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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  K d   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.

plos one

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