Expression of a rat liver microsomal cytochrome P-450 catalyzing testosterone 16 alpha-hydroxylation in Saccharomyces cerevisiae: vitamin D3 25-hydroxylase and testosterone 16 alpha-hydroxylase are distinct forms of cytochrome P-450.
Rat cytochrome P-450(M-1) cDNA was expressed in Saccharomyces cerevisiae TD1 cells by using a yeast-Escherichia coli shuttle vector consisting of P-450(M-1) cDNA, yeast alcohol dehydrogenase promoter and yeast cytochrome c terminator. The yeast cells synthesized up to 2 X 10(5) molecules of P-450(M-1) per cell. The microsomal fraction prepared from the transformed cells contained 0.1 nmol of cytochrome P-450 per mg of protein. The expressed cytochrome P-450 catalyzed 16 alpha- and 2 alpha-hydroxylations of testosterone in accordance with the catalytic activity of P-450(M-1), but did not hydroxylate vitamin D3 or 1 alpha-hydroxycholecalciferol at the 25 position. The expressed cytochrome P-450 also catalyzed the oxidation of several drugs and did not show 25-hydroxylation activity toward 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. However, it cross-reacted with the polyclonal and monoclonal antibodies elicited against purified P-450cc25 which catalyzed the 25-hydroxylation of vitamin D3. These results indicated that P-450(M-1) cDNA coded the 2 alpha- and 16 alpha-hydroxylase of testosterone, and that these two positions of testosterone are hydroxylated by a single form of cytochrome P-450. Vitamin D3 25-hydroxylase and testosterone 16 alpha- and 2 alpha-hydroxylase are different gene products, although these two hydroxylase activities are immunochemically indistinguishable.
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D015153:Blotting, Western; D002384:Catalysis; D053493:Cholestanetriol 26-Monooxygenase; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D000072467:Cytochrome P450 Family 2; D005822:Genetic Vectors; D006900:Hydroxylation; D008861:Microsomes / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology*; D010957:Plasmids; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000235:genetics*; D014170:Transformation, Genetic
D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C450279:CYP2C11 protein, rat; D000072467:Cytochrome P450 Family 2; D039181:Steroid 16-alpha-Hydroxylase; C022724:testosterone 7-alpha-hydroxylase, hamster; D053493:Cholestanetriol 26-Monooxygenase