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  • br Introduction The principal pathways of adrenal and gonada

    2024-05-18


    Introduction The principal pathways of adrenal and gonadal steroidogenesis have been known for over 50 years (reviewed by Miller and Auchus, 2011). Cholesterol is first converted to pregnenolone via three reactions, 20-hydroxylation, 22-hydroxylation and scission of the 20,22 carbon–carbon bond, all catalyzed by mitochondrial P450scc. Pregnenolone may then be converted to other Δ5 steroids: 17α-OH-pregnenolone (17-Preg) and DHEA by P450c17, and thence to androstenediol by several forms of HSD17B. These Δ5 steroids may be converted to the corresponding Δ4 steroids, progesterone, 17α-OH-progesterone (17OHP), androstenedione and testosterone, by adrenal or gonadal 3β-hydroxysteroid dehydrogenase, type 2 (3βHSD2). P450c17 readily catalyzes the 17α-hydroxylation of progesterone and pregnenolone to 17-Preg and 17OHP, respectively. When progesterone is combined with P450c17, about 30% is 16α-hydroxylated (Swart et al, 1993, Swart et al, 2010) and about 1% is 21-hydroxylated (Yoshimoto et al., 2012), but physiologic relevance has not been established for these reactions. P450c17 then may also catalyze the 17,20 lyase activity that converts 21-carbon (C21) 17-hydroxy steroids such as 17-Preg or 17OHP to 19-carbon (C19) precursors of sex steroids. Much effort has centered on the inhibition of 17,20 lyase activity for therapy of sex steroid dependent malignancies, such as breast and prostate cancer (DeVore, Scott, 2012, O'Donnell et al, 2004, Salvador et al, 2013). P450c17 is the microsomal P450 enzyme that catalyzes both 17α-hydroxylase and 17,20 lyase activities (reviewed by Miller and Auchus, 2011). These two activities were once thought to be catalyzed by separate enzymes that differed in the adrenals and gonads; however, the two activities co-purify (Nakajin et al., 1981), expression of bovine P450c17 in nonsteroidogenic COS-1 SC75741 conferred both 17α-hydroxylase and 17,20 lyase activities (Zuber et al., 1986), and the cloning of identical cDNAs from adrenal and testis (Chung et al., 1987) showed that both came from the same gene (Picado-Leonard and Miller, 1987). Thus both activities had to be catalyzed by a single enzyme, and the distinction between 17α-hydroxylase and 17,20 lyase had to be functional and not genetic or structural. Nevertheless, clinical observations showed that adrenal 17α-hydroxylase activity (reflected by serum cortisol concentrations) was fairly constant throughout life, whereas adrenal 17,20 lyase activity (reflected by serum DHEA and DHEAS concentrations) was low in early childhood but rose abruptly during adrenarche beginning at ages 8–10 years and reaching maximal values in the mid 20s (Apter et al, 1979, Orentreich et al, 1984) (Fig. 1). As P450c17 has both 17α-hydroxylase and 17,20 lyase activities, it is the key branch point in steroidogenesis: P450c17 is not expressed in the zona glomerulosa, hence neither activity is present and pregnenolone is converted to mineralocorticoids; the zona fasciculata has 17α-hydroxylase activity but not 17,20 lyase activity, hence pregnenolone is converted to cortisol; in the zona reticularis, both activities are present, so that pregnenolone is converted to precursors of sex steroids. Research on the 17,20 lyase activity of P450c17 has been confounded by differences among various species: the predominant pathway to C19 steroids for human (Auchus et al, 1998, Lin et al, 1991, Lin et al, 1993) and bovine (Zuber et al., 1986) P450c17 is via the Δ5 pathway, but pig (Nakajin et al, 1981, Yanagibashi, Hall, 1986), frog (Lutz et al., 2001), and trout (Sakai et al., 1992) P450c17 have readily measured Δ4 17, 20-lyase activity, and the Δ4 pathway predominates with the rat (Fevold et al., 1989), hamster (Mathieu et al., 2002) and guinea pig (Kominami et al, 1992, Tremblay et al, 1994).
    Cell biology and enzymology of P450c17 The principal factor regulating the 17,20 lyase activity of P450c17 is electron transfer (reviewed by Miller, 2005). Like all microsomal P450 enzymes, P450c17 receives electrons from reduced nicotinamide adenine dinucleotide phosphate (NADPH) via the membrane-bound flavoprotein, P450 oxidoreductase (POR) and mediates catalysis via the iron atom in the P450 heme ring. POR receives two electrons from NADPH and transfers them one at a time to the P450 (Wang et al., 1997). The crystal structures of N-terminally truncated forms of rat POR (Hubbard et al, 2001, Wang et al, 1997), dynamic modeling based on nuclear magnetic resonance and small-angle X-ray scattering (Ellis et al., 2009), crystallography of disulfide cross-linked mutants (Xia et al., 2011), and computational modeling (Pandey and Flück, 2013) show that POR is a butterfly-shaped protein: one ‘wing’ contains a flavin adenine dinucleotide (FAD) moiety, and the other contains a flavin mononucleotide (FMN) moiety; these two domains are separated by a flexible hinge region (Fig. 2). When the FAD moiety of POR receives two electrons from NADPH there is a conformational change in the hinge region, bringing the two ‘wings’ together and aligning the isoalloxazine rings of the FAD and the FMN, permitting the electrons to flow from the FAD to the FMN. The POR then returns to its initial, more open conformation, allowing the FMN domain to associate with the redox-partner binding site of the P450 by electrostatic interactions: the surface of the electron-donating FMN domain has numerous acidic residues, whereas the redox-partner binding site of P450 enzymes contain numerous basic residues (Auchus and Miller, 1999). The electrons from the FMN domain then flow to the heme iron atom of the P450 (where catalysis is mediated) and the POR dissociates from the P450 (reviewed by Miller, 2005).