Isoleucine-15 of rainbow trout carbonyl reductase-like 20β-hydroxysteroid dehydrogenase is critical for coenzyme (NADPH) binding

Carbonyl reductase-like 20 beta-hydroxysteroid dehydrogenase (CR/ 20 beta-HSD) is an enzyme that converts 17 alpha-hydroxyprogesterone to 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (the maturation-inducing hormone of salmonid fish). We have previously isolated two types of CR/20 beta-HSD cDNAs from ovarian follicle of rainbow trout (Oncorhynchus mykiss), Recombinant proteins produced by expression in Escherichia coli in vitro showed that one (type A) had CR and 20 beta-HSD activity but that the other (type B) did not. Among the three distinct residues between the protein products encoded by the two cDNAs, two residues (positions 15 and 27) are located in the N-terminal Rossmann fold, the coenzyme binding site. To investigate the structure/function relationships of CR/20 beta-HSDs, we generated mutants by site-directed mutagenesis at the following positions: MutA/l15T, MutB/T15I, and MutB/Q27K. Enzyme activity of wild-type A was abolished by substitution of Ile-15 by Thr (MutA/I15T). Conversely, enzyme activity was acquired by the replacement of Thr-15 with lie in type B (MutB/T15I), MutB/T15I mutant showed properties similar to the wild-type A in every aspect tested, Mutation MutB/Q27K had only partial enzyme activity, indicating that Ile-15 plays an important role in enzyme binding of cofactor NADPH.