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Literature summary extracted from

  • Chang, Y.H.; Huang, T.J.; Chuang, L.Y.; Hwang, C.C.
    Role of S114 in the NADH-induced conformational change and catalysis of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni (2009), Biochim. Biophys. Acta, 1794, 1459-1466.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.50 expressed in Escherichia coli BL21 (DE3) cells Comamonas testosteroni

Protein Variants

EC Number Protein Variants Comment Organism
1.1.1.50 S114A the mutation eliminates the hydrogen bonding interaction with P185, causing a conformational change in a nonproductive binding of NADH and a significant loss of activity, the mutant enzyme decreases 3100fold in V/Et value with no apparent change in Km value for substrates Comamonas testosteroni
1.1.1.50 S114A mutant enzyme exhibits a pronounced increase in the magnitude of ellipticity at 222 nm. S114A mutant enzyme decreases 3100fold in catalytic efficiency with no apparent change in Km for substrates. Addition of NADH to S114A mutant enzyme induces a secondary structural change Comamonas testosteroni

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.1.1.50 NADH
-
 Comamonas testosteroni

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.50 0.0012
-
 5alpha-androstane-3,17-dione wild-type, pH 7.5, 25°C Comamonas testosteroni
1.1.1.50 0.0012
-
 5alpha-androstan-3,17-dione wild type enzyme, in 0.1 M HEPES at pH 7.5 Comamonas testosteroni
1.1.1.50 0.0031
-
 5alpha-androstan-3,17-dione mutant enzyme S114A, in 0.1 M HEPES at pH 7.5 Comamonas testosteroni
1.1.1.50 0.0031
-
 5alpha-androstane-3,17-dione mutant S114A, pH 7.5, 25°C Comamonas testosteroni
1.1.1.50 0.004
-
 NADH wild-type, pH 7.5, 25°C Comamonas testosteroni
1.1.1.50 0.004
-
 NADH wild type enzyme, in 0.1 M HEPES at pH 7.5 Comamonas testosteroni
1.1.1.50 0.0065
-
 NADH mutant enzyme S114A, in 0.1 M HEPES at pH 7.5 Comamonas testosteroni
1.1.1.50 0.0065
-
 NADH mutant S114A, pH 7.5, 25°C Comamonas testosteroni

Organism

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

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.1.50 Ni-NTA column chromatography Comamonas testosteroni

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.50 5alpha-androstane-3,17-dione + NADH + H+
-
 Comamonas testosteroni androsterone + NAD+
-
 ?
1.1.1.50 androsterone + NAD+
-
 Comamonas testosteroni 5alpha-androstan-3,17-dione + NADH + H+
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.1.1.50 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase
-
 Comamonas testosteroni

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.1.50 0.02
-
 NADH mutant S114A, pH 7.5, 25°C Comamonas testosteroni
1.1.1.50 63
-
 NADH wild-type, pH 7.5, 25°C Comamonas testosteroni

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.50 NAD+
-
 Comamonas testosteroni
1.1.1.50 NADH NADH association to enzyme involves a bimolecular binding step and isomerization. The binding of NADH into a hydrophobic pocket in the active site restricts its motion and shields the fluorescence quenching from solvent Comamonas testosteroni

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
1.1.1.50 0.0034
-
 NADH wild-type, pH 7.5, 25°C Comamonas testosteroni
1.1.1.50 0.006
-
 NADH mutant S114A, pH 7.5, 25°C 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
1.1.1.50 3.8
-
 NADH mutant S114A, pH 7.5, 25°C Comamonas testosteroni
1.1.1.50 6.5
-
 5alpha-androstane-3,17-dione mutant S114A, pH 7.5, 25°C Comamonas testosteroni
1.1.1.50 15000
-
 NADH wild-type, pH 7.5, 25°C Comamonas testosteroni
1.1.1.50 54000
-
 5alpha-androstane-3,17-dione wild-type, pH 7.5, 25°C Comamonas testosteroni