Expression of bovine cytochrome P450c17 cDNA in Saccharomyces cerevisiae.
We constructed expression plasmids for bovine adrenal cytochrome P450c17 (P450c17) by inserting the corresponding cDNA between the yeast alcohol dehydrogenase I promoter and terminator of the expression vector pAAH5. Plasmids pA alpha 1 and pA alpha 2 contained the entire coding region for bovine P450c17, whereas pAC alpha 1 included the cDNA coding for chimeric P450c alpha consisting of the amino-terminal 45 amino acid residues of rat P450c and the carboxy-terminal 482 amino acid residues of bovine P450c17. The transformed Saccharomyces cerevisiae AH22/pA alpha 1, AH22/pA alpha 2, and AH22/pAC alpha 1 cells produced about 1 x 10(5), 1 x 10(5), and 2 x 10(4) molecules per cell of the corresponding P450 hemoproteins, respectively. On incubation with the cultures of each of the three strains, progesterone was specifically converted into 17 alpha-hydroxyprogesterone, which was not further converted into androstenedione, indicating that the three strains showed 17 alpha-hydroxylase activity, but almost no C17,20-lyase activity. The microsomal fraction prepared from the AH22/pA alpha 1 cells showed 17 alpha-hydroxylase activity toward progesterone and pregnenolone to higher extents, and exhibited C17,20-lyase activity toward 17 alpha-hydroxypregnenolone to a lesser extent and almost no C17,20-lyase activity toward 17 alpha-hydroxyprogesterone. These results indicated that bovine P450c17 synthesized in S. cerevisiae cells manifests the 17 alpha-hydroxylase activity, but not the C17,20-lyase activity.
Sakaki
T
T
Takarazuka Research Center, Sumitomo Chemical Co., Ltd., Hyogo, Japan.
Shibata
M
M
Yabusaki
Y
Y
Murakami
H
H
Ohkawa
H
H
D000311:Adrenal Glands / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D008969:Molecular Sequence Data; D010957:Plasmids; D011374:Progesterone / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D011374:Progesterone; D004247:DNA; D003577:Cytochrome P-450 Enzyme System