Assessment of porcine and human 16-ene-synthase, a third activity of P450c17, in the formation of an androstenol precursor

Role of recombinant cytochrome b₅ and P450 reductase

Recently, we have shown that the biosynthesis of androstenol, a potential endogenous ligand for the orphan receptors constitutive androstane receptor and pregnane-X-receptor, requires the presence of enzymes of the steroidogenic pathway, such as 3β-hydroxysteroid dehydrogenase, 5α-reductase and 3α-hydroxysteroid dehydrogenase. In this report, we examine at the molecular level whether the enzyme 17α-hydroxylase/17,20-lyase (P450c17), which possesses dual 17α-hydroxylase and 17,20-lyase activities and catalyzes the production of precursors for glucocorticoids and sex steroids, is also able to catalyze the formation of a third class of active steroids, 16-ene steroids (including androstenol). The role of components of the P450 complex is also assessed. We transfected human embryonic kidney (HEK-293) cells with various amounts of vectors expressing P450c17, NADPH-cytochrome P450 reductase, and cytochrome b₅. Our results showed that P450c17 possesses a 16-ene-synthase activity able to transform pregnenolone into 5,16-androstadien-3β-ol, without the formation of the precursor 17-hydroxypregnenolone. Cytochrome b₅ has a much stronger effect on the 16-ene-synthase activity than on the 17α-hydroxylase/17,20-lyase activities. On the other hand, P450reductase has a drastic effect on the latter, but a negligible one on 5,16-androstadien-3β-ol synthesis. Our results therefore demonstrate that human P450c17, as other enzymes of the classical steroidogenic pathway, is involved in the biosynthetic pathway leading to the formation of androstenol.