title: Expression of rat liver vitamin D3 25-hydroxylase cDNA in Saccharomyces cerevisiae.
abstract: 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.
authors.affiliation: Biotechnology Laboratory, Takarazuka Research Center, Sumitomo Chemical Co., Hyogo, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Akiyoshi-Shibata
authors.forename: E
authors.initials: E
authors.lastname: Usui
authors.forename: T
authors.initials: T
authors.lastname: Sakaki
authors.forename: Y
authors.initials: Y
authors.lastname: Yabusaki
authors.forename: M
authors.initials: M
authors.lastname: Noshiro
authors.forename: K
authors.initials: K
authors.lastname: Okuda
authors.forename: H
authors.initials: H
authors.lastname: Ohkawa
mesh_terms: 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
chemical_list: D004272:DNA, Mitochondrial; D003577:Cytochrome P-450 Enzyme System; D000426:Alcohol Dehydrogenase; D013250:Steroid Hydroxylases; D053493:Cholestanetriol 26-Monooxygenase