Dataset Information


The catalytic roles of P185 and T188 and substrate-binding loop flexibility in 3?-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni.

ABSTRACT: 3?-Hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni reversibly catalyzes the oxidation of androsterone with NAD(+) to form androstanedione and NADH. 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. This study determines the functional roles of the flexible substrate-binding loop in conformational changes and enzyme catalysis. A stopped-flow study reveals that the rate-limiting step in the reaction is the release of the NADH. The mutation at P185 in the hinge region and T188 in the loop causes a significant increase in the Kd value for NADH by fluorescence titration. A kinetic study of the mutants of P185A, P185G, T188A and T188S shows an increase in k(cat), K(androsterone) and K(iNAD) and equal primary isotope effects of (D)V and (D) (V/K). Therefore, these mutants 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. 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.

PROVIDER: S-EPMC3662788 | BioStudies |

REPOSITORIES: biostudies

Similar Datasets

1991-01-01 | S-EPMC1151422 | BioStudies
| S-EPMC5264612 | BioStudies
| S-EPMC6295900 | BioStudies
| S-EPMC6319916 | BioStudies
| S-EPMC7084900 | BioStudies
| S-EPMC5235360 | BioStudies
| S-EPMC2858024 | BioStudies
| S-EPMC3995211 | BioStudies
| S-EPMC5816668 | BioStudies
1969-01-01 | S-EPMC1184647 | BioStudies