Molecular characterization of specifically active recombinant fused enzymes consisting of CYP3A4, NADPH-cytochrome P450 oxidoreductase, and cytochrome b5.
Microsomal cytochrome P450 3A4 (CYP3A4) catalyzes monooxygenase reactions toward a diverse group of exogenous and endogenous substrates and requires cytochrome b5 (b5) in the oxidation of the typical substrate testosterone. To analyze the molecular interaction among CYP3A4, NADPH-cytochrome P450 oxidoreductase (P450 reductase), and b5, we constructed several fused enzyme genes and expressed them in Saccharomyces cerevisiae. The recombinant fused enzymes CYP3A4-truncated (t)-P450 reductase-t-b5 (3RB) and CYP3A4-t-b5-t-P450 reductase (3BR) in yeast microsomes showed a higher specific activity in 6beta-hydroxylation of testosterone than did the reconstitution premixes of CYP3A4, P450 reductase, and b5. The purified fused enzymes exhibited lower Km values and substantially increased Vmax values in 6beta-hydroxylation of testosterone and oxidation of nifedipine. Moreover, the fused enzymes showed significantly higher activities in cytochrome c reduction than the reconstitution premixes. Although the affinity of 3RB toward cytochrome c was twice as high as that of 3BR, 3BR and 3RB showed nearly the same affinity toward NADPH/NADH. In addition, the heme of the CYP3A4 moiety of 3RB was reduced preferentially and more rapidly than that of 3BR, whereas the heme of the b5 moiety of 3BR was selectively reduced compared with that of 3RB. These results suggest that the conformation of the 3RB molecule was the most suitable for high activity because of appropriate ordering of the CYP3A4, P450 reductase, and b5 moieties for efficient electron flow. Thus, we believe that the b5 moiety plays an important role in the efficient transfer of the second electron in the vicinity of the CYP3A4 moiety.
Inui
Hideyuki
H
Research Center for Environmental Genomics, Kobe University, Nada-ku, Kobe 657-8501, Japan.
Maeda
Atsuyo
A
Ohkawa
Hideo
H
D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D015786:Cytochromes b5 / Q000235:genetics* / Q000378:metabolism; D004579:Electron Transport; D006801:Humans; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D009543:Nifedipine / Q000378:metabolism; D010084:Oxidation-Reduction; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D013739:Testosterone; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; D009543:Nifedipine