Literature summary for 1.8.2.2 extracted from

Brito, J.A.; Denkmann, K.; Pereira, I.A.; Archer, M.; Dahl, C.
Thiosulfate dehydrogenase (TsdA) from Allochromatium vinosum structural and functional insights into thiosulfate oxidation (2015), J. Biol. Chem., 290, 9222-9238 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene tsdA, recombinant expression of Strep-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain DH5alpha	Allochromatium vinosum

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant wild-type and K208N and K208G mutant enzymes, freeor in complex with tetrathionate, dithionite, or bisulfit, sitting drop vapour diffusion method, mixing of 0.003 ml of 8 mg/ml protein in 20 mM Bis-Tris-HCl, pH 6.5, with 0.0015 ml of reservoir solution containing 23.5% w/v PEG 3350, 0.2 M (NH4)2SO4, 0.1 M Bis-Tris, pH 6.28, and 0.1 M NaI, and equilibration against 0.120 ml of reservoir solution, the complex crystals are formed by soaking in an excess of ligands, tetrathionate, dithionite, and bisulfite, 20°C, X-ray diffraction structure determination and analysis at 1.40-1.98 A resolution, modelling	Allochromatium vinosum

Crystallization (Comment)

Organism

purified recombinant wild-type and K208N and K208G mutant enzymes, freeor in complex with tetrathionate, dithionite, or bisulfit, sitting drop vapour diffusion method, mixing of 0.003 ml of 8 mg/ml protein in 20 mM Bis-Tris-HCl, pH 6.5, with 0.0015 ml of reservoir solution containing 23.5% w/v PEG 3350, 0.2 M (NH4)2SO4, 0.1 M Bis-Tris, pH 6.28, and 0.1 M NaI, and equilibration against 0.120 ml of reservoir solution, the complex crystals are formed by soaking in an excess of ligands, tetrathionate, dithionite, and bisulfite, 20°C, X-ray diffraction structure determination and analysis at 1.40-1.98 A resolution, modelling

Allochromatium vinosum

Protein Variants

Protein Variants	Comment	Organism
C96G	site-directed mutagenesis, inactive mutant	Allochromatium vinosum
C96H	site-directed mutagenesis, inactive mutant	Allochromatium vinosum
C96M	site-directed mutagenesis, inactive mutant	Allochromatium vinosum
K208G	site-directed mutagenesis, the mutant is catalytically active in thiosulfate oxidation as well as in tetrathionate reduction	Allochromatium vinosum
K208N	site-directed mutagenesis, the mutant is catalytically active in thiosulfate oxidation as well as in tetrathionate reduction	Allochromatium vinosum
M209G	site-directed mutagenesis, the mutant is catalytically active in thiosulfate oxidation as well as in tetrathionate reduction	Allochromatium vinosum

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.14	-	thiosulfate	recombinant wild-type enzyme, pH 5.0, 30°C, with ferricyanide	Allochromatium vinosum
0.17	-	thiosulfate	recombinant mutant K208N, pH 5.0, 30°C, with ferricyanide	Allochromatium vinosum
0.46	-	thiosulfate	recombinant mutant K208G, pH 5.0, 30°C, with ferricyanide	Allochromatium vinosum
0.9	-	thiosulfate	recombinant mutant M209G, pH 4.0, 30°C, with ferricyanide	Allochromatium vinosum
1.08	-	thiosulfate	recombinant mutant M209G, pH 5.0, 30°C, with ferricyanide	Allochromatium vinosum
1.1	-	thiosulfate	recombinant mutant K208G, pH 4.0, 30°C, with ferricyanide	Allochromatium vinosum
1.1	-	thiosulfate	recombinant wild-type enzyme, pH 4.0, 30°C, with ferricyanide	Allochromatium vinosum
1.16	-	tetrathionate	recombinant mutant M209G, pH 5.0, 30°C, with methylviologen	Allochromatium vinosum
1.17	-	tetrathionate	recombinant mutant K208G, pH 5.0, 30°C, with methylviologen	Allochromatium vinosum
1.3	-	thiosulfate	recombinant mutant K208N, pH 4.0, 30°C, with ferricyanide	Allochromatium vinosum
1.98	-	tetrathionate	recombinant wild-type enzyme, pH 5.0, 30°C, with methylviologen	Allochromatium vinosum
3.2	-	tetrathionate	recombinant mutant K208N, pH 5.0, 30°C, with methylviologen	Allochromatium vinosum

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
periplasm	-	Allochromatium vinosum	-	-

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
2 thiosulfate + 2 ferricytochrome c	Allochromatium vinosum	in Allochromatium vinosum, enzyme TsdA operates in the direction of tetrathionate formation	tetrathionate + 2 ferrocytochrome c	-	r

Organism

Organism	UniProt	Comment	Textmining
Allochromatium vinosum	D3RVD4	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant Strep-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by affinity chromatography and gel filtration	Allochromatium vinosum

Reaction

Reaction	Comment	Organism	Reaction ID
2 thiosulfate + 2 ferricytochrome c = tetrathionate + 2 ferrocytochrome c + 4 H+	Cys96 is an essential residue for catalysis, the TsdA reaction cycle involves the transient presence of heme 1 in the high-spin state caused by movement of the S atom of Cys96 out of the iron coordination sphere	Allochromatium vinosum	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
2 thiosulfate + 2 ferricyanide	-	Allochromatium vinosum	tetrathionate + 2 ferrocyanide	-	r
2 thiosulfate + 2 ferricytochrome c	in Allochromatium vinosum, enzyme TsdA operates in the direction of tetrathionate formation	Allochromatium vinosum	tetrathionate + 2 ferrocytochrome c	-	r
2 thiosulfate + 2 ferricytochrome c	in Allochromatium vinosum, enzyme TsdA operates in the direction of tetrathionate formation. A ligand switch from Lys208 to Met209 is observed upon reduction of the enzyme	Allochromatium vinosum	tetrathionate + 2 ferrocytochrome c	-	r
additional information	tetrathionate-dependent methylviologen oxidation and thiosulfate-dependent ferricyanide reduction are measured	Allochromatium vinosum	?	-	?
tetrathionate + reduced methylviologen	-	Allochromatium vinosum	2 thiosulfate + methylviologen	-	r

Subunits

Subunits	Comment	Organism
More	three-dimensional structure analysis of wild-type and mutant enzymes, modelling, overview	Allochromatium vinosum

Synonyms

Synonyms	Comment	Organism
TsdA	-	Allochromatium vinosum

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Allochromatium vinosum

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
3	6	tetrathionate	recombinant mutant K208N, pH 5.0, 30°C, with methylviologen	Allochromatium vinosum
37	-	tetrathionate	recombinant wild-type enzyme, pH 5.0, 30°C, with methylviologen	Allochromatium vinosum
42	-	tetrathionate	recombinant mutant K208G, pH 5.0, 30°C, with methylviologen	Allochromatium vinosum
54	-	tetrathionate	recombinant mutant M209G, pH 5.0, 30°C, with methylviologen	Allochromatium vinosum
350	-	thiosulfate	recombinant mutant K208N, pH 5.0, 30°C, with ferricyanide	Allochromatium vinosum
370	-	thiosulfate	recombinant mutant K208G, pH 5.0, 30°C, with ferricyanide	Allochromatium vinosum
660	-	thiosulfate	recombinant mutant K208N, pH 4.0, 30°C, with ferricyanide	Allochromatium vinosum
760	-	thiosulfate	recombinant mutant M209G, pH 5.0, 30°C, with ferricyanide	Allochromatium vinosum
1070	-	thiosulfate	recombinant mutant K208G, pH 4.0, 30°C, with ferricyanide	Allochromatium vinosum
1330	-	thiosulfate	recombinant mutant M209G, pH 4.0, 30°C, with ferricyanide	Allochromatium vinosum
2100	-	thiosulfate	recombinant wild-type enzyme, pH 5.0, 30°C, with ferricyanide	Allochromatium vinosum
14000	-	thiosulfate	recombinant wild-type enzyme, pH 4.0, 30°C, with ferricyanide	Allochromatium vinosum

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
4	5	assay at	Allochromatium vinosum

Cofactor

Cofactor	Comment	Organism	Structure
cytochrome c	a diheme c-type cytochrome. The enzyme contains two typical class I c-type cytochrome domains wrapped around two hemes axially coordinated by His53/Cys96 and His164/Lys208. These domains are very similar, suggesting a gene duplication event during evolution. A ligand switch from Lys208 to Met209 is observed upon reduction of the enzyme. Heme 2 is the electron exit point	Allochromatium vinosum
heme	ferric heme, the enzyme shows unusual histidine-cysteine axial heme coordination. Heme structure analysis, detailed overview	Allochromatium vinosum

General Information

General Information	Comment	Organism
additional information	Cys96 is an essential residue for catalysis	Allochromatium vinosum

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
11.3	-	tetrathionate	recombinant mutant K208N, pH 5.0, 30°C, with methylviologen	Allochromatium vinosum
18.7	-	tetrathionate	recombinant wild-type enzyme, pH 5.0, 30°C, with methylviologen	Allochromatium vinosum
35.9	-	tetrathionate	recombinant mutant K208G, pH 5.0, 30°C, with methylviologen	Allochromatium vinosum
46.7	-	tetrathionate	recombinant mutant M209G, pH 5.0, 30°C, with methylviologen	Allochromatium vinosum
510	-	thiosulfate	recombinant mutant K208N, pH 4.0, 30°C, with ferricyanide	Allochromatium vinosum
710	-	thiosulfate	recombinant mutant M209G, pH 5.0, 30°C, with ferricyanide	Allochromatium vinosum
790	-	thiosulfate	recombinant mutant K208G, pH 5.0, 30°C, with ferricyanide	Allochromatium vinosum
970	-	thiosulfate	recombinant mutant K208G, pH 4.0, 30°C, with ferricyanide	Allochromatium vinosum
1480	-	thiosulfate	recombinant mutant M209G, pH 4.0, 30°C, with ferricyanide	Allochromatium vinosum
2100	-	thiosulfate	recombinant mutant K208N, pH 5.0, 30°C, with ferricyanide	Allochromatium vinosum
13000	-	thiosulfate	recombinant wild-type enzyme, pH 4.0, 30°C, with ferricyanide	Allochromatium vinosum
14100	-	thiosulfate	recombinant wild-type enzyme, pH 5.0, 30°C, with ferricyanide	Allochromatium vinosum