Expression of Housefly CYP6A1 and NADPH-Cytochrome P450 Reductase in Escherichia coli and Reconstitution of an Insecticide-Metabolizing P450 System

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Abstract
The house fly (Musca domestica) cytochrome P450 gene CYP6Al was expressed in Escherichia coli. The native protein was produced at a level of 0.25-0.34 mu mol/L (15-20 mg/L) of culture with approximately 50% of the P450 being associated with the membrane fraction. The CYP6Al 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 CYP6Al 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 CYP6Al 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 CYP6Al 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.