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Functional Expression of Mosquito NADPH-Cytochrome P450 Reductase in Escherichia coli

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Abstract:

A complete NADPH-cytochrome P450 reductase (CPR) cDNA was isolated from Anopheles minimus Theobald mosquitoes by using reverse transcription-polymerase chain reaction-based methods. The complete cDNA contains 2,040 bp encoding the protein of 679 amino acids, with a calculated molecular mass of 77.3 kDa. The deduced amino acid sequence had a typical feature of CPR by possessing conserved domains involved in binding of flavin mononucleotide, flavin adenine dinucleotide, and NADPH cofactors. The complete CPR cDNA was expressed as 6x His-tagged fusion protein in both membrane and cytosolic fractions in Escherichia coli, both fractions contained NADPH-cytochrome c reducing activity. The membrane-bound form containing N-terminal membrane anchor was subjected to purification, and K m values were determined for NADPH and cytochrome c. The purified CPR enzyme was functionally active, as demonstrated by its ability to support CYP6AA3-mediated metabolism in the reconstituted reaction in vitro. Initial test suggested that CYP6AA3 could play a role in deltamethrin metabolism.
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  • Journal of Economic Entomology is published bimonthly in February, April, June, August, October, and December. The journal publishes articles on the economic significance of insects and is divided into the following sections: apiculture & social insects; arthropods in relation to plant disease; forum; insecticide resistance and resistance management; ecotoxicology; biological and microbial control; ecology and behavior; sampling and biostatistics; household and structural insects; medical entomology; molecular entomology; veterinary entomology; forest entomology; horticultural entomology; field and forage crops, and small grains; stored-product; commodity treatment and quarantine entomology; and plant resistance. In addition to research papers, Journal of Economic Entomology publishes Letters to the Editor, interpretive articles in a Forum section, Short Communications, Rapid Communications, and Book Reviews.
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