title: The catalytic roles of P185 and T188 and substrate-binding loop flexibility in 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni.
abstract: 3α-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.
authors.affiliation: Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan. cchwang@kmu.edu.tw
authors.forename: Chi-Ching
authors.initials: CC
authors.lastname: Hwang
authors.forename: Yi-Hsun
authors.initials: YH
authors.lastname: Chang
authors.forename: Hwei-Jen
authors.initials: HJ
authors.lastname: Lee
authors.forename: Tzu-Pin
authors.initials: TP
authors.lastname: Wang
authors.forename: Yu-Mei
authors.initials: YM
authors.lastname: Su
authors.forename: Hsin-Wei
authors.initials: HW
authors.lastname: Chen
authors.forename: Po-Huang
authors.initials: PH
authors.lastname: Liang
mesh_terms: D020816:Amino Acid Motifs; D000738:Androsterone / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D055162:Biocatalysis; D020134:Catalytic Domain; D002942:Circular Dichroism; D020604:Comamonas testosteroni / Q000201:enzymology*; D006913:Hydroxysteroid Dehydrogenases / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D009243:NAD / Q000737:chemistry; D010084:Oxidation-Reduction; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D013816:Thermodynamics; D013912:Threonine / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D009243:NAD; D013912:Threonine; D011392:Proline; D000738:Androsterone; C425306:3alpha-hydroxysteroid dehydrogenase-carbonyl reductase; D006913:Hydroxysteroid Dehydrogenases