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 (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 | 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 Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
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 | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
periplasm | - |
Allochromatium vinosum | - |
- |
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 | UniProt | Comment | Textmining |
---|---|---|---|
Allochromatium vinosum | D3RVD4 | - |
- |
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 | 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 |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | Comment | Organism |
---|---|---|
More | three-dimensional structure analysis of wild-type and mutant enzymes, modelling, overview | Allochromatium vinosum |
Synonyms | Comment | Organism |
---|---|---|
TsdA | - |
Allochromatium vinosum |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Allochromatium vinosum |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
4 | 5 | assay at | Allochromatium vinosum |
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 | Comment | Organism |
---|---|---|
additional information | Cys96 is an essential residue for catalysis | Allochromatium vinosum |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
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 |