Characterization of a fusion protein between human cytochrome P450 1A1 and rat NADPH-P450 oxidoreductase in Escherichia coli.
A cDNA of fusion protein between human cytochrome P450 1A1 and rat NADPH-P450 reductase was genetically engineered and expressed in Escherichia coli DH5alpha cells under the control of an inducible tac promoter (Y. J. Chun, T. Shimada, and F. P. Guengerich, (1996) Arch. Biochem. Biophys. 330, 48-58). E. coli membranes of transformed cells showed much higher P450 1Al-dependent monooxygenase and NADPH-P450 reductase activities than pCW control vector or P450 1A1 expression vector-transformed cells. Ethoxyresorufin O-deethylase and methoxyresorufin O-demethylase were 22-fold and 11-fold higher than the control activity, respectively. alpha-Naphthoflavone and beta-naphthoflavone strongly inhibited P450 1A1 activity of the fusion protein, with alpha-naphthoflavone being more potent than beta-naphthoflavone. Divalent cations (e.g. Ca2+ and Mg2+) increased P450 1A1 activity as well as NADPH-P450 reductase activity. These results demonstrate that this fusion protein in E. coli membrane may be a useful model for elucidating details of protein-protein interactions between P450 and NADPH-P450 reductase in the endoplasmic reticulum of mammalian cells.
Chun
Y J
YJ
Toxicology Research Center, Korea Research Institute of Chemical Technology (KRICT), Taejon.
Jeong
T C
TC
Roh
J K
JK
Guengerich
F P
FP
D000818:Animals; D001571:Benzoflavones / Q000378:metabolism; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism*; D018076:DNA, Complementary; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010078:Oxazines / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D019324:beta-Naphthoflavone / Q000378:metabolism
D001571:Benzoflavones; D018076:DNA, Complementary; D010078:Oxazines; D011993:Recombinant Fusion Proteins; C076580:7-methoxyresorufin; C007649:ethoxyresorufin; C011512:alpha-naphthoflavone; D019324:beta-Naphthoflavone; D019363:Cytochrome P-450 CYP1A1; D009251:NADPH-Ferrihemoprotein Reductase