Roles of NADPH-P450 reductase and apo- and holo-cytochrome b5 on xenobiotic oxidations catalyzed by 12 recombinant human cytochrome P450s expressed in membranes of Escherichia coli.
Drug oxidation activities of 12 recombinant human cytochrome P450s (P450) coexpressed with human NADPH-P450 reductase (NPR) in bacterial membranes (P450/NPR membranes) were determined and compared with those of other recombinant systems and those of human liver microsomes. Addition of exogenous membrane-bound NPR to the P450/NPR membranes enhanced the catalytic activities of CYP2C8, CYP2C9, CYP2C19, CYP3A4, and CYP3A5. Enhancement of activities of CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2D6, and CYP2E1 in membranes was not observed after the addition of NPR (4 molar excess to each P450). Exogenous purified human cytochrome b5 (b5) further enhanced catalytic activities of CYP2A6, CYP2B6, CYP2C8, CYP2E1, CYP3A4, and CYP3A5/NPR membranes. Catalytic activities of CYP2C9 and CYP2C19 were enhanced by addition of b5 in reconstituted systems but not in the P450/NPR membranes. Apo b5 (devoid of heme) enhanced catalytic activities when added to both membrane and reconstituted systems, except for CYP2E1/NPR membranes and the reconstituted system containing purified CYP2E1 and NPR. Catalytic activities in P450/NPR membranes fortified with b5 were roughly similar to those measured with microsomes of insect cells coexpressing P450 with NPR (and b5) and/or human liver microsomes, based on equivalent P450 contents. These results suggest that interactions of P450 and NPR coexpressed in membranes or mixed in reconstituted systems appear to be different in some human CYP2 family enzymes, possibly due to a conformational role of b5. P450/NPR membrane systems containing b5 are useful models for prediction of the rates for liver microsomal P450-dependent drug oxidations.
Yamazaki
Hiroshi
H
Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-0934, Japan. yamazaki@pharm.hokudai.ac.jp
Nakamura
Mami
M
Komatsu
Tomoko
T
Ohyama
Katsuhiro
K
Hatanaka
Naoya
N
Asahi
Satoru
S
Shimada
Noriaki
N
Guengerich
F Peter
FP
Shimada
Tsutomu
T
Nakajima
Miki
M
Yokoi
Tsuyoshi
T
D002462:Cell Membrane / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D015786:Cytochromes b5 / Q000378:metabolism*; D004926:Escherichia coli; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000096:biosynthesis; D015262:Xenobiotics / Q000494:pharmacology*
D011994:Recombinant Proteins; D015262:Xenobiotics; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase