Literature summary for 1.1.1.50 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.

Search for more literature for 1.1.1.50

Cloned(Commentary)

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

Protein Variants

Protein Variants	Comment	Organism
P185A	site-directed mutagenesis, analysis of kinetics and structure compared to the wild-type enzyme	Comamonas testosteroni
P185G	site-directed mutagenesis, analysis of kinetics and structure compared to the wild-type enzyme	Comamonas testosteroni
T188A	site-directed mutagenesis, analysis of kinetics and structure compared to the wild-type enzyme	Comamonas testosteroni
T188S	site-directed mutagenesis, analysis of kinetics and structure compared to the wild-type enzyme	Comamonas testosteroni
W173F/P185W	site-directed mutagenesis, analysis of kinetics and structure compared to the wild-type enzyme	Comamonas testosteroni
W173F/T188W	site-directed mutagenesis, analysis of kinetics and structure compared to the wild-type enzyme	Comamonas testosteroni

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
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)

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

Organism

Organism	UniProt	Comment	Textmining
Comamonas testosteroni	P80702	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant wild-type and mutant enzymes	Comamonas testosteroni

Reaction

Reaction	Comment	Organism	Reaction ID
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)

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

Subunits

Subunits	Comment	Organism
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

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

Temperature Optimum [°C]

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

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
10.5	-	assay at	Comamonas testosteroni

Cofactor

Cofactor	Comment	Organism	Structure
NAD+	-	Comamonas testosteroni
NADH	-	Comamonas testosteroni

General Information

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

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Release 2023.1 (January 2023)