report here for the first time that the ectoine synthase is a metal-dependent enzyme.	DISCUSS
65	70	metal	chemical	We report here for the first time that the ectoine synthase is a metal-dependent enzyme.	DISCUSS
0	6	ICP-MS	experimental_method	ICP-MS, metal-depletion and reconstitution experiments (Fig 3) consistently identify iron as the biologically most relevant metal for the EctC-catalyzed cyclocondensation reaction.	DISCUSS
8	54	metal-depletion and reconstitution experiments	experimental_method	ICP-MS, metal-depletion and reconstitution experiments (Fig 3) consistently identify iron as the biologically most relevant metal for the EctC-catalyzed cyclocondensation reaction.	DISCUSS
85	89	iron	chemical	ICP-MS, metal-depletion and reconstitution experiments (Fig 3) consistently identify iron as the biologically most relevant metal for the EctC-catalyzed cyclocondensation reaction.	DISCUSS
124	129	metal	chemical	ICP-MS, metal-depletion and reconstitution experiments (Fig 3) consistently identify iron as the biologically most relevant metal for the EctC-catalyzed cyclocondensation reaction.	DISCUSS
138	142	EctC	protein	ICP-MS, metal-depletion and reconstitution experiments (Fig 3) consistently identify iron as the biologically most relevant metal for the EctC-catalyzed cyclocondensation reaction.	DISCUSS
32	38	cupins	protein_type	However, as observed with other cupins, EctC is a somewhat promiscuous enzyme as far as the catalytically important metal is concerned when they are provided in large molar excess (Fig 3c).	DISCUSS
40	44	EctC	protein	However, as observed with other cupins, EctC is a somewhat promiscuous enzyme as far as the catalytically important metal is concerned when they are provided in large molar excess (Fig 3c).	DISCUSS
116	121	metal	chemical	However, as observed with other cupins, EctC is a somewhat promiscuous enzyme as far as the catalytically important metal is concerned when they are provided in large molar excess (Fig 3c).	DISCUSS
114	119	metal	chemical	Although some uncertainty remains with respect to the precise identity of amino acid residues that participate in metal binding by (Sa)EctC, our structure-guided site-directed mutagenesis experiments targeting the presumptive iron-binding residues (Fig 6a and 6b) demonstrate that none of them can be spared (Table 1).	DISCUSS
132	134	Sa	species	Although some uncertainty remains with respect to the precise identity of amino acid residues that participate in metal binding by (Sa)EctC, our structure-guided site-directed mutagenesis experiments targeting the presumptive iron-binding residues (Fig 6a and 6b) demonstrate that none of them can be spared (Table 1).	DISCUSS
135	139	EctC	protein	Although some uncertainty remains with respect to the precise identity of amino acid residues that participate in metal binding by (Sa)EctC, our structure-guided site-directed mutagenesis experiments targeting the presumptive iron-binding residues (Fig 6a and 6b) demonstrate that none of them can be spared (Table 1).	DISCUSS
145	187	structure-guided site-directed mutagenesis	experimental_method	Although some uncertainty remains with respect to the precise identity of amino acid residues that participate in metal binding by (Sa)EctC, our structure-guided site-directed mutagenesis experiments targeting the presumptive iron-binding residues (Fig 6a and 6b) demonstrate that none of them can be spared (Table 1).	DISCUSS
226	247	iron-binding residues	site	Although some uncertainty remains with respect to the precise identity of amino acid residues that participate in metal binding by (Sa)EctC, our structure-guided site-directed mutagenesis experiments targeting the presumptive iron-binding residues (Fig 6a and 6b) demonstrate that none of them can be spared (Table 1).	DISCUSS
24	36	metal center	site	The architecture of the metal center of ectoine synthase seems to be subjected to considerable evolutionary constraints.	DISCUSS
40	56	ectoine synthase	protein_type	The architecture of the metal center of ectoine synthase seems to be subjected to considerable evolutionary constraints.	DISCUSS
20	26	Glu-57	residue_name_number	The three residues (Glu-57, Tyr-85, His-93) that we deem to form it (Figs 6 and 7b) are strictly conserved in a large collection of EctC-type proteins originating from 16 bacterial and three archaeal phyla (Fig 2).	DISCUSS
28	34	Tyr-85	residue_name_number	The three residues (Glu-57, Tyr-85, His-93) that we deem to form it (Figs 6 and 7b) are strictly conserved in a large collection of EctC-type proteins originating from 16 bacterial and three archaeal phyla (Fig 2).	DISCUSS
36	42	His-93	residue_name_number	The three residues (Glu-57, Tyr-85, His-93) that we deem to form it (Figs 6 and 7b) are strictly conserved in a large collection of EctC-type proteins originating from 16 bacterial and three archaeal phyla (Fig 2).	DISCUSS
88	106	strictly conserved	protein_state	The three residues (Glu-57, Tyr-85, His-93) that we deem to form it (Figs 6 and 7b) are strictly conserved in a large collection of EctC-type proteins originating from 16 bacterial and three archaeal phyla (Fig 2).	DISCUSS
132	150	EctC-type proteins	protein_type	The three residues (Glu-57, Tyr-85, His-93) that we deem to form it (Figs 6 and 7b) are strictly conserved in a large collection of EctC-type proteins originating from 16 bacterial and three archaeal phyla (Fig 2).	DISCUSS
171	180	bacterial	taxonomy_domain	The three residues (Glu-57, Tyr-85, His-93) that we deem to form it (Figs 6 and 7b) are strictly conserved in a large collection of EctC-type proteins originating from 16 bacterial and three archaeal phyla (Fig 2).	DISCUSS
191	199	archaeal	taxonomy_domain	The three residues (Glu-57, Tyr-85, His-93) that we deem to form it (Figs 6 and 7b) are strictly conserved in a large collection of EctC-type proteins originating from 16 bacterial and three archaeal phyla (Fig 2).	DISCUSS
80	89	N-γ-ADABA	chemical	We also show here for the first time that, in addition to its natural substrate N-γ-ADABA, EctC also converts the isomer N-α-ADABA into ectoine, albeit with a 73-fold reduced catalytic efficiency (S3a and S3b Fig).	DISCUSS
91	95	EctC	protein	We also show here for the first time that, in addition to its natural substrate N-γ-ADABA, EctC also converts the isomer N-α-ADABA into ectoine, albeit with a 73-fold reduced catalytic efficiency (S3a and S3b Fig).	DISCUSS
121	130	N-α-ADABA	chemical	We also show here for the first time that, in addition to its natural substrate N-γ-ADABA, EctC also converts the isomer N-α-ADABA into ectoine, albeit with a 73-fold reduced catalytic efficiency (S3a and S3b Fig).	DISCUSS
136	143	ectoine	chemical	We also show here for the first time that, in addition to its natural substrate N-γ-ADABA, EctC also converts the isomer N-α-ADABA into ectoine, albeit with a 73-fold reduced catalytic efficiency (S3a and S3b Fig).	DISCUSS
175	195	catalytic efficiency	evidence	We also show here for the first time that, in addition to its natural substrate N-γ-ADABA, EctC also converts the isomer N-α-ADABA into ectoine, albeit with a 73-fold reduced catalytic efficiency (S3a and S3b Fig).	DISCUSS
11	22	active site	site	Hence, the active site of ectoine synthase must possess a certain degree of structural plasticity, a notion that is supported by the report on the EctC-catalyzed formation of the synthetic compatible solute ADPC through the cyclic condensation of two glutamine molecules.	DISCUSS
26	42	ectoine synthase	protein_type	Hence, the active site of ectoine synthase must possess a certain degree of structural plasticity, a notion that is supported by the report on the EctC-catalyzed formation of the synthetic compatible solute ADPC through the cyclic condensation of two glutamine molecules.	DISCUSS
147	151	EctC	protein	Hence, the active site of ectoine synthase must possess a certain degree of structural plasticity, a notion that is supported by the report on the EctC-catalyzed formation of the synthetic compatible solute ADPC through the cyclic condensation of two glutamine molecules.	DISCUSS
207	211	ADPC	chemical	Hence, the active site of ectoine synthase must possess a certain degree of structural plasticity, a notion that is supported by the report on the EctC-catalyzed formation of the synthetic compatible solute ADPC through the cyclic condensation of two glutamine molecules.	DISCUSS
251	260	glutamine	chemical	Hence, the active site of ectoine synthase must possess a certain degree of structural plasticity, a notion that is supported by the report on the EctC-catalyzed formation of the synthetic compatible solute ADPC through the cyclic condensation of two glutamine molecules.	DISCUSS
17	26	N-α-ADABA	chemical	Our finding that N-α-ADABA serves as a substrate for ectoine synthase has physiologically relevant ramifications for those microorganisms that can both synthesize and catabolize ectoine, since they need to prevent a futile cycle of synthesis and degradation when N-α-ADABA is produced as an intermediate in the catabolic route.	DISCUSS
53	69	ectoine synthase	protein_type	Our finding that N-α-ADABA serves as a substrate for ectoine synthase has physiologically relevant ramifications for those microorganisms that can both synthesize and catabolize ectoine, since they need to prevent a futile cycle of synthesis and degradation when N-α-ADABA is produced as an intermediate in the catabolic route.	DISCUSS
123	137	microorganisms	taxonomy_domain	Our finding that N-α-ADABA serves as a substrate for ectoine synthase has physiologically relevant ramifications for those microorganisms that can both synthesize and catabolize ectoine, since they need to prevent a futile cycle of synthesis and degradation when N-α-ADABA is produced as an intermediate in the catabolic route.	DISCUSS
178	185	ectoine	chemical	Our finding that N-α-ADABA serves as a substrate for ectoine synthase has physiologically relevant ramifications for those microorganisms that can both synthesize and catabolize ectoine, since they need to prevent a futile cycle of synthesis and degradation when N-α-ADABA is produced as an intermediate in the catabolic route.	DISCUSS
263	272	N-α-ADABA	chemical	Our finding that N-α-ADABA serves as a substrate for ectoine synthase has physiologically relevant ramifications for those microorganisms that can both synthesize and catabolize ectoine, since they need to prevent a futile cycle of synthesis and degradation when N-α-ADABA is produced as an intermediate in the catabolic route.	DISCUSS
60	62	C6	chemical	Although we cannot identify the true chemical nature of the C6 compound that was trapped in the (Sa)EctC structure nor its precise origin, we treated this compound as a proxy for the natural substrate of ectoine synthase, which is a C6 compound as well (Fig 7a).	DISCUSS
97	99	Sa	species	Although we cannot identify the true chemical nature of the C6 compound that was trapped in the (Sa)EctC structure nor its precise origin, we treated this compound as a proxy for the natural substrate of ectoine synthase, which is a C6 compound as well (Fig 7a).	DISCUSS
100	104	EctC	protein	Although we cannot identify the true chemical nature of the C6 compound that was trapped in the (Sa)EctC structure nor its precise origin, we treated this compound as a proxy for the natural substrate of ectoine synthase, which is a C6 compound as well (Fig 7a).	DISCUSS
105	114	structure	evidence	Although we cannot identify the true chemical nature of the C6 compound that was trapped in the (Sa)EctC structure nor its precise origin, we treated this compound as a proxy for the natural substrate of ectoine synthase, which is a C6 compound as well (Fig 7a).	DISCUSS
204	220	ectoine synthase	protein_type	Although we cannot identify the true chemical nature of the C6 compound that was trapped in the (Sa)EctC structure nor its precise origin, we treated this compound as a proxy for the natural substrate of ectoine synthase, which is a C6 compound as well (Fig 7a).	DISCUSS
122	131	N-γ-ADABA	chemical	We assumed that its location and mode of binding gives, in all likelihood, clues as to the position of the true substrate N-γ-ADABA within the EctC active site.	DISCUSS
143	147	EctC	protein	We assumed that its location and mode of binding gives, in all likelihood, clues as to the position of the true substrate N-γ-ADABA within the EctC active site.	DISCUSS
148	159	active site	site	We assumed that its location and mode of binding gives, in all likelihood, clues as to the position of the true substrate N-γ-ADABA within the EctC active site.	DISCUSS
8	33	site-directed mutagenesis	experimental_method	Indeed, site-directed mutagenesis of those five residues that contact the unknown C6 compound (Fig 7b) yielded (Sa)EctC variants with strongly impaired enzyme function but near wild-type levels of iron (Table 1).	DISCUSS
112	114	Sa	species	Indeed, site-directed mutagenesis of those five residues that contact the unknown C6 compound (Fig 7b) yielded (Sa)EctC variants with strongly impaired enzyme function but near wild-type levels of iron (Table 1).	DISCUSS
115	119	EctC	protein	Indeed, site-directed mutagenesis of those five residues that contact the unknown C6 compound (Fig 7b) yielded (Sa)EctC variants with strongly impaired enzyme function but near wild-type levels of iron (Table 1).	DISCUSS
177	186	wild-type	protein_state	Indeed, site-directed mutagenesis of those five residues that contact the unknown C6 compound (Fig 7b) yielded (Sa)EctC variants with strongly impaired enzyme function but near wild-type levels of iron (Table 1).	DISCUSS
197	201	iron	chemical	Indeed, site-directed mutagenesis of those five residues that contact the unknown C6 compound (Fig 7b) yielded (Sa)EctC variants with strongly impaired enzyme function but near wild-type levels of iron (Table 1).	DISCUSS
61	79	strongly conserved	protein_state	This set of data and the fact that the targeted residues are strongly conserved among EctC-type proteins (Fig 2) is consistent with their potential role in N-γ-ADABA binding or enzyme catalysis.	DISCUSS
86	104	EctC-type proteins	protein_type	This set of data and the fact that the targeted residues are strongly conserved among EctC-type proteins (Fig 2) is consistent with their potential role in N-γ-ADABA binding or enzyme catalysis.	DISCUSS
156	165	N-γ-ADABA	chemical	This set of data and the fact that the targeted residues are strongly conserved among EctC-type proteins (Fig 2) is consistent with their potential role in N-γ-ADABA binding or enzyme catalysis.	DISCUSS
30	51	crystallographic data	evidence	We therefore surmise that our crystallographic data and the site-directed mutagenesis study reported here provide a structural and functional view into the architecture of the EctC active site (Fig 7b).	DISCUSS
60	91	site-directed mutagenesis study	experimental_method	We therefore surmise that our crystallographic data and the site-directed mutagenesis study reported here provide a structural and functional view into the architecture of the EctC active site (Fig 7b).	DISCUSS
176	180	EctC	protein	We therefore surmise that our crystallographic data and the site-directed mutagenesis study reported here provide a structural and functional view into the architecture of the EctC active site (Fig 7b).	DISCUSS
181	192	active site	site	We therefore surmise that our crystallographic data and the site-directed mutagenesis study reported here provide a structural and functional view into the architecture of the EctC active site (Fig 7b).	DISCUSS
4	20	ectoine synthase	protein_type	The ectoine synthase from the cold-adapted marine bacterium S. alaskensis can be considered as a psychrophilic enzyme (S3a Fig), types of proteins with a considerable structural flexibility.	DISCUSS
43	59	marine bacterium	taxonomy_domain	The ectoine synthase from the cold-adapted marine bacterium S. alaskensis can be considered as a psychrophilic enzyme (S3a Fig), types of proteins with a considerable structural flexibility.	DISCUSS
60	73	S. alaskensis	species	The ectoine synthase from the cold-adapted marine bacterium S. alaskensis can be considered as a psychrophilic enzyme (S3a Fig), types of proteins with a considerable structural flexibility.	DISCUSS
64	82	crystal structures	evidence	This probably worked to the detriment of our efforts in solving crystal structures of the full-length (Sa)EctC protein in complex with either N-γ-ADABA or ectoine.	DISCUSS
90	101	full-length	protein_state	This probably worked to the detriment of our efforts in solving crystal structures of the full-length (Sa)EctC protein in complex with either N-γ-ADABA or ectoine.	DISCUSS
103	105	Sa	species	This probably worked to the detriment of our efforts in solving crystal structures of the full-length (Sa)EctC protein in complex with either N-γ-ADABA or ectoine.	DISCUSS
106	110	EctC	protein	This probably worked to the detriment of our efforts in solving crystal structures of the full-length (Sa)EctC protein in complex with either N-γ-ADABA or ectoine.	DISCUSS
119	134	in complex with	protein_state	This probably worked to the detriment of our efforts in solving crystal structures of the full-length (Sa)EctC protein in complex with either N-γ-ADABA or ectoine.	DISCUSS
142	151	N-γ-ADABA	chemical	This probably worked to the detriment of our efforts in solving crystal structures of the full-length (Sa)EctC protein in complex with either N-γ-ADABA or ectoine.	DISCUSS
155	162	ectoine	chemical	This probably worked to the detriment of our efforts in solving crystal structures of the full-length (Sa)EctC protein in complex with either N-γ-ADABA or ectoine.	DISCUSS
8	17	microbial	taxonomy_domain	Because microbial ectoine producers can colonize ecological niches with rather different physicochemical attributes, it seems promising to exploit this considerable biodiversity to identify EctC proteins with enhanced protein stability.	DISCUSS
18	25	ectoine	chemical	Because microbial ectoine producers can colonize ecological niches with rather different physicochemical attributes, it seems promising to exploit this considerable biodiversity to identify EctC proteins with enhanced protein stability.	DISCUSS
190	203	EctC proteins	protein_type	Because microbial ectoine producers can colonize ecological niches with rather different physicochemical attributes, it seems promising to exploit this considerable biodiversity to identify EctC proteins with enhanced protein stability.	DISCUSS
57	61	EctC	protein	It is hoped that these can be further employed to obtain EctC crystal structures with either the substrate or the reaction product.	DISCUSS
62	80	crystal structures	evidence	It is hoped that these can be further employed to obtain EctC crystal structures with either the substrate or the reaction product.	DISCUSS
31	47	ectoine synthase	protein_type	Together with our finding that ectoine synthase is metal dependent, these crystal structures should allow a more detailed understanding of the chemistry underlying the EctC-catalyzed cyclocondensation reaction.	DISCUSS
51	66	metal dependent	protein_state	Together with our finding that ectoine synthase is metal dependent, these crystal structures should allow a more detailed understanding of the chemistry underlying the EctC-catalyzed cyclocondensation reaction.	DISCUSS
74	92	crystal structures	evidence	Together with our finding that ectoine synthase is metal dependent, these crystal structures should allow a more detailed understanding of the chemistry underlying the EctC-catalyzed cyclocondensation reaction.	DISCUSS
168	172	EctC	protein	Together with our finding that ectoine synthase is metal dependent, these crystal structures should allow a more detailed understanding of the chemistry underlying the EctC-catalyzed cyclocondensation reaction.	DISCUSS