title: Engineering mouse P450coh to a novel corticosterone 15 alpha-hydroxylase and modeling steroid-binding orientation in the substrate pocket.
abstract: The F209L mutation alters specificity of P450coh from coumarin 7-hydroxylation to 15 alpha-hydroxylation of 11-deoxysteroids such as testosterone and 11-deoxycorticosterone. Neither the wild-type nor F209L exhibits activity toward 11 beta-hydroxysteroids including corticosterone. Mutation of Phe-209 to Asn, however, confers on mutant F209N a high corticosterone 15 alpha-hydroxylase activity. F209V also exhibits low corticosterone 15 alpha-hydroxylase activity; Km and Vmax are 10-fold higher and lower, respectively, than for F209N. The results are consistent with the hypothesis that direct interaction of Asn-209 with 11OH is responsible for high corticosterone 15 alpha-hydroxylase activity. To support this hypothesis, a possible steroid-binding orientation is modeled in the substrate pocket of P450cam. Our weighted homology and constrained alignments map residue 209 of P450coh to Met-184 and Met-191 of P450cam. Energy minimization of corticosterone in the substrate pocket results in the 11OH of the steroid directed toward Met-184 (7 A) and Met-191 (16 A), and in C15 located near the sixth axial position of the heme. The steroid-binding model suggests that the P450cam's substrate pocket may be conserved in the mammalian P450 and can accommodate a steroid molecule, and that residue 209 appears to be located at the critical site that determines the steroid-substrate specificity of a P450 depending on the type of group at the 11-position of steroid molecule.
authors.affiliation: Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
authors.forename: M
authors.initials: M
authors.lastname: Iwasaki
authors.forename: T A
authors.initials: TA
authors.lastname: Darden
authors.forename: L G
authors.initials: LG
authors.lastname: Pedersen
authors.forename: D G
authors.initials: DG
authors.lastname: Davis
authors.forename: R O
authors.initials: RO
authors.lastname: Juvonen
authors.forename: T
authors.initials: T
authors.lastname: Sueyoshi
authors.forename: M
authors.initials: M
authors.lastname: Negishi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D003900:Desoxycorticosterone / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D011487:Protein Conformation; D015202:Protein Engineering; D017386:Sequence Homology, Amino Acid; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D003900:Desoxycorticosterone; D010649:Phenylalanine; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C460223:Cyp2a4 protein, mouse; D000072467:Cytochrome P450 Family 2; C050356:steroid 15-alpha-hydroxylase