SUBSTRATE-SPECIFICITY FOR THE EPOXIDATION OF TERPENOIDS AND ACTIVE-SITE TOPOLOGY OF HOUSE-FLY CYTOCHROME-P450 6A1

Heterologous expression in Escherichia coli, purification, and reconst itution of house fly P450 6A1 and NADPH-cytochrome P450 reductase were used to study the metabolism of terpenoids. In addition to the epoxid ation 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 n eryl methyl esters, juvenile hormones I and III, and farnesal but not farnesol or farnesoic acid. P450 6A1 reconstituted with NADPH-cytochro me P450 reductase and phosphatidylcholine did not metabolize alpha-pin ene, limonene, or the insect growth regulators hydroprene and methopre ne. The four geometric isomers of methyl farnesoate were metabolized p redominantly to the 10,11-epoxides, but also to the 6,7-epoxides and t o the diepoxides. The 10,11-epoxide of methyl (2E,6E)-farnesoate was p roduced in a 3:1 ratio of the (10S) and (10R) enantiomers. Monoepoxide s of methyl farnesoate were metabolized efficiently to the diepoxides. Methyl farnesoate epoxidation was strongly inhibited by a bulky subst ituted imidazole. The active site topology of P450 6A1 was studied by the reaction of the enzyme with phenyldiazene to form a phenyl-iron co mplex. Ferricyanide-induced in situ migration of the phenyl group show ed formation of the N-phenylprotoporphyrinporphyrin IX adducts in a 17 :25:33:24 ratio of the N-B:N-A:N-C:N-D isomers. These experiments sugg est that metabolism of xenobiotics by this P450, constitutively overex pressed in insecticide-resistant strains of the house fly, is not seve rely limited by stereochemically constrained access to the active site .