Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.1.1.3 (HSD)
 3,464 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mammalian 3 alpha-hydroxysteroid dehydrogenases (3 alpha-HSDs) regulate steroid hormone levels. cDNA cloning indicates that the rat and human liver isoforms display high sequence identity and that they belong to the aldo-keto reductase (AKR) superfamily. Of these the most extensively characterized is rat liver 3 alpha-HSD. The recently solved X-ray crystal structure shows that this enzyme adopts an (alpha/beta)8-barrel scaffold (Hoog et al. 1994). NAD(P)H binds in an extended anti-conformation and lies along the inner surface of the barrel. The nicotinamide ring is stabilized by interaction with Y216. The 4-pro(R)-hydrogen transferred in the reaction is in close proximity to Y55. K84, D50 and H117 which are implicated in catalysis. These residues are located at the base of a hydrophobic pocket which is presumed to be involved in binding steroid hormone. This catalytic tetrad is conserved in members of the AKR superfamily. Mutant enzymes support roles for Y55 in steroid binding and for K84 as the general acid involved in catalysis. The gene for rat 3 alpha-HSD has been cloned and is 47 kb in length and contains 9 exon-intron boundaries which are highly conserved in the human gene(s). The 5'-flanking regions of the rat and human genes contain consensus sequences for AP-1, Oct-1 and multiple copies of perfect and imperfect steroid hormone response elements (REs) (estrogen, glucocorticoid (GRE), and progesterone) which may comprise a steroid response unit (SRU) (Lin & Penning 1995). Constitutive and regulated expression of the rat 3 alpha-HSD gene has been studied by transiently transfecting reporter gene (chloramphenicol acetyltransferase, CAT) constructs into human hepatoma (HepG2) cells. With respect to the transcription start-site (+1), a proximal (-498 to -199bp) and distal (-20 to -4.0kb) enhancer, as well as a powerful silencer (-755 to -498 bp) were located in the promoter. Band-shift and supershift assays provide evidence that Oct-1 binds to the silencer. Tandem repeats of the imperfect proximal and distal GREs that reside in the SRU were inserted into tk-CAT vectors and transiently transfected. Stimulation of transfected cells with dexamethasone resulted in robust CAT activity. These data indicate that glucocorticoids may positively regulate transcription of the rat 3 alpha-HSD gene from the SRU.
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PMID:3 alpha-hydroxysteroid dehydrogenase: three dimensional structure and gene regulation. 894 1

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.
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PMID:Structure and function of 3 alpha-hydroxysteroid dehydrogenase. 902 23