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Literature summary extracted from

  • Hwang, C.C.; Chang, P.R.; Wang, T.P.
    Contribution of remote substrate binding energy to the enzymatic rate acceleration for 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (2017), Chem. Biol. Interact., 276, 133-140.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.357 recombinant His-tagged enzyme overexpression in Escherichia coli strain BL21(DE3) Comamonas testosteroni

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.357 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
1.1.1.357 0.0012
-
 androstenol reombinant His-tagged enzyme, pH 10.5, 25°C Comamonas testosteroni
1.1.1.357 0.0028
-
 androsterone reombinant His-tagged enzyme, pH 10.5, 25°C Comamonas testosteroni
1.1.1.357 0.14
-
 NAD+ reombinant His-tagged enzyme, pH 10.5, 25°C, with androstenol Comamonas testosteroni
1.1.1.357 0.39
-
 NAD+ reombinant His-tagged enzyme, pH 10.5, 25°C, with androsterone Comamonas testosteroni
1.1.1.357 0.6
-
 2-decalol reombinant His-tagged enzyme, pH 10.5, 25°C Comamonas testosteroni
1.1.1.357 27
-
 Cyclohexanol reombinant His-tagged enzyme, pH 10.5, 25°C Comamonas testosteroni

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.1.357 androsterone + NAD+ Comamonas testosteroni
-
 5alpha-androstan-3,17-dione + NADH + H+
-
 r

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.357 Comamonas testosteroni P80702
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.1.357 recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography to homogeneity Comamonas testosteroni

Reaction

EC Number Reaction Comment Organism Reaction ID
1.1.1.357 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
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.357 2-decalol + NAD+
-
 Comamonas testosteroni 2-decalone + NADH + H+
-
 r
1.1.1.357 androstenol + NAD+
-
 Comamonas testosteroni 5alpha-androst-16-en-3-one + NADH + H+
-
 r
1.1.1.357 androsterone + NAD+
-
 Comamonas testosteroni 5alpha-androstan-3,17-dione + NADH + H+
-
 r
1.1.1.357 cyclohexanol + NAD+
-
 Comamonas testosteroni cyclohexanone + NADH + H+
-
 r
1.1.1.357 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

EC Number Synonyms Comment Organism
1.1.1.357 3alpha-HSD/CR
-
 Comamonas testosteroni
1.1.1.357 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase
-
 Comamonas testosteroni

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.357 25
-
 assay at Comamonas testosteroni

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.1.357 2.2
-
 2-decalol reombinant His-tagged enzyme, pH 10.5, 25°C Comamonas testosteroni
1.1.1.357 2.5
-
 Cyclohexanol reombinant His-tagged enzyme, pH 10.5, 25°C Comamonas testosteroni
1.1.1.357 230
-
 androstenol reombinant His-tagged enzyme, pH 10.5, 25°C Comamonas testosteroni
1.1.1.357 500
-
 androsterone reombinant His-tagged enzyme, pH 10.5, 25°C Comamonas testosteroni

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.357 9 10.5 assay at Comamonas testosteroni

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.357 NAD+
-
 Comamonas testosteroni
1.1.1.357 NADH
-
 Comamonas testosteroni

Expression

EC Number Organism Comment Expression
1.1.1.357 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

EC Number General Information Comment Organism
1.1.1.357 evolution enzyme 3alpha-HSD/CR belongs to the short chain dehydrogenase/ reductase (SDR) superfamily Comamonas testosteroni

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.1.1.357 0.1
-
 Cyclohexanol reombinant His-tagged enzyme, pH 10.5, 25°C Comamonas testosteroni
1.1.1.357 3.7
-
 2-decalol reombinant His-tagged enzyme, pH 10.5, 25°C Comamonas testosteroni
1.1.1.357 178571
-
 androsterone reombinant His-tagged enzyme, pH 10.5, 25°C Comamonas testosteroni
1.1.1.357 191667
-
 androstenol reombinant His-tagged enzyme, pH 10.5, 25°C Comamonas testosteroni