title: Coexpression of genetically engineered fused enzyme between yeast NADPH-P450 reductase and human cytochrome P450 3A4 and human cytochrome b5 in yeast.
abstract: Human hepatic cytochrome P450 3A4 (CYP3A4) was expressed in yeast Saccharomyces cerevisiae. While the expression level was high as compared with other human hepatic cytochrome P450s, CYP3A4 showed almost no catalytic activity toward testosterone. Coexpression of CYP3A4 with yeast NADPH-P450 reductase did not give a full activity. Low monooxygenase activity of CYP3A4 was attributed to the insufficient reduction of heme iron of CYP3A4 by NADPH-P450 reductase. To enhance the efficiency of electron transfer from NADPH-P450 reductase to CYP3A4, a fused enzyme was constructed between CYP3A4 and yeast NADPH-P450 reductase. The rapid reduction of the heme iron of the fused enzyme by NADPH was observed. The fused enzyme showed a high testosterone 6beta-hydroxylation activity with a sigmoidal velocity saturation curve. However, the coupling efficiency between NADPH utilization and testosterone 6beta-hydroxylation was only 10%. Finally, coexpression of the fused enzyme and human cytochrome b5 was examined. A significant decrease in the Km value and a remarkable increase in the coupling efficiency were observed. Substrate-induced spectra revealed that the dissociation constant of the fused enzyme for testosterone significantly decreased with coexpression of human cytochrome b5. These results strongly suggest that human cytochrome b5 directly interacts with the CYP3A4 domain of the fused enzyme and modifies the tertiary structure of substrate binding pocket, resulting in tight binding of the substrate and high coupling efficiency.
authors.affiliation: Biotechnology Laboratory, Sumitomo Chemical Company, Ltd., Hyogo, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Hayashi
authors.forename: T
authors.initials: T
authors.lastname: Sakaki
authors.forename: S
authors.initials: S
authors.lastname: Kominami
authors.forename: K
authors.initials: K
authors.lastname: Inouye
authors.forename: Y
authors.initials: Y
authors.lastname: Yabusaki
mesh_terms: D001483:Base Sequence; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D015786:Cytochromes b5 / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D006900:Hydroxylation; D066298:In Vitro Techniques; 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; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism
chemical_list: D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D013739:Testosterone; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase