Expression of house fly CYP6A1 and NADPH-cytochrome P450 reductase in Escherichia coli and reconstitution of an insecticide-metabolizing P450 system.
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.
Andersen
J F
JF
Department of Entomology, University of Arizona, Tucson 85721.
Utermohlen
J G
JG
Feyereisen
R
R
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary; D004926:Escherichia coli; D006793:Houseflies / Q000201:enzymology*; D007306:Insecticides / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000378:metabolism; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics* / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013057:Spectrum Analysis
D018076:DNA, Complementary; D007306:Insecticides; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; C019647:aldrin epoxidase; C023306:heptachlor epoxidase; D009251:NADPH-Ferrihemoprotein Reductase