Characterization of NADPH-cytochrome P450 reductase gene from the cotton bollworm, Helicoverpa armigera.
A complete cDNA encoding the NADPH-cytochrome P450 reductase (haCPR) and its genomic sequence from the cotton bollworm Helicoverpa armigera were cloned and sequenced. The open reading frame of haCPR codes for a protein of 687 amino acid residues with a calculated molecular mass of 77.4kDa. The haCPR gene spans over 11 kb and its coding region is interrupted by 11 introns. haCPR is ubiquitously expressed in various tissues and at various stages of development. Escherichia coli produced haCPR enzyme exhibited catalytic activity for NADPH-dependent reduction of cytochrome c, following Michaelis-Menten kinetics. The functionality of CPR was further demonstrated by its capacity to support cytochrome P450 (e.g. haCYP9A14 and chicken CYP1A5) mediated O-dealkylation activity of alkoxyresorufins. The flavoprotein-specific inhibitor (diphenyleneiodonium chloride, DPI) showed a potent inhibition to haCPR activity (IC50=1.69 μM). Inhibitory effect of secondary metabolites in the host plants (tannic acid, quercetin and gossypol) on CPR activity (with an IC50 value ranged from 15 to 90 μM) was also observed.
State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
Dong
D
Liu
State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; Beijing Center for Disease Control and Prevention, Institute of Disinfection and Vector Control, Beijing 100013, China.
Xiaojie
X
Zhou
State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
Mei
M
Li
State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
Shunyi
S
Zhu
State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address: qiuxh@ioz.ac.cn.
Xinghui
X
Qiu
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D015870:Gene Expression; D023061:Gene Order; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D009036:Moths / Q000201:enzymology / Q000235:genetics*; D009251:NADPH-Ferrihemoprotein Reductase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D009251:NADPH-Ferrihemoprotein Reductase
Enzymatic kinetics; Gene structure; Helicoverpa armigera; Inhibition; NADPH–cytochrome P-450 reductase; Spatial and temporal expression