Expression of rat NADPH-cytochrome P-450 reductase cDNA in Saccharomyces cerevisiae.
A full-length cDNA for rat NADPH-cytochrome P-450 reductase was cloned by the procedure of Okayama and Berg (1982) from hepatic poly(A)RNA prepared from phenobarbital-induced rats. Both cDNA and amino acid sequences agreed with the sequences reported by Porter and Kasper (1985) except for four single base differences. Three expression plasmids were constructed by insertion of the reductase cDNA between yeast alcohol dehydrogenase I (ADH) promoter and terminator regions. Plasmids pARF1 and pTRF2 were constructed with slightly different lengths between the ADH promoter and the initiation codon; on introduction into Saccharomyces cerevisiae AH22 cells, they synthesized about 1 X 10(3) and 5 X 10(3) reductase protein molecules per cell, respectively. A third plasmid, pARM1, containing a cytochrome P-450MC cDNA expression unit located between two reductase cDNA expression units synthesized 4 X 10(5) cytochrome P-450MC hemoprotein and 1 X 10(4) reductase protein molecules per cell. The cellular extracts of the AH22/pARM1 strain, which synthesized both rat enzymes, showed higher cytochrome c reductase and cytochrome P-450MC-dependent 7-ethoxycoumarin O-deethylation activities as compared to extracts of the AH22/pAMC1 strain, which synthesized only rat cytochrome P-450MC. 7-Ethoxycoumarin O-deethylation activity in the cellular extract of AH22/pARM1 strain was partly inhibited by the addition of anti-rat reductase IgG. In addition, whole AH22/pARM1 cells exhibited higher monooxygenase activity toward acetanilide and 7-ethoxycoumarin than control AH22/pAMC1 cells. These results indicated that a functional electron-transport chain consisting of rat NADPH-cytochrome P-450 reductase and rat cytochrome P-450MC was constructed in S. cerevisiae cells.
Murakami
H
H
Yabusaki
Y
Y
Ohkawa
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D005786:Gene Expression Regulation; D008970:Molecular Weight; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D012333:RNA, Messenger; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase