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Literature summary for 1.1.1.357 extracted from
- Effects of active site residues of 3alpha-hydroxysteroid dehydrogenase from Pseudomonas sp. b-0831 on its catalysis and cofactor binding (2018), Biosci. Biotechnol. Biochem., 82, 1702-1707 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| recombinant expression of N-terminally His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3). The N-terminal His-tag has little effect on the enzyme function | Pseudomonas sp. B-0831 |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| K157A | site-directed mutagenesis of a catalytic site residue, the NADH binding affinity of K157A mutant is much lower than that of the wild-type resulting in a decreased kcat | Pseudomonas sp. B-0831 |
| S114A | site-directed mutagenesis of a catalytic site residue, the mutant shows higher Km and lower kcat values in both oxidation and reduction reactions compared to wild-type | Pseudomonas sp. B-0831 |
| S114A/Y153A | site-directed mutagenesis of a catalytic site residue, the double mutant shows higher Km and a highly reduced kcat values in both oxidation and reduction reactions compared to wild-type | Pseudomonas sp. B-0831 |
| Y153A | site-directed mutagenesis of a catalytic site residue, the mutant shows higher Km and lower kcat values in both oxidation and reduction reactions compared to wild-type | Pseudomonas sp. B-0831 |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | steady-state kinetics | Pseudomonas sp. B-0831 | |
| 51 | - |
Dehydrocholic acid | recombinant His-tagged wild-type enzyme, pH 8.0, 37°C | Pseudomonas sp. B-0831 |  |
| 56 | - |
cholic acid | recombinant His-tagged wild-type enzyme, pH 8.0, 37°C | Pseudomonas sp. B-0831 | |
| 78 | - |
cholic acid | recombinant His-tagged mutant S114A/Y153F, pH 8.0, 37°C | Pseudomonas sp. B-0831 | |
| 96 | - |
cholic acid | recombinant His-tagged mutant K157A, pH 8.0, 37°C | Pseudomonas sp. B-0831 | |
| 239 | - |
Dehydrocholic acid | recombinant His-tagged mutant S114A, pH 8.0, 37°C | Pseudomonas sp. B-0831 | |
| 468 | - |
cholic acid | recombinant His-tagged mutant Y153F, pH 8.0, 37°C | Pseudomonas sp. B-0831 | |
| 586 | - |
cholic acid | recombinant His-tagged mutant S114A, pH 8.0, 37°C | Pseudomonas sp. B-0831 | |
| 2489 | - |
Dehydrocholic acid | recombinant His-tagged mutant Y153F, pH 8.0, 37°C | Pseudomonas sp. B-0831 | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| cholic acid + NAD+ | Pseudomonas sp. B-0831 | - |
dehydrocholic acid + NADH + H+ | - |
r | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Pseudomonas sp. B-0831 | - |
- |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and dialysis | Pseudomonas sp. B-0831 |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| cholic acid + NAD+ | - |
Pseudomonas sp. B-0831 | dehydrocholic acid + NADH + H+ | - |
r | |
| dehydrocholic acid + NADH + H+ | - |
Pseudomonas sp. B-0831 | cholic acid + NAD+ | - |
r | |
| additional information | NADH binding to the recombinant enzyme Ps3alphaHSD and its mutants is thermodynamically analyzed using isothermal titration calorimetry | Pseudomonas sp. B-0831 | ? | - |
- |
|
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| 3alpha-hydroxysteroid dehydrogenase | - |
Pseudomonas sp. B-0831 |
| Ps3alphaHSD | - |
Pseudomonas sp. B-0831 |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 37 | - |
assay at, both reaction directions | Pseudomonas sp. B-0831 |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.012 | - |
cholic acid | recombinant His-tagged mutant S114A/Y153F, pH 8.0, 37°C | Pseudomonas sp. B-0831 | |
| 0.045 | - |
cholic acid | recombinant His-tagged mutant K157A, pH 8.0, 37°C | Pseudomonas sp. B-0831 | |
| 0.62 | - |
cholic acid | recombinant His-tagged mutant Y153F, pH 8.0, 37°C | Pseudomonas sp. B-0831 | |
| 0.8 | - |
cholic acid | recombinant His-tagged mutant S114A, pH 8.0, 37°C | Pseudomonas sp. B-0831 | |
| 0.89 | - |
Dehydrocholic acid | recombinant His-tagged mutant S114A, pH 8.0, 37°C | Pseudomonas sp. B-0831 | |
| 1.1 | - |
Dehydrocholic acid | recombinant His-tagged mutant Y153F, pH 8.0, 37°C | Pseudomonas sp. B-0831 | |
| 23 | - |
cholic acid | recombinant His-tagged wild-type enzyme, pH 8.0, 37°C | Pseudomonas sp. B-0831 | |
| 48 | - |
Dehydrocholic acid | recombinant His-tagged wild-type enzyme, pH 8.0, 37°C | Pseudomonas sp. B-0831 | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 8 | - |
assay at, both reaction directions | Pseudomonas sp. B-0831 |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| NAD+ | - |
Pseudomonas sp. B-0831 | |
| NADH | - |
Pseudomonas sp. B-0831 | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| malfunction | loss of hydrogen bonding with NADH upon the Y153F mutation results in increased enthalpy change, partially compensated by increased entropy change. The NADH binding affinity of K157A mutant is much lower than that of the wild-type, mainly due to loss of a hydrogen bond. The decreased affinity results in decreased kcat. Compared to the wild-type, the mutants S114A and Y153F show higher Km and lower kcat values in both oxidation and reduction reactions. Simultaneous mutation of S114A and Y153F results in a significant decrease in kcat relative to the single mutant | Pseudomonas sp. B-0831 |
| additional information | residues Ser114, Tyr153, and Lys157 form the catalytic triad. Tyr153 is a catalytic base and Ser114 is a substitute | Pseudomonas sp. B-0831 |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.00015 | - |
cholic acid | recombinant His-tagged mutant S114A/Y153F, pH 8.0, 37°C | Pseudomonas sp. B-0831 | |
| 0.00044 | - |
Dehydrocholic acid | recombinant His-tagged mutant Y153F, pH 8.0, 37°C | Pseudomonas sp. B-0831 | |
| 0.0005 | - |
cholic acid | recombinant His-tagged mutant K157A, pH 8.0, 37°C | Pseudomonas sp. B-0831 | |
| 0.0013 | - |
cholic acid | recombinant His-tagged mutant Y153F, pH 8.0, 37°C | Pseudomonas sp. B-0831 | |
| 0.0014 | - |
cholic acid | recombinant His-tagged mutant S114A, pH 8.0, 37°C | Pseudomonas sp. B-0831 | |
| 0.0037 | - |
Dehydrocholic acid | recombinant His-tagged mutant S114A, pH 8.0, 37°C | Pseudomonas sp. B-0831 | |
| 0.411 | - |
cholic acid | recombinant His-tagged wild-type enzyme, pH 8.0, 37°C | Pseudomonas sp. B-0831 | |
| 0.94 | - |
Dehydrocholic acid | recombinant His-tagged wild-type enzyme, pH 8.0, 37°C | Pseudomonas sp. B-0831 | |
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