title: Alteration of mouse cytochrome P450coh 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 P45015 alpha and P450coh, for example, which are expressed in female liver and male kidney cells, catalyse 15 alpha-hydroxylation of delta 4 3-ketone steroids, such as testosterone and 7-hydroxylation of coumarin, respectively. In spite of their divergent catalytic activities, however, these enzymes differ by only 11 amino acids within their 494 residues. To determine the structural basis of the different substrate specificities of P45015 alpha and P450coh 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 P450coh from coumarin to steroid hydroxylation.
authors.affiliation: Pharmacogenetics Section, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
authors.forename: R L
authors.initials: RL
authors.lastname: Lindberg
authors.forename: M
authors.initials: M
authors.lastname: Negishi
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics; D000818:Animals; D002460:Cell Line; D003374:Coumarins / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D013250:Steroid Hydroxylases / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D003374:Coumarins; D003577:Cytochrome P-450 Enzyme System; C030123:coumarin; D013250:Steroid Hydroxylases