Brian P. Schlegel

Structure and function of 3α-hydroxysteroid dehydrogenase

Mammalian 3 alpha-hydroxysteroid dehydrogenases (3 alpha-HSDs) inactivate circulating steroid hormones, and in target tissues regulate the occupancy of steroid hormone receptors. Molecular cloning indicates that 3 alpha-HSDs are members of the aldo-keto reductase (AKR) superfamily and display high sequence identity (> 60%). Of these, the most extensively characterized is rat liver 3 alpha-HSD. X-ray crystal structures of the apoenzyme and the E.NADP+ complex have been determined and serve as structural templates for other 3 alpha-HSDs. These structures reveal that rat liver 3 alpha-HSD adopts an (alpha/beta)8-barrel protein fold. NAD(P)(H) lies perpendicular to the barrel axis in an extended conformation, with the nicotinamide ring at the core of the barrel, and the adenine ring at the periphery of the structure. The nicotinamide ring is stabilized by interaction with Y216, S166, D167, and Q190, so that the A-face points into the vacant active site. The 4-pro-(R) hydrogen transferred in the oxidoreduction of steroids is in close proximity to a catalytic tetrad that consists of D50, Y55, K84, and H117. A water molecule is within hydrogen bond distance of H117 and Y55, and its position may mimic the position of the carbonyl of a 3-ketosteroid substrate. The catalytic tetrad is conserved in members of the AKR superfamily and resides at the base of an apolar cleft implicated in binding steroid hormone. The apolar cleft consists of a side of apolar residues (L54, W86, F128, and F129), and opposing this side is a flexible loop that contains W227. These constraints suggest that the alpha-face of the steroid would orient itself along that side of the cleft containing W86. Site-directed mutagenesis of the catalytic tetrad indicates that Y55 and K84 are essential for catalysis. Y55S and Y55F mutants are catalytically inactive, but still form binary (E.NADPH) and ternary (E.NADH.Testosterone) complexes; by contrast K84R and K84M mutants are catalytically inactive, but do not bind steroid hormone. The reliance on a Tyr/Lys pair is reminiscent of catalytic mechanisms proposed for other AKR members as well as for HSDs that belong to the short-chain dehydrogenase/reductase (SDR) family, in which Tyr is the general acid, with its pKa being lowered by Lys. Superimposition of the nicotinamide rings in the structures of 3 alpha-HSD (an AKR) and 3 alpha, 20 beta-HSD (an SDR) show that the Tyr/Lys pairs are positionally conserved, suggesting convergent evolution across protein families to a common mechanism for HSD catalysis. W86Y and W227Y mutants bind testosterone to the E.NADH complex, with effective increases in Kd of 8- and 20-fold. These data provide the first evidence that the side of the apolar cleft containing W86 and the opposing flexible loop containing W227 are parts of the steroid-binding site. Detailed mutagenesis studies of the apolar cleft and elucidation of a ternary complex structure will ultimately provide details of the determinants that govern steroid hormone recognition. These determinants could provide a rational basis for structure-based inhibitor design.

Mammalian 3α-hydroxysteroid dehydrogenases

Abstract Mammalian 3α-hydroxysteroid dehydrogenases (3α-HSDs) regulate steroid hormone levels. For example, hepatic 3α-HSDs inactivate circulating androgens, progestins, and glucocorticoids. In target tissues they regulate access of steroid hormones to steroid hormone receptors. For example, in the prostate 3α-HSD acts as a molecular switch and controls the amount of 5α-dihydrotestosterone that can bind to the androgen receptor, while in the brain 3α-HSD can regulate the amount of tetrahydrosteroids that can alter GABA a receptor function. Molecular cloning indicates that these mammalian 3α-HSDs belong to the aldo-keto reductase superfamily and that they are highly homologous proteins. Using the three-dimensional structure of rat liver 3α-HSD as a template for site-directed mutagenesis, details regarding structure-function relationships, including catalysis and cofactor and steroid hormone recognition have been elucidated. These details may be relevant to all mammalian 3α-HSDs.

Characterization of the Substrate Binding Site in Rat Liver 3α-Hydroxysteroid/Dihydrodiol Dehydrogenase THE ROLES OF TRYPTOPHANS IN LIGAND BINDING AND PROTEIN FLUORESCENCE

Rat liver 3α-hydroxysteroid dehydrogenase (3α-HSD), a member of the aldoketoreductase superfamily, inactivates circulating steroid hormones using NAD(P)H as cofactor. Despite determination of the 3α-HSD·NADP+ binary complex structure, the functional elements that dictate the binding of steroids remain unclear (Bennett, M.J., Schlegel, B.P., Jez, J.M., Penning, T.M., and Lewis, M. (1996) Biochemistry 35, 10702-10711). Two tryptophans (Trp86 and Trp227) near the active site may have roles in substrate binding, and their fluorescence may be quenched upon binding of NADPH. Trp86 is located within an apolar cleft, while Trp227 is found on an opposing loop near the active site. A third tryptophan, Trp148, is on the periphery of the structure. To investigate the roles of these tryptophans in protein fluorescence and ligand binding, we generated three mutant enzymes (W86Y, W148Y, and W227Y) by site-directed mutagenesis. Spectroscopic measurements on these proteins showed that Trp148 contributed the most to the enzyme fluorescence spectra, with Trp227 adding the least. Trp86 was identified as the tryptophan quenched by bound NADPH through an energy transfer mechanism. The W86Y mutant altered binding of cofactor (a 3-fold increase in Kd for NADPH) and steroid (a 7-fold increase in Kd for testosterone). This mutation also dramatically decreased the catalytic efficiency observed with one-, two-, and three-ring substrates and decreased the binding affinity for nonsteroidal anti-inflammatory drugs but had little effect on the binding of aldose reductase inhibitors. Interestingly, mutation of Trp227 significantly impaired steroid binding (a 22-fold increase in Kd for testosterone), but did not alter binding of cofactor, smaller substrates, or inhibitors. Kinetically, the W148Y mutant was similar to wild-type enzyme. Our results demonstrate that Trp86 and the apolar cleft is part of the substrate binding pocket. In addition, we propose a role for Trp227 and its associated loop in binding steroids, but not small substrates or inhibitors, most likely through interaction with the C- and D-rings of the steroid. This work provides the first evidence that tryptophans on opposite sides of the apolar cleft are part of the steroid binding pocket and suggests how the enzyme may discriminate between nonsteroidal anti-inflammatory drugs and aldose reductase inhibitors like zopolrestat. A model of how androstanedione binds in the apolar cleft is developed. These data provide further evidence that loop structures in members of the aldoketoreductase superfamily are critical determinants of ligand binding.