Andersen J F, Walding J K, Evans P H, Bowers W S, Feyereisen R
Department of Entomology, University of Arizona, Tucson 85721, USA.
Chem Res Toxicol. 1997 Feb;10(2):156-64. doi: 10.1021/tx9601162.
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.
利用家蝇P450 6A1和NADPH-细胞色素P450还原酶在大肠杆菌中的异源表达、纯化及重组来研究萜类化合物的代谢。除了之前已证明的环二烯类杀虫剂的环氧化反应[安德森等人（1994年），《生物化学》33卷，2171 - 2177页]，这种细胞色素P450还能使多种萜类化合物发生环氧化，如法呢基、香叶基和橙花基甲酯、保幼激素I和III以及法呢醛，但不能代谢法呢醇或法呢酸。用NADPH-细胞色素P450还原酶和磷脂酰胆碱重组的P450 6A1不能代谢α-蒎烯、柠檬烯以及昆虫生长调节剂烯虫酯和烯虫炔酯。法呢酸甲酯的四种几何异构体主要代谢为10,11-环氧化物，但也有6,7-环氧化物和二环氧物。(2E,6E)-法呢酸甲酯的10,11-环氧化物以(10S)和(10R)对映体3:1的比例生成。法呢酸甲酯的单环氧化物能有效地代谢为二环氧物。法呢酸甲酯的环氧化反应受到一种体积较大的取代咪唑的强烈抑制。通过酶与苯二氮烯反应形成苯基-铁配合物来研究P450 6A1的活性位点拓扑结构。铁氰化物诱导苯基的原位迁移显示形成了N-苯基原卟啉-卟啉IX加合物，其NB:NA:NC:ND异构体的比例为17:25:33:24。这些实验表明，在家蝇抗杀虫剂品系中组成性过表达的这种P450对外源化合物的代谢，不会受到活性位点立体化学限制进入的严重限制。
