title: Substrate specificity for the epoxidation of terpenoids and active site topology of house fly cytochrome P450 6A1.
abstract: Heterologous expression in Escherichia coli, purification, and reconstitution of house fly P450 6A1 and NADPH-cytochrome P450 reductase were used to study the metabolism of terpenoids. In addition to the epoxidation of cyclodiene insecticides demonstrated previously [Andersen et al. (1994) Biochemistry 33, 2171-2177], this cytochrome P450 was shown to epoxidize a variety of terpenoids such as farnesyl, geranyl, and neryl methyl esters, juvenile hormones I and III, and farnesal but not farnesol or farnesoic acid. P450 6A1 reconstituted with NADPH-cytochrome P450 reductase and phosphatidylcholine did not metabolize alpha-pinene, limonene, of the insect growth regulators hydroprene and methoprene. The four geometric isomers of methyl farnesoate were metabolized predominantly to the 10,11-epoxides, but also the 6,7-epoxides and to the diepoxides. The 10,11-epoxide of methyl (2E,6E)-farnesoate was produced in a 3:1 ratio of the (10S) and (10R) enantiomers. Monoepoxides of methyl farnesoate were metabolized efficiently to the diepoxides. Methyl farnesoate epoxidation was strongly inhibited by a bulky substituted imidazole. The active site topology of P450 6A1 was studied by the reaction of the enzyme with phenyldiazene to form a phenyl-iron complex. Ferricyanide-induced in situ migration of the phenyl group showed formation of the N-phenylprotopor-phyrinporphyrin IX adducts in a 17:25:33:24 ratio of the NB:NA:NC:ND isomers. These experiments suggest that metabolism of xenobiotics by this P450, constitutively overexpressed in insecticide-resistant strains of the house fly, is not severely limited by stereochemically constrained access to the active site.
authors.affiliation: Department of Entomology, University of Arizona, Tucson 85721, USA.
authors.forename: J F
authors.initials: JF
authors.lastname: Andersen
authors.forename: J K
authors.initials: JK
authors.lastname: Walding
authors.forename: P H
authors.initials: PH
authors.lastname: Evans
authors.forename: W S
authors.initials: WS
authors.lastname: Bowers
authors.forename: R
authors.initials: R
authors.lastname: Feyereisen
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002384:Catalysis; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D004852:Epoxy Compounds / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D006793:Houseflies / Q000201:enzymology*; D007536:Isomerism; D010084:Oxidation-Reduction; D013379:Substrate Specificity / Q000187:drug effects; D013729:Terpenes / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D004852:Epoxy Compounds; D005231:Fatty Acids, Unsaturated; D013729:Terpenes; C032429:methyl farnesoate; D003577:Cytochrome P-450 Enzyme System