Functional expression of recombinant spiny dogfish shark (Squalus acanthias) cytochrome P450c17 (17 alpha-hydroxylase/C17,20-lyase) in yeast (Pichia pastoris).
The cDNA encoding the spiny dogfish shark (Squalus acanthias) testicular form of cytochrome P450c17 (CYP17) was used to direct the heterologous expression of a functional enzyme in yeast (Pichia pastoris). This protein possesses two enzymatic activities: 17 alpha-hydroxylase and C17,20-lyase reactions. Cytochrome P450c17 is a key steroidogenic enzyme for the production of sex steroids in gonadal tissue and for cortisol production in adrenal tissue. This study describes the culture conditions and the enzymatic activity of recombinant shark cytochrome P450c17. The shark enzyme was compatible with the endogenous yeast NADPH-cytochrome P450 reductase and was bioactive within the living yeast cell. Progesterone (at 15 microM) was metabolized (51 pmol/min/10(9) cells) faster than pregnenolone (36 pmol/min/10(9) cells). Both progesterone and pregnenolone were completely metabolized to their respective androgens (androstenedione and dehydroepiandrosterone). Although 11 beta-hydroxy-progesterone was readily 17 alpha-hydroxylated by the shark P450, the lyase reaction was not evident. Alterations to the 2-carbon sidechain of progesterone (21-hydroxylation or 20 beta-reduction) prevented metabolism. High-density cultures (> 1.5 x 10(9) cells/ml) yielded the greatest quantity of recombinant protein but cultures of lower density produced more recombinant protein per cell. This is the first report of heterologous expression in yeast of a steroidogenic cytochrome P450 from a lower vertebrate.
Trant
J M
JM
Department of Zoology and Physiology, Louisiana State University, Baton Rouge 70803, USA. trant@umbi.umd.edu
D000446:Aldehyde-Lyases / Q000096:biosynthesis / Q000235:genetics*; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics*; D004284:Dogfish; D008297:Male; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013254:Steroid 17-alpha-Hydroxylase; D013737:Testis / Q000201:enzymology
D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D013254:Steroid 17-alpha-Hydroxylase; D000446:Aldehyde-Lyases