Expression of rat liver vitamin D3 25-hydroxylase cDNA in Saccharomyces cerevisiae.
The cDNA coding for the precursor protein of rat liver mitochondrial vitamin D3 25-hydroxylase, cytochrome P450LMT25, was expressed under the control of the yeast alcohol dehydrogenase I promoter and terminator in Saccharomyces cerevisiae AH22 cells. The transformed yeast cells produced a P450LMT25 protein with an almost similar apparent molecular weight as compared with that of the authentic mature enzyme. The expression level of the P450LMT25 hemoprotein was about 5 x 10(4) molecules per cell as determined by reduced CO-difference spectra. The mitochondrial fraction prepared from the transformed yeast cells exhibited both 25-hydroxylase activity toward 1 alpha-hydroxyvitamin D3 and 27-hydroxylase activity toward 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol in a reconstituted system containing bovine adrenodoxin and NADPH-adrenodoxin reductase.
Akiyoshi-Shibata
M
M
Biotechnology Laboratory, Takarazuka Research Center, Sumitomo Chemical Co., Hyogo, Japan.
Usui
E
E
Sakaki
T
T
Yabusaki
Y
Y
Noshiro
M
M
Okuda
K
K
Ohkawa
H
H
D000426:Alcohol Dehydrogenase / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D053493:Cholestanetriol 26-Monooxygenase; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics; D004272:DNA, Mitochondrial / Q000096:biosynthesis*; D015870:Gene Expression; D008099:Liver / Q000201:enzymology*; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D051381:Rats; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013250:Steroid Hydroxylases / Q000096:biosynthesis / Q000235:genetics*; D014170:Transformation, Genetic
D004272:DNA, Mitochondrial; D003577:Cytochrome P-450 Enzyme System; D000426:Alcohol Dehydrogenase; D013250:Steroid Hydroxylases; D053493:Cholestanetriol 26-Monooxygenase