Cloned (Comment) | Organism |
---|---|
recombinant expression of wild-type and mutant enzymes | Comamonas testosteroni |
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 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 | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
androsterone + NAD+ | Comamonas testosteroni | - |
5alpha-androstan-3,17-dione + NADH + H+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Comamonas testosteroni | P80702 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and mutant enzymes | Comamonas testosteroni |
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 |
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 | 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 | Comment | Organism |
---|---|---|
3alpha-HSD/CR | - |
Comamonas testosteroni |
3alpha-hydroxysteroid dehydrogenase/carbonyl reductase | - |
Comamonas testosteroni |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Comamonas testosteroni |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
10.5 | - |
assay at | Comamonas testosteroni |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NAD+ | - |
Comamonas testosteroni | |
NADH | - |
Comamonas testosteroni |
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 |