Modulation of 17α-Hydroxylase/17,20-Lyase Activity of Guinea Pig Cytochrome P450c17 by Site-Directed Mutagenesis

Abstract

Microsomal cytochrome P450c17 (17α-hydroxylase/17,20-lyase) catalyzes two reactions in the Δ⁵ and Δ⁴ pathways leading to the production of C₁₉ steroids. Transient expression of human, bovine, porcine, rat, and mouse P450c17 cDNAs showed that the protein has 17α-hydroxylase and 17,20-lyase activities, converting pregnenolone and progesterone into Δ⁵- and Δ⁴-C₁₉ steroids, respectively, although the rat and mouse proteins have a preferential pathway toward the Δ⁴ steroids. The guinea pig (gp) P450c17 shares 46% to 70% amino acid identity with the corresponding proteins of other species, and further characterization indicated that the guinea pig enzyme only converts progesterone to androstenedione. In this study, we have tried to identify amino acid(s) in the gpP450c17 that governs such a steroid specificity. Among the various mutants that we have created, change of the arginine (R) residue at position 200 to an asparagine (N) (R200N) in the gpP450c17 protein increased reactivity toward pregnenolone compared with the wild-type enzyme. Pregnenolone was converted into 17α-hydroxypregnenolone and dehydroepiandrosterone. However, this gain occurred at the expense of the 17,20-lyase activity toward 17α-hydroxyprogesterone. The R200N mutation in the gpP450c17 protein introduced a potential N-linked glycosylation site (²⁰⁰Asn-X-Thr²⁰²); however, substitution of the Thr²⁰² residue by an asparagine (R200N/T202N), which abolishes the site, did not change the preference of the gpP450c17 mutant for pregnenolone. Furthermore, introduction of a putative glycosylation site at amino acid 185 in the gpP450c17 enzyme did not alter substrate specificity. The properties of the amino acid were also investigated, and neither the charge nor the size of the sidechain elicited change in the substrate specificity of gpP450c17. Thus, our results demonstrate that the mutation of arginine to asparagine at position 200 changes the substrate specificity of the gpP450c17 enzyme.