Alteration of mouse cytochrome P450_coh substrate specificity by mutation of a single amino-acid residue

Abstract

AS a family of structurally-related enzymes, cytochrome P450 (P450) monooxygenases exhibit paradoxical characteristics: although collectively the enzymes display a broad range of substrate specificities, individually they are characterized by a high degree of substrate and product selectivity. Mouse P450_15α, and P450_coh, for example, which are expressed in female liver and male kidney cells^1,2, catalyse 15α-hydroxylation of ^Δ4 3-ketone steroids, such as testosterone and 7-hydroxylation of coumarin, respectively^3–6. In spite of their divergent catalytic activities, however, these enzymes differ by only 11 amino acids within their 494 residues⁵. To determine the structural basis of the different substrate specificities of P450_15α and P450_coh we therefore altered each of these 11 residues by site-directed mutagenesis, expressing the mutant cytochromes in COS-1 cells. We report that the activities of both cytochromes depend critically on the identities of the amino acids at positions 117, 209 and 365 and, moreover, that a single mutation in which Phe 209 is substituted by Leu is sufficient to convert the specificity of P450_coh from coumarin to steroid hydroxylation.