The Catalytic Roles of P185 and T188 and Substrate-Binding Loop Flexibility in 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase from <i>Comamonas testosteroni</i>

<div><p>3α-Hydroxysteroid dehydrogenase/carbonyl reductase from <i>Comamonas testosteroni</i> 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 <i>K_d</i> value for NADH by fluorescence titration. A kinetic study of the mutants of P185A, P185G, T188A and T188S shows an increase in <i>k_cat, K_androsterone</i> and <i>K_iNAD</i> and equal primary isotope effects of <i>^DV</i> and <i>^D</i>(<i>V/K</i>). 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.</p>