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Literature summary extracted from

  • Hwang, C.; Chang, Y.; Hsu, C.; Hsu, H.; Li, C.; Pon, H.
    Mechanistic roles of Ser-114, Tyr-155, and Lys-159 in 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni (2005), J. Biol. Chem., 280, 3522-3528.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.50 overexpression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Comamonas testosteroni
1.1.1.213 overexpression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Comamonas testosteroni

Protein Variants

EC Number Protein Variants Comment Organism
1.1.1.50 K159A site-directed mutagenesis, the mutant shows altered kinetics and pH profile, and 200fold reduced activity compared to the wild-type Comamonas testosteroni
1.1.1.50 S114A site-directed mutagenesis, the mutant shows altered kinetics and pH profile, and 3400fold reduced activity compared to the wild-type Comamonas testosteroni
1.1.1.50 S114A/Y155F site-directed mutagenesis, the mutant shows altered kinetics and pH profile, and 200000fold reduced activity compared to the wild-type Comamonas testosteroni
1.1.1.50 Y155F site-directed mutagenesis, the mutant shows altered kinetics and pH profile, and 2800fold reduced activity compared to the wild-type Comamonas testosteroni
1.1.1.50 Y155F/K159A site-directed mutagenesis, the mutant shows altered kinetics and pH profile, and 9400fold reduced activity compared to the wild-type Comamonas testosteroni
1.1.1.213 K159A site-directed mutagenesis, altered kinetics in comparison to the wild-type enzyme Comamonas testosteroni
1.1.1.213 S114A site-directed mutagenesis, altered kinetics in comparison to the wild-type enzyme Comamonas testosteroni
1.1.1.213 S114A/Y155F site-directed mutagenesis, altered kinetics in comparison to the wild-type enzyme Comamonas testosteroni
1.1.1.213 Y155F site-directed mutagenesis, altered kinetics in comparison to the wild-type enzyme Comamonas testosteroni
1.1.1.213 Y155F/K159A site-directed mutagenesis, altered kinetics in comparison to the wild-type enzyme Comamonas testosteroni

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.50 additional information
-
 additional information steady-state kinetics Comamonas testosteroni
1.1.1.50 0.00044
-
 androsterone pH 9.0, 25°C, recombinant His-tagged mutant S114A/Y155F Comamonas testosteroni
1.1.1.50 0.0055
-
 androsterone pH 9.0, 25°C, recombinant His-tagged wild-type Comamonas testosteroni
1.1.1.50 0.007
-
 androsterone pH 9.0, 25°C, recombinant His-tagged mutant S114A Comamonas testosteroni
1.1.1.50 0.065
-
 androsterone pH 9.0, 25°C, recombinant His-tagged mutant K159A Comamonas testosteroni
1.1.1.50 0.074
-
 androsterone pH 9.0, 25°C, recombinant His-tagged mutant Y155F Comamonas testosteroni
1.1.1.50 0.1
-
 androsterone pH 9.0, 25°C, recombinant His-tagged mutant Y155F/K159A Comamonas testosteroni
1.1.1.213 additional information
-
 additional information steady-state kinetics Comamonas testosteroni
1.1.1.213 0.00044
-
 androsterone pH 9.0, 25°C, recombinant mutant S114A/Y155F Comamonas testosteroni
1.1.1.213 0.0055
-
 androsterone pH 9.0, 25°C, recombinant wild-type enzyme Comamonas testosteroni
1.1.1.213 0.007
-
 androsterone pH 9.0, 25°C, recombinant mutant S114A Comamonas testosteroni
1.1.1.213 0.065
-
 androsterone pH 9.0, 25°C, recombinant mutant K159A Comamonas testosteroni
1.1.1.213 0.074
-
 androsterone pH 9.0, 25°C, recombinant mutant Y155F Comamonas testosteroni
1.1.1.213 0.1
-
 androsterone pH 9.0, 25°C, recombinant mutant Y155F/K159A Comamonas testosteroni

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.1.1.50 70000
-
 about, recombinant His-tagged wild-type and mutant enzymes, gel filtration Comamonas testosteroni
1.1.1.213 32000
-
 x * 32000, about, recombinant His-tagged wild-type and mutant enzymes, SDS-PAGE Comamonas testosteroni

Natural Substrates/ Products (Substrates)

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

Organism

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

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.1.50 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, and gel filtration to homogeneity Comamonas testosteroni
1.1.1.213 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain Bl21(DE3) by nickel affinity chromatography, to homogeneity Comamonas testosteroni

Reaction

EC Number Reaction Comment Organism Reaction ID
1.1.1.50 a 3alpha-hydroxysteroid + NAD(P)+ = a 3-oxosteroid + NAD(P)H + H+ the catalytic triad is composed of Ser-114, Tyr-155, and Lys-159, structural analysis and sequence alignment of the short chain dehydrogenase/reductase family Comamonas testosteroni
a
1.1.1.213 a 3alpha-hydroxysteroid + NAD(P)+ = a 3-oxosteroid + NAD(P)H + H+ catalytic triad residues Ser114, Tyr155, and Lys159 have mechanistic roles in the catalytic reaction Comamonas testosteroni
a

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.1.1.50 additional information
-
-
 Comamonas testosteroni

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.50 androsterone + NAD+
-
 Comamonas testosteroni 5alpha-androstane-3,17-dione + NADH
-
 r
1.1.1.50 additional information bifunctional enzyme also catalyzing the reaction of carbonyl reductase, EC 1.1.1.184 Comamonas testosteroni ?
-
 ?
1.1.1.213 androsterone + NAD+
-
 Comamonas testosteroni androstanedione + NADH
-
 r
1.1.1.213 androsterone + NAD+
-
 Comamonas testosteroni 5alpha-androstane-3,17-dione + NADH
-
 r
1.1.1.213 additional information bifunctional enzyme exhibiting 3alpha-hydroxysteroid dehydrogenase activity and carbonyl reductase activity Comamonas testosteroni ?
-
 ?

Subunits

EC Number Subunits Comment Organism
1.1.1.50 dimer 2 * 28349-28425, recombinant His-tagged wild-type and mutant enzymes, mass spectrometry Comamonas testosteroni
1.1.1.213 ? x * 32000, about, recombinant His-tagged wild-type and mutant enzymes, SDS-PAGE Comamonas testosteroni

Synonyms

EC Number Synonyms Comment Organism
1.1.1.50 3alpha-HSD/CR
-
 Comamonas testosteroni
1.1.1.50 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase
-
 Comamonas testosteroni
1.1.1.50 More cf. EC 1.1.1.184 Comamonas testosteroni
1.1.1.213 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase
-
 Comamonas testosteroni
1.1.1.213 alpha-HSD/CR
-
 Comamonas testosteroni
1.1.1.213 More the enzyme belongs to the short-chain dehydrogenase/reductase family Comamonas testosteroni

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.50 25
-
 assay at Comamonas testosteroni
1.1.1.213 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
1.1.1.50 0.0028
-
 androsterone pH 9.0, 25°C, recombinant His-tagged mutant S114A/Y155F Comamonas testosteroni
1.1.1.50 0.003
-
 androsterone pH 9.0, 25°C, recombinant His-tagged mutant Y155F/K159A Comamonas testosteroni
1.1.1.50 0.12
-
 androsterone pH 9.0, 25°C, recombinant His-tagged mutant S114A Comamonas testosteroni
1.1.1.50 1.5
-
 androsterone pH 9.0, 25°C, recombinant His-tagged mutant Y155F Comamonas testosteroni
1.1.1.50 18.9
-
 androsterone pH 9.0, 25°C, recombinant His-tagged mutant K159A Comamonas testosteroni
1.1.1.50 318
-
 androsterone pH 9.0, 25°C, recombinant His-tagged wild-type Comamonas testosteroni
1.1.1.213 0.0028
-
 androsterone pH 9.0, 25°C, recombinant mutant S114A/Y155F Comamonas testosteroni
1.1.1.213 0.003
-
 androsterone pH 9.0, 25°C, recombinant mutant Y155F/K159A Comamonas testosteroni
1.1.1.213 0.12
-
 androsterone pH 9.0, 25°C, recombinant mutant S114A Comamonas testosteroni
1.1.1.213 1.5
-
 androsterone pH 9.0, 25°C, recombinant mutant Y155F Comamonas testosteroni
1.1.1.213 18.9
-
 androsterone pH 9.0, 25°C, recombinant mutant K159A Comamonas testosteroni
1.1.1.213 318
-
 androsterone pH 9.0, 25°C, recombinant wild-type enzyme Comamonas testosteroni

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.50 9
-
 assay at Comamonas testosteroni
1.1.1.213 9
-
 assay at Comamonas testosteroni

pH Range

EC Number pH Minimum pH Maximum Comment Organism
1.1.1.50 5 11 pH profile, recombinant His-tagged wild-type and mutant enzymes Comamonas testosteroni
1.1.1.213 5.5 11 pH profile Comamonas testosteroni

Cofactor

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