CYP17A1 exhibits 17αhydroxylase/17,20-lyase activity towards 11β-hydroxyprogesterone and 11-ketoprogesterone metabolites in the C11-oxy backdoor pathway.
Cytochrome P450 17α-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 C21 and C19 steroids which include 17α-hydroxyprogesterone and androgens, as well as C11-oxy C21 and C11-oxy C19 steroids. We previously reported that 11β-hydroxyprogesterone (11OHP4), 21-deoxycortisol (21dF) and their keto derivatives are converted by 5α-reductases and hydroxysteroid dehydrogenases yielding C19 steroids in the backdoor pathway. In this study the 17α-hydroxylase and 17,20-lyase activity of CYP17A1 towards the unconventional C11-oxy C21 steroid substrates and their 5α- and 3α,5α-reduced metabolites was investigated in transfected HEK-293 cells. CYP17A1 catalysed the 17α-hydroxylation of 11OHP4 to 21dF and 11-ketoprogesterone (11KP4) to 21-deoxycortisone (21dE) with negligible hydroxylation of their 5α-reduced metabolites while no lyase activity was detected. The 3α,5α-reduced C11-oxy C21 steroids-5α-pregnan-3α,11β-diol-20-one (3,11diOH-DHP4) and 5α-pregnan-3α-ol-11,20-dione (alfaxalone) were rapidly hydroxylated to 5α-pregnan-3α,11β,17α-triol-20-one (11OH-Pdiol) and 5α-pregnan-3α,17α-diol-11,20-dione (11K-Pdiol), with the lyase activity subsequently catalysing to conversion to the C11-oxy C19 steroids, 11β-hydroxyandrosterone and 11-ketoandrosterone, respectively. Docking of 11OHP4, 11KP4 and the 5α-reduced metabolites, 5α-pregnan-11β-ol-3,20-dione (11OH-DHP4) and 5α-pregnan-3,11,20-trione (11K-DHP4) with human CYP17A1 showed minimal changes in the orientation of these C11-oxy C21 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, 17α-hydroxypregnenolone potentially allowing more orientational freedom and facilitating the conversion of the C11-oxy C21 to C11-oxy C19 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 C21 steroid products. Although CYP17A1 exhibited no catalytic activity towards the 5α-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 C21 steroids could potentially contribute to the androgen pool in tissue expressing steroidogenic enzymes in the backdoor pathway.
van Rooyen D
，Yadav R
，Scott EE
，Swart AC
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The in vitro metabolism of 11β-hydroxyprogesterone and 11-ketoprogesterone to 11-ketodihydrotestosterone in the backdoor pathway.
Increased circulating 11β-hydroxyprogesterone (11OHP4), biosynthesised in the human adrenal, is associated with 21-hydroxylase deficiency in congenital adrenal hyperplasia. 17α-hydroxyprogesterone levels are also increased, with the steroid's metabolism to dihydrotestosterone in the backdoor pathway contributing to hyperandrogenic clinical conditions. In this study we investigated the in vitro biosynthesis and downstream metabolism of 11OHP4. Both cytochrome P450 11β-hydroxylase and aldosterone synthase catalyse the biosynthesis of 11OHP4 from progesterone (P4) which is converted to 11-ketoprogesterone (11KP4) by 11β-hydroxysteroid dehydrogenase type 2, while type 1 readily catalysed the reverse reaction. We showed in HEK-293 cells that these C11-oxy C21 steroids were metabolised by steroidogenic enzymes in the backdoor pathway-5α-reductase (SRD5A) and 3α-hydroxysteroid type 3 (AKR1C2) converted 11OHP4 to 5α-pregnan-11β-ol,3,20-dione and 5α-pregnan-3α,11β-diol-20-one, while 11KP4 was converted to 5α-pregnan-3,11,20-trione and 5α-pregnan-3α-ol-11,20-dione (alfaxalone), respectively. Cytochrome P450 17α-hydroxylase/17,20-lyase catalysed the hydroxylase and lyase reaction to produce the C11-oxy C19 steroids demonstrated in the conversion of alfaxalone to 11-oxy steroids demonstrated in the conversion of alfaxalone to 11ketoandrosterone. In LNCaP cells, a prostate cancer cell model endogenously expressing the relevant enzymes, 11OHP4 and 11KP4 were metabolised to the potent androgen, 11-ketodihydrotestosterone (11KDHT), thus suggesting the C11-oxy C21 steroids contribute to the pool of validating the in vitro biosynthesis of C11-oxy C19 steroids from C11-oxy C21 steroids. The in vitro reduction of 11KP4 at C3 and C5 by AKR1C2 and SRD5A has confirmed the metabolic route of the urinary metabolite, 3α,20α-dihydroxy-5β-pregnan-11-one. Although our assays have demonstrated the conversion of 11OHP4 and 11KP4 by steroidogenic enzymes in the backdoor pathway yielding 11KDHT, thus suggesting the C11-oxy C21 steroids contribute to the pool of potent androgens, the in vivo confirmation of this metabolic route remains challenging.
van Rooyen D
，Gent R
，Barnard L
，Swart AC
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The 11β-hydroxysteroid dehydrogenase isoforms: pivotal catalytic activities yield potent C11-oxy C(19) steroids with 11βHSD2 favouring 11-ketotestosterone, 11-ketoandrostenedione and 11-ketoprogesterone biosynthesis.
The 11β-hydroxysteroid dehydrogenase (11βHSD) types 1 and 2 are primarily associated with glucocorticoid inactivation and reactivation. Several adrenal C11-oxy C19 and C11-oxy C21 steroids, which have been identified in prostate cancer, 21-hydroxylase deficiency and polycystic ovary syndrome, are substrates for these isozymes. This study describes the kinetic parameters of 11βHSD1 and 11βHSD2 towards the C11-keto and C11-hydroxy derivatives of the C19 and C21 steroids. The apparent Km and Vmax values indicate the more prominent 11βHSD2 activity towards 11β-hydroxy androstenedione, 11β-hydroxytestosterone and 11β-hydroxyprogesterone in contrast to the 11βHSD1 reduction of the C11-keto steroids, as was demonstrated in the LNCaP cell model in the production of 11-ketotestosterone and 11-ketodihydrotestosterone. Data highlighted the role of 11βHSD2 and cytochrome P450 17A1 in the contribution of C11-oxy C21 steroids to the C11-oxy C19 steroid pool in the C11-oxy backdoor pathway. In addition, 11βHSD2 activity, catalysing 11-ketotestosterone biosynthesis, was shown to be key in the production of prostate specific antigen and in the progression of prostate cancer to castration resistant prostate cancer. The study at hand thus provides evidence that 11βHSD isozymes play key roles in pathophysiological states, more so than was previously put forward.
Gent R
，du Toit T
，Bloem LM
，Swart AC
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