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Literature summary for 1.1.1.357 extracted from
- Contribution of remote substrate binding energy to the enzymatic rate acceleration for 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (2017), Chem. Biol. Interact., 276, 133-140.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| recombinant His-tagged enzyme overexpression in Escherichia coli strain BL21(DE3) | Comamonas testosteroni |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | steady-state kinetics of 3alpha-HSD/CR catalyzed reaction of NADþ with androsterone and the truncated analogues, overview. The uniform binding energy from the B-ring of steroids with the active site of 3alpha-HSD/CR equally contributes 2.1 kcal/mol to stabilize both the transition state and ground state of the ternary complex, leading to the similarity in kcat for 2-decalol and cyclohexanol. Differential binding interactions of the remote BCD-ring and CD-ring of androsterone with the active site of 3alpha-HSD/CR contribute 8.5 and 6.4 kcal/mol to the stabilization of the transition state, respectively. The removal of the carbonyl group at C17 of androsterone has small effects on catalysis. Both uniform and differential binding energies from the remote sites of androsterone compared to cyclohexanol contribute to the 3a-HSD/CR catalysis, resulting in the increases in kcat and kcat/KB | Comamonas testosteroni | |
| 0.0012 | - |
androstenol | reombinant His-tagged enzyme, pH 10.5, 25°C | Comamonas testosteroni |  |
| 0.0028 | - |
androsterone | reombinant His-tagged enzyme, pH 10.5, 25°C | Comamonas testosteroni | |
| 0.14 | - |
NAD+ | reombinant His-tagged enzyme, pH 10.5, 25°C, with androstenol | Comamonas testosteroni | |
| 0.39 | - |
NAD+ | reombinant His-tagged enzyme, pH 10.5, 25°C, with androsterone | Comamonas testosteroni | |
| 0.6 | - |
2-decalol | reombinant His-tagged enzyme, pH 10.5, 25°C | Comamonas testosteroni | |
| 27 | - |
Cyclohexanol | reombinant His-tagged enzyme, pH 10.5, 25°C | Comamonas testosteroni | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| androsterone + NAD+ | Comamonas testosteroni | - |
5alpha-androstan-3,17-dione + NADH + H+ | - |
r | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Comamonas testosteroni | P80702 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography to homogeneity | Comamonas testosteroni |
Reaction
| Reaction | Comment | Organism | Reaction ID |
|---|---|---|---|
| a 3alpha-hydroxysteroid + NAD(P)+ = a 3-oxosteroid + NAD(P)H + H+ | the reaction catalyzed by the enzyme shows an ordered bi bi kinetic mechanism with NAD+ being the first substrate to be bound and NADH the last product to be released. The rate-limiting steps along the reaction pathway is the release of NADH for reaction with andosterone or androsterol, while the hydride transfer for 2-decalol is rate limiting for the overall reaction. Structure of the transition state by measuring the rate constant of a reaction upon substitution of a heavy atom for a light one at or adjacent to the bond cleavage during the reaction | Comamonas testosteroni |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 2-decalol + NAD+ | - |
Comamonas testosteroni | 2-decalone + NADH + H+ | - |
r | |
| androstenol + NAD+ | - |
Comamonas testosteroni | 5alpha-androst-16-en-3-one + NADH + H+ | - |
r | |
| androsterone + NAD+ | - |
Comamonas testosteroni | 5alpha-androstan-3,17-dione + NADH + H+ | - |
r | |
| cyclohexanol + NAD+ | - |
Comamonas testosteroni | cyclohexanone + NADH + H+ | - |
r | |
| additional information | 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase catalyzes the oxidation of androsterone with NAD+ to form androstanedione and NADH with the rate-limiting step being the release of NADH in the overall reaction. No enzyme activity is detected for methanol, ethanol, and 2-propanol, which lack the steroid scaffold of androsterone, implying that the steroid scaffold plays an important role in enzyme catalytic specificity. Role of remote substrate binding interactions contributing to the rate enhancement by 3apha-HSD/CR, overview. Uniform binding improves catalysis with simple cyclic alcohols, differential binding improves catalysis of steroid substrates. Reaction product identifcation by LC-MS/MS analysis | Comamonas testosteroni | ? | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| 3alpha-HSD/CR | - |
Comamonas testosteroni |
| 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase | - |
Comamonas testosteroni |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 25 | - |
assay at | Comamonas testosteroni |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 2.2 | - |
2-decalol | reombinant His-tagged enzyme, pH 10.5, 25°C | Comamonas testosteroni | |
| 2.5 | - |
Cyclohexanol | reombinant His-tagged enzyme, pH 10.5, 25°C | Comamonas testosteroni | |
| 230 | - |
androstenol | reombinant His-tagged enzyme, pH 10.5, 25°C | Comamonas testosteroni | |
| 500 | - |
androsterone | reombinant His-tagged enzyme, pH 10.5, 25°C | Comamonas testosteroni | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 9 | 10.5 | assay at | Comamonas testosteroni |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| NAD+ | - |
Comamonas testosteroni | |
| NADH | - |
Comamonas testosteroni | |
Expression
| Organism | Comment | Expression |
|---|---|---|
| Comamonas testosteroni | the enzyme is induced in the presence of testosterone and progesterone due to the steroids preventing repressor repA from binding the regulatory regions | up |
General Information
| General Information | Comment | Organism |
|---|---|---|
| evolution | enzyme 3alpha-HSD/CR belongs to the short chain dehydrogenase/ reductase (SDR) superfamily | Comamonas testosteroni |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.1 | - |
Cyclohexanol | reombinant His-tagged enzyme, pH 10.5, 25°C | Comamonas testosteroni | |
| 3.7 | - |
2-decalol | reombinant His-tagged enzyme, pH 10.5, 25°C | Comamonas testosteroni | |
| 178571 | - |
androsterone | reombinant His-tagged enzyme, pH 10.5, 25°C | Comamonas testosteroni | |
| 191667 | - |
androstenol | reombinant His-tagged enzyme, pH 10.5, 25°C | Comamonas testosteroni | |
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