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.

Xenobiotic response in Drosophila melanogaster: Sex dependence of P450 and GST gene induction

“…Little is known of the physiological substrates of the induced enzymes but Cyp6a2 was previously shown to be ecdysone-inducible (Spiegelman et al, 1997; see also Dunkov et al, 1997;Dombrowski et al, 1998) and to metabolize JH III and related sesquiterpenoids (Andersen et al 1997), and Cyp18a1 was initially cloned as a result of its ecdysone inducibility (Hurban and Thummel, 1993). Lobster CYP45, a CYP6/9-like P450 is induced by phenobarbital and by 20-hydroxyecdysone (Snyder 1998).…”

CYP15A1, the cytochrome P450 that catalyzes epoxidation of methyl farnesoate to juvenile hormone III in cockroach corpora allata

“…Other enzymes are certainly capable of epoxidation of methyl farnesoate, e.g., house fly CYP6A1 (24), which is expressed in the gut and fat body (25), and D. punctata CYP9E1, which is highly expressed in the fat body (J. F. Andersen, G.C.U., J.F.K., and R.F., unpublished results). Male accessory glands and ovaries of mosquitoes apparently also express an epoxidase (26,27).…”

Structure and stereochemistry of products of hydroxylation of human steroid hormones by a housefly cytochrome P450 (CYP6A1)

“…Such potential application in turn requires detailed characterization of the substrate specificity and efficient, sensitive techniques for product identification and characterization. In this study we have used CYP6A1, an insect cytochrome P450 with very wide substrate specificity, 2,3 as a model enzyme to characterize oxidation of a different class of substrates, the human steroid hormones testosterone, progesterone and androstenedione. Identification of the products of these hydroxylation reactions by NMR spectroscopy is the purpose of the present paper.…”