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

Abstract

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.

Cloning and characterization of 60S ribosomal protein L22 (RPL22) from Culex pipiens pallens

“…In larvae the difference reached 12 folds, and c. 10 folds in adults. Andersen et al (1994) found that a periplasmically directed reductase supports monooxygenase reactions with CYP6A1 in a reconstituted system, and suggested that CYP6A1 is a major cyclodiene epoxidase and multiple P450 forms were responsible for the elevated monooxygenase activities in insecticide resistance (Andersen et al, 1994). In present study, the CYP6A1 gene in C6/36-RPL22 cells was also detected, and the results showed that mRNA level of CYP6A1 was lower than that of control.…”

“…Viability of C6/36-RPL22 cells in the presence of deltamethrin was compared with control cells to observe variance of deltamethrin resistance of these cells. Because CYP6A1 is an important insecticide resistant gene (Andersen et al, 1994), we also analyzed the mRNA levels of CYP6A1 between the C6/36-RPL22 cells and the control when RPL22 was overexpressed.…”

Comparative molecular modeling of Anopheles gambiae CYP6Z1, a mosquito P450 capable of metabolizing DDT

“…Although higher expression in insecticide-resistant strains does not necessarily guarantee relevance to insecticide resistance (12), CYP6Z1 has been repeatedly highlighted as overexpressed in adult males and females of the permethrin-resistant RSP strain collected in West Kenya (East Africa) and constitutively expressed at low levels in larvae and pupae of the RSP strain (13,14). And CYP6Z2 (but not CYP6Z1) has been documented as overexpressed in adult males and females of the permethrin-resistant Odumasy strain collected in Southern Ghana (West Africa) (15).Noting that DDT-metabolizing and cypermethrin-metabolizing P450s have already been identified in the CYP6 family of dipterans (Drosophila melanogaster and Musca domestica) and lepidopterans (Helicoverpa zea) (16)(17)(18)(19)(20)(21)(22), the overall purpose of this study was to determine whether any of CYP6 family transcripts expressed at higher levels in DDT-resistant strains have potential to code for a P450 capable of metabolizing DDT. Because we were specifically interested in comparing the metabolic profiles of the closely related CYP6Z1 and CYP6Z2 (69.6% amino acid identity), molecular modelings were carried out to predict their catalytic site geometries.…”

“…Noting that DDT-metabolizing and cypermethrin-metabolizing P450s have already been identified in the CYP6 family of dipterans (Drosophila melanogaster and Musca domestica) and lepidopterans (Helicoverpa zea) (16)(17)(18)(19)(20)(21)(22), the overall purpose of this study was to determine whether any of CYP6 family transcripts expressed at higher levels in DDT-resistant strains have potential to code for a P450 capable of metabolizing DDT. Because we were specifically interested in comparing the metabolic profiles of the closely related CYP6Z1 and CYP6Z2 (69.6% amino acid identity), molecular modelings were carried out to predict their catalytic site geometries.…”

Strategies for the construction of insect P450 fusion enzymes

“…The first purified insect P450 was the xenobiotic-degrading enzyme CYP6A1 also from the housefly, which was reconstituted in an Escherichia coli system allowing the identification of aldrin and heptachlor as substrates [26]. CYP6A1 has a spacious heme active site which is compatible with several substrate geometries and orientations [27].…”