Expression of bovine adrenodoxin and NADPH-adrenodoxin reductase cDNAs in Saccharomyces cerevisiae.
Expression of both bovine adrenodoxin (ADX) and NADPH-adrenodoxin reductase (ADR) were examined in Saccharomyces cerevisiae. Three ADX and two ADR expression plasmids were constructed by inserting each of the corresponding cDNA fragments between the yeast alcohol dehydrogenase I promoter and terminator of the expression vector pAAH5N. Plasmids pAX and pMX contained the coding region for the precursor and mature ADX, respectively, while pCMX carried the mature ADX preceded by the mitochondrial signal of yeast cytochrome c oxidase subunit IV (COX IV). Similarly, pMR and pCMR coded for mature ADR without and with the mitochondrial signal of yeast COX IV, respectively. Transformed S. cerevisiae AH22[rho 0]/pAX cells produced the ADX precursor, while AH22[rho 0]/pMX and AH22[rho 0]/pCMX cells produced mature ADX (mat-ADX) and modified ADX (mat-COX/ADX), respectively. Mat-ADX and mat-COX/ADX were found mainly in the cytosolic and mitochondrial fractions, respectively, and showed cytochrome c reductase activity. AH22[rho+]/pMR and AH22[rho+]/pCMR cells produced mature ADR (mat-ADR) and modified ADR (mat-COX/ADR), respectively. Mat-ADR lacking the mitochondrial signal was found in the cytosolic fraction and exhibited cytochrome c reductase activity, while mat-COX/ADR was localized in the mitochondrial fraction, but showed no reductase activity. In an in vitro reconstituted system consisting of both mat-COX/ADX- and mat-ADR-containing fractions, bovine P450scc converted cholesterol into pregnenolone. Thus mat-COX/ADX and mat-ADR produced in the yeast can transfer electrons from NADPH to P450scc.
Akiyoshi-Shibata
M
M
Takarazuka Research Center, Sumitomo Chemical Co., Ltd., Takarazuka, Hyogo, Japan.
Sakaki
T
T
Yabusaki
Y
Y
Murakami
H
H
Ohkawa
H
H
D000325:Adrenodoxin / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002417:Cattle; D002784:Cholesterol / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000235:genetics*; D005287:Ferredoxin-NADP Reductase / Q000235:genetics*; D015870:Gene Expression; D005800:Genes, Fungal; D006868:Hydrolysis; D008291:Malate Dehydrogenase / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism; D008969:Molecular Sequence Data; D009245:NADH Dehydrogenase / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010957:Plasmids; D012333:RNA, Messenger / Q000032:analysis; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*
D012333:RNA, Messenger; D000325:Adrenodoxin; D009249:NADP; D004247:DNA; D002784:Cholesterol; D006899:Mixed Function Oxygenases; D008291:Malate Dehydrogenase; D005287:Ferredoxin-NADP Reductase; D009245:NADH Dehydrogenase