CYP17A1 exhibits 17αhydroxylase/17,20-lyase activity towards 11β-hydroxyprogesterone and 11-ketoprogesterone metabolites in the C11-oxy backdoor pathway

Cytochrome P450 17 alpha-hydroxylase/17,20-lyase (CYP17A1) plays a pivotal role in the regulation of adrenal and gonadal steroid hormone biosynthesis. More recent studies highlighted the enzyme's role in the backdoor pathway leading to androgen production. Increased CYP17A1 activity in endocrine disorders and diseases are associated with elevated C-21 and C-19 steroids which include 17 alpha-hydroxyprogesterone and androgens, as well as C11-oxy C-21 and C11-oxy C-19 steroids. We previously reported that 11 beta-hydroxyprogesterone (11OHP4), 21-deoxycortisol (21dF) and their keto derivatives are converted by 5 alpha-reductases and hydroxysteroid dehydrogenases yielding C-19 steroids in the backdoor pathway. In this study the 17 alpha-hydroxylase and 17,20-lyase activity of CYP17A1 towards the unconventional C11-oxy C-21 steroid substrates and their 5 alpha- and 3 alpha,5 alpha-reduced metabolites was investigated in transfected HEK-293 cells. CYP17A1 catalysed the 17 alpha-hydroxylation of 11OHP4 to 21dF and 11-ketoprogesterone (11KP4) to 21-deoxycortisone (21dE) with negligible hydroxylation of their 5 alpha-reduced metabolites while no lyase activity was detected. The 3 alpha,5 alpha-reduced C11-oxy C-21 steroids-5 alpha-pregnan-3 alpha,11 beta-diol-20-one (3,11diOH-DHP4) and 5 alpha-pregnan-3a-ol-11,20-dione (alfaxalone) were rapidly hydroxylated to 5 alpha-pregnan-3 alpha,11 beta,17 alpha-triol-20-one (11OH-Pdiol) and 5 alpha-pregnan-3 alpha,17 alpha-diol-11,20-dione (11K-Pdiol), with the lyase activity subsequently catalysing to conversion to the C-11-oxy C-19 steroids, 11 beta-hydroxyandrosterone and 11-ketoandrosterone, respectively. Docking of 11OHP4, 11KP4 and the 5 alpha-reduced metabolites, 5 alpha-pregnan-11 beta-ol-3,20-dione (11OH-DHP4) and 5 alpha-pregnan-3,11,20-trione (11K-DHP4) with human CYP17A1 showed minimal changes in the orientation of these C11-oxy C-21 steroids in the active pocket when compared with the binding of progesterone suggesting the 17,20-lyase is impaired by the C11-hydroxyl and keto moieties. The structurally similar 3,11diOH-DHP4 and alfaxalone showed a greater distance between C17 and the heme group compared to the natural substrate, 17 alpha-hydroxypregnenolone potentially allowing more orientational freedom and facilitating the conversion of the C11-oxy C-21 to C11-oxy C-19 steroids. In summary, our in vitro assays showed that while CYP17A1 readily hydroxylated 11OHP4 and 11KP4, the enzyme was unable to catalyse the 17,20-lyase reaction of these C11-oxy C-21 steroid products. Although CYP17A1 exhibited no catalytic activity towards the 5 alpha-reduced intermediates, once the C4-C5 double bond and the keto group at C3 were reduced, both the hydroxylation and lyase reactions proceeded efficiently. These findings show that the C11-oxy C-21 steroids could potentially contribute to the androgen pool in tissue expressing steroidogenic enzymes in the backdoor pathway