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Literature summary 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 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.8.2.2 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)

EC Number Crystallization (Comment) Organism
1.8.2.2 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

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

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.8.2.2 0.14
-
 thiosulfate recombinant wild-type enzyme, pH 5.0, 30°C, with ferricyanide Allochromatium vinosum
1.8.2.2 0.17
-
 thiosulfate recombinant mutant K208N, pH 5.0, 30°C, with ferricyanide Allochromatium vinosum
1.8.2.2 0.46
-
 thiosulfate recombinant mutant K208G, pH 5.0, 30°C, with ferricyanide Allochromatium vinosum
1.8.2.2 0.9
-
 thiosulfate recombinant mutant M209G, pH 4.0, 30°C, with ferricyanide Allochromatium vinosum
1.8.2.2 1.08
-
 thiosulfate recombinant mutant M209G, pH 5.0, 30°C, with ferricyanide Allochromatium vinosum
1.8.2.2 1.1
-
 thiosulfate recombinant mutant K208G, pH 4.0, 30°C, with ferricyanide Allochromatium vinosum
1.8.2.2 1.1
-
 thiosulfate recombinant wild-type enzyme, pH 4.0, 30°C, with ferricyanide Allochromatium vinosum
1.8.2.2 1.16
-
 tetrathionate recombinant mutant M209G, pH 5.0, 30°C, with methylviologen Allochromatium vinosum
1.8.2.2 1.17
-
 tetrathionate recombinant mutant K208G, pH 5.0, 30°C, with methylviologen Allochromatium vinosum
1.8.2.2 1.3
-
 thiosulfate recombinant mutant K208N, pH 4.0, 30°C, with ferricyanide Allochromatium vinosum
1.8.2.2 1.98
-
 tetrathionate recombinant wild-type enzyme, pH 5.0, 30°C, with methylviologen Allochromatium vinosum
1.8.2.2 3.2
-
 tetrathionate recombinant mutant K208N, pH 5.0, 30°C, with methylviologen Allochromatium vinosum

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
1.8.2.2 periplasm
-
 Allochromatium vinosum
-
-

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.8.2.2 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

EC Number Organism UniProt Comment Textmining
1.8.2.2 Allochromatium vinosum D3RVD4
-
-

Purification (Commentary)

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

Reaction

EC Number Reaction Comment Organism Reaction ID
1.8.2.2 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)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.8.2.2 2 thiosulfate + 2 ferricyanide
-
 Allochromatium vinosum tetrathionate + 2 ferrocyanide
-
 r
1.8.2.2 2 thiosulfate + 2 ferricytochrome c in Allochromatium vinosum, enzyme TsdA operates in the direction of tetrathionate formation Allochromatium vinosum tetrathionate + 2 ferrocytochrome c
-
 r
1.8.2.2 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
1.8.2.2 additional information tetrathionate-dependent methylviologen oxidation and thiosulfate-dependent ferricyanide reduction are measured Allochromatium vinosum ?
-
 ?
1.8.2.2 tetrathionate + reduced methylviologen
-
 Allochromatium vinosum 2 thiosulfate + methylviologen
-
 r

Subunits

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

Synonyms

EC Number Synonyms Comment Organism
1.8.2.2 TsdA
-
 Allochromatium vinosum

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.8.2.2 30
-
 assay at Allochromatium vinosum

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.8.2.2 3 6 tetrathionate recombinant mutant K208N, pH 5.0, 30°C, with methylviologen Allochromatium vinosum
1.8.2.2 37
-
 tetrathionate recombinant wild-type enzyme, pH 5.0, 30°C, with methylviologen Allochromatium vinosum
1.8.2.2 42
-
 tetrathionate recombinant mutant K208G, pH 5.0, 30°C, with methylviologen Allochromatium vinosum
1.8.2.2 54
-
 tetrathionate recombinant mutant M209G, pH 5.0, 30°C, with methylviologen Allochromatium vinosum
1.8.2.2 350
-
 thiosulfate recombinant mutant K208N, pH 5.0, 30°C, with ferricyanide Allochromatium vinosum
1.8.2.2 370
-
 thiosulfate recombinant mutant K208G, pH 5.0, 30°C, with ferricyanide Allochromatium vinosum
1.8.2.2 660
-
 thiosulfate recombinant mutant K208N, pH 4.0, 30°C, with ferricyanide Allochromatium vinosum
1.8.2.2 760
-
 thiosulfate recombinant mutant M209G, pH 5.0, 30°C, with ferricyanide Allochromatium vinosum
1.8.2.2 1070
-
 thiosulfate recombinant mutant K208G, pH 4.0, 30°C, with ferricyanide Allochromatium vinosum
1.8.2.2 1330
-
 thiosulfate recombinant mutant M209G, pH 4.0, 30°C, with ferricyanide Allochromatium vinosum
1.8.2.2 2100
-
 thiosulfate recombinant wild-type enzyme, pH 5.0, 30°C, with ferricyanide Allochromatium vinosum
1.8.2.2 14000
-
 thiosulfate recombinant wild-type enzyme, pH 4.0, 30°C, with ferricyanide Allochromatium vinosum

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.8.2.2 4 5 assay at Allochromatium vinosum

Cofactor

EC Number Cofactor Comment Organism Structure
1.8.2.2 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
1.8.2.2 heme ferric heme, the enzyme shows unusual histidine-cysteine axial heme coordination. Heme structure analysis, detailed overview Allochromatium vinosum

General Information

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

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.8.2.2 11.3
-
 tetrathionate recombinant mutant K208N, pH 5.0, 30°C, with methylviologen Allochromatium vinosum
1.8.2.2 18.7
-
 tetrathionate recombinant wild-type enzyme, pH 5.0, 30°C, with methylviologen Allochromatium vinosum
1.8.2.2 35.9
-
 tetrathionate recombinant mutant K208G, pH 5.0, 30°C, with methylviologen Allochromatium vinosum
1.8.2.2 46.7
-
 tetrathionate recombinant mutant M209G, pH 5.0, 30°C, with methylviologen Allochromatium vinosum
1.8.2.2 510
-
 thiosulfate recombinant mutant K208N, pH 4.0, 30°C, with ferricyanide Allochromatium vinosum
1.8.2.2 710
-
 thiosulfate recombinant mutant M209G, pH 5.0, 30°C, with ferricyanide Allochromatium vinosum
1.8.2.2 790
-
 thiosulfate recombinant mutant K208G, pH 5.0, 30°C, with ferricyanide Allochromatium vinosum
1.8.2.2 970
-
 thiosulfate recombinant mutant K208G, pH 4.0, 30°C, with ferricyanide Allochromatium vinosum
1.8.2.2 1480
-
 thiosulfate recombinant mutant M209G, pH 4.0, 30°C, with ferricyanide Allochromatium vinosum
1.8.2.2 2100
-
 thiosulfate recombinant mutant K208N, pH 5.0, 30°C, with ferricyanide Allochromatium vinosum
1.8.2.2 13000
-
 thiosulfate recombinant wild-type enzyme, pH 4.0, 30°C, with ferricyanide Allochromatium vinosum
1.8.2.2 14100
-
 thiosulfate recombinant wild-type enzyme, pH 5.0, 30°C, with ferricyanide Allochromatium vinosum