Functional expression of mosquito NADPH-cytochrome P450 reductase in Escherichia coli.
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, 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.
Kaewpa
Dolnapa
D
Department of Biochemistry, Faculty of Science, Mahidol University, Rama 6 Road, Phyatai, Bangkok 10400, Thailand.
Boonsuepsakul
Soamrutai
S
Rongnoparut
Pornpimol
P
D000595:Amino Acid Sequence; D000818:Animals; D000852:Anopheles / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase