Andersen J F, Utermohlen J G, Feyereisen R
Department of Entomology, University of Arizona, Tucson 85721.
Biochemistry. 1994 Mar 1;33(8):2171-7. doi: 10.1021/bi00174a025.
The house fly (Musca domestica) cytochrome P450 gene CYP6A1 was expressed in Escherichia coli. The native protein was produced at a level of 0.25-0.34 mumol/L (15-20 mg/L) of culture with approximately 50% of the P450 being associated with the membrane fraction. The CYP6A1 protein was characterized spectrally and purified by a combination of hydrophobic interaction and hydroxyapatite chromatography. The house fly NADPH-cytochrome P450 reductase gene was also expressed in E. coli. Expression of a cytoplasmically directed reductase resulted in a protein that reduced cytochrome c but did not support P450 monooxygenase reactions. However, a periplasmically directed reductase was found to support monooxygenase reactions with CYP6A1 in a reconstituted system. The reconstituted system was effective in the epoxidation of the cyclodiene insecticides aldrin and heptachlor, with turnover rates of 12 and 34 min-1, respectively. The enzyme showed little detectable activity in the O-dealkylation and N-dealkylation of various compounds that are metabolized by house fly microsomes. Incubation with polyclonal antisera raised against purified CYP6A1 inhibited the microsomal epoxidation of heptachlor by 65%. Under the same conditions, the metabolism of 7-methoxy-4-methylcoumarin was inhibited only slightly. The results suggest that CYP6A1 is a major cyclodiene epoxidase in the house fly and that multiple P450 forms are responsible for the elevated monooxygenase activities in insecticide-resistant flies.
家蝇（Musca domestica）细胞色素P450基因CYP6A1在大肠杆菌中表达。天然蛋白的产量为每升培养物0.25 - 0.34 μmol/L（15 - 20 mg/L），约50%的P450与膜部分相关。通过光谱对CYP6A1蛋白进行了表征，并通过疏水相互作用和羟基磷灰石色谱相结合的方法进行了纯化。家蝇NADPH - 细胞色素P450还原酶基因也在大肠杆菌中表达。细胞质导向还原酶的表达产生了一种能还原细胞色素c但不支持P450单加氧酶反应的蛋白。然而，发现周质导向还原酶在重组系统中支持与CYP6A1的单加氧酶反应。该重组系统对环二烯类杀虫剂艾氏剂和七氯的环氧化有效，周转率分别为12和34 min⁻¹。该酶在被家蝇微粒体代谢的各种化合物的O - 脱烷基化和N - 脱烷基化反应中几乎没有可检测到的活性。用针对纯化的CYP6A1产生的多克隆抗血清孵育可使七氯的微粒体环氧化受到65%的抑制。在相同条件下，7 - 甲氧基 - 4 - 甲基香豆素的代谢仅受到轻微抑制。结果表明，CYP6A1是家蝇中的一种主要环二烯环氧化酶，多种P450形式导致了抗杀虫剂家蝇中单加氧酶活性的升高。
