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Literature summary extracted from

  • Chang, Y.H.; Chuang, L.Y.; Hwang, C.C.
    Mechanism of proton transfer in the 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni (2007), J. Biol. Chem., 282, 34306-34314.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.50 expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Comamonas testosteroni
1.1.1.213 expression of 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 mutation changes the rate-limiting step to the hydride transfer, proton transfer is blocked in the mutant but can be rescued using exogenous proton acceptors, such as buffers, small primary amines, and azide, overview Comamonas testosteroni
1.1.1.50 K159M site-directed mutagenesis, the mutation changes the rate-limiting step to the hydride transfer, proton transfer is blocked in the mutant but can be rescued using exogenous proton acceptors, such as buffers, small primary amines, and azide, overview Comamonas testosteroni
1.1.1.213 K159A site-directed mutagenesis, the mutation changes the rate-limiting step to the hydride transfer, proton transfer is blocked in the mutant but can be rescued using exogenous proton acceptors, such as buffers, small primary amines, and azide, overview Comamonas testosteroni
1.1.1.213 K159M site-directed mutagenesis, the mutation changes the rate-limiting step to the hydride transfer, proton transfer is blocked in the mutant but can be rescued using exogenous proton acceptors, such as buffers, small primary amines, and azide, overview 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 kinetics of wild-type and mutant enzymes in presence or absence of CAPS and methylamine, overview Comamonas testosteroni
1.1.1.50 0.0051
-
 androsterone pH 10.4, 25°C, recombinant wild-type enzyme Comamonas testosteroni
1.1.1.50 0.033
-
 androsterone pH 10.4, 25°C, recombinant mutant K159A Comamonas testosteroni
1.1.1.213 additional information
-
 additional information kinetics of wild-type and mutant enzymes in presence or absence of CAPS and methylamine, overview Comamonas testosteroni
1.1.1.213 0.0051
-
 androsterone pH 10.4, 25°C, recombinant wild-type enzyme Comamonas testosteroni
1.1.1.213 0.033
-
 androsterone pH 10.4, 25°C, recombinant mutant K159A 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
-
 androstanedione + 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 P80702
-
-
1.1.1.213 Comamonas testosteroni
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.1.50 recombinant wild-type and mutant enzymes ifrom Escherichia coli strain BL21(DE3) Comamonas testosteroni
1.1.1.213 recombinant wild-type and mutant enzymes ifrom Escherichia coli strain BL21(DE3) 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 overall oxidoreductive reaction comprises the deprotonation of tyrosine, proton abstraction by the tyrosinate anion, and hydride transfer from the hydroxysteroid to NAD+, followed by the release of a proton from the hydroxy group of tyrosine to the solution. The proton transfer to the external base, from Tyr155 to Lys159 via 2'-OH of ribose, with a late transition state is the rate-limiting step, mechanism, overview Comamonas testosteroni
a
1.1.1.213 a 3alpha-hydroxysteroid + NAD(P)+ = a 3-oxosteroid + NAD(P)H + H+ The overall oxidoreductive reaction comprises the deprotonation of tyrosine, proton abstraction by the tyrosinate anion, and hydride transfer from the hydroxysteroid to NAD+, followed by the release of a proton from the hydroxy group of tyrosine to the solution., The proton transfer to the external base, from Tyr155 to Lys159 via 2'-OH of ribose, with a late transition state is the rate-limiting step, mechanism, overview 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
1.1.1.213 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 androstanedione + NADH
-
 r
1.1.1.50 androsterone + NAD+ the intramolecular proton transfer is a rate-limiting step, with a concomitant releasing of protons to bulk solvent Comamonas testosteroni androstanedione + NADH
-
 r
1.1.1.213 androsterone + NAD+
-
 Comamonas testosteroni androstanedione + NADH
-
 r
1.1.1.213 androsterone + NAD+ the intramolecular proton transfer is a rate-limiting step, with a concomitant releasing of protons to bulk solvent Comamonas testosteroni androstanedione + NADH
-
 r

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 1.1.1.213 Comamonas testosteroni
1.1.1.213 3alpha-HSD/CR
-
 Comamonas testosteroni
1.1.1.213 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase
-
 Comamonas testosteroni
1.1.1.213 More cf. EC 1.1.1.50 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

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.50 10.4
-
 assay at Comamonas testosteroni
1.1.1.213 10.4
-
 assay at 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