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Literature summary extracted from

  • Hwang, C.C.; Chang, Y.H.; Lee, H.J.; Wang, T.P.; Su, Y.M.; Chen, H.W.; Liang, P.H.
    The catalytic roles of P185 and T188 and substrate-binding loop flexibility in 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni (2013), PLoS ONE, 8, e63594.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.50 recombinant expression of wild-type and mutant enzymes Comamonas testosteroni

Protein Variants

EC Number Protein Variants Comment Organism
1.1.1.50 P185A site-directed mutagenesis, analysis of kinetics and structure compared to the wild-type enzyme Comamonas testosteroni
1.1.1.50 P185G site-directed mutagenesis, analysis of kinetics and structure compared to the wild-type enzyme Comamonas testosteroni
1.1.1.50 T188A site-directed mutagenesis, analysis of kinetics and structure compared to the wild-type enzyme Comamonas testosteroni
1.1.1.50 T188S site-directed mutagenesis, analysis of kinetics and structure compared to the wild-type enzyme Comamonas testosteroni
1.1.1.50 W173F/P185W site-directed mutagenesis, analysis of kinetics and structure compared to the wild-type enzyme Comamonas testosteroni
1.1.1.50 W173F/T188W site-directed mutagenesis, analysis of kinetics and structure compared 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, and stopped-flow study, kinetics of enzyme mutants P185A, P185G, T188A, and T188S showing an increase in kcat, Ka drosterone and KiNAD and equal primary isotope effects of DV and D(V/K) 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-androstan-3,17-dione + NADH + H+
-
 r

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 recombinant wild-type and mutant enzymes 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+ rate-limiting step in the reaction is the release of NADH and proton. P185, T188 and the flexible substrate-binding loop are involved in binding with the nucleotide cofactor and with androsterone and are also involved in catalysis, catalytic mechanism, overview Comamonas testosteroni
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.50 androsterone + NAD+
-
 Comamonas testosteroni 5alpha-androstan-3,17-dione + NADH + H+
-
 r
1.1.1.50 androsterone + NAD+ rate-limiting step in the reaction is the release of NADH Comamonas testosteroni 5alpha-androstan-3,17-dione + NADH + H+
-
 r

Subunits

EC Number Subunits Comment Organism
1.1.1.50 More secondary structures of the wild-type and mutants of P185A, P185G, T188A and T188S 3alpha-HSD/CRs are assessed by CD spectroscopy by measuring the ellipticity in the 190-250 nm range at room temperature 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

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.50 25
-
 assay at Comamonas testosteroni

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.50 10.5
-
 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

General Information

EC Number General Information Comment Organism
1.1.1.50 evolution enzyme 3alpha-HSD/CR belongs to the short chain dehydrogenase/reductase (SDR) superfamily Comamonas testosteroni
1.1.1.50 malfunction mutation at P185 in the hinge region and T188 in the loop causes a significant increase in the Kd value for NADH. Mutants P185A, P185G, T188A, and T188S show an increase the dissociation of the nucleotide cofactor, thereby increasing the rate of release of the product and producing the rate-limiting step in the hydride transfer Comamonas testosteroni
1.1.1.50 additional information catalytic tetrad N86-S114-Y155-K159, catalytic roles of P185 and T188 and substrate-binding loop flexibility in 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase. Structurally the substrate-binding loop of the residues, T188-K208, is unresolved, while binding with NAD+ causes the appearance of T188-P191 in the binary complex, functional roles of the flexible substrate-binding loop in conformational changes and enzyme catalysis, overview. Simulated molecular modeling gives results that are consistent with the conformational change in the substrate-binding loop after NAD+ binding. These results indicate that P185, T188 and the flexible substrate-binding loop are involved in binding with the nucleotide cofactor and with androsterone and are also involved in catalysis. Homology structure modeling, overview Comamonas testosteroni