Mechanism of inhibition of 3 alpha, 20 beta-hydroxysteroid dehydrogenase by a licorice-derived steroidal inhibitor.

BACKGROUND: Bacterial 3 alpha, 20 beta-hydroxysteroid dehydrogenase (3 alpha, 20 beta-HSD) reversibly oxidizes the 3 alpha and 20 beta hydroxyl groups of androstanes and pregnanes and uses nicotinamide adenine dinucleotide as a cofactor. 3 alpha, 20 beta-HSD belongs to a family of short-chain dehydrogenases that has a highly conserved Tyr-X-X-X-Lys sequence. The family includes mammalian enzymes involved in hypertension, digestion, fertility and spermatogenesis. Several members of the enzyme family, including 3 alpha, 20 beta-HSD, are competitively inhibited by glycyrrhizic acid, a steroidal compound found in licorice, and its derivative, carbenoxolone, an anti-inflammatory glucocorticoid. RESULTS: The three-dimensional structure of the enzyme-carbenoxolone complex has been determined and refined at 2.2 A resolution to a crystallographic R-factor of 19.4%. The hemisuccinate side chain of carbenoxolone makes a hydrogen bond with the hydroxyl group of the conserved residue Tyr152 and occupies the position of the nicotinamide ring of the cofactor. The occupancies of the inhibitor in four independent catalytic sites refine to 100%, 95%, 54% and 36%. CONCLUSIONS: The steroid binds at the catalytic site in a mode much like the previously proposed mode of binding of the substrate cortisone. No bound cofactor molecules were found. The varying occupancy of steroid molecules observed in the four catalytic sites is either due to packing differences or indicates a cooperative effect among the four sites. The observed binding accounts for the inhibition of 3 alpha, 20 beta-HSD.

The refined three-dimensional structure of 3 alpha,20 beta-hydroxysteroid dehydrogenase and possible roles of the residues conserved in short-chain dehydrogenases.

BACKGROUND: Bacterial 3 alpha,20 beta-hydroxysteroid dehydrogenase reversibly oxidizes the 3 alpha and 20 beta hydroxyl groups of steroids derived from androstanes and pregnanes. It was the first short-chain dehydrogenase to be studied by X-ray crystallography. The previous description of the structure of this enzyme, at 2.6 A resolution, did not permit unambiguous assignment of several important groups. We have further refined the structure of the complex of the enzyme with its cofactor, nicotinamide adenine dinucleotide (NAD), and solvent molecules, at the same resolution. RESULTS: The asymmetric unit of the crystal contains four monomers, each with 253 amino acid residues, 38 water molecules, and 176 cofactor atoms belonging to four NAD molecules--one for each subunit. The positioning of the cofactor molecule has been modified from our previous model and is deeper in the catalytic cavity as observed for other members of both the long-chain and short-chain dehydrogenase families. The nicotinamide-ribose end of the cofactor has several possible conformations or is dynamically disordered. CONCLUSIONS: The catalytic site contains residues Tyr152 and Lys156. These two amino acids are strictly conserved in the short-chain dehydrogenase superfamily. Modeling studies with a cortisone molecule in the catalytic site suggest that the Tyr152, Lys156 and Ser139 side chains promote electrophilic attack on the (C20-O) carbonyl oxygen atom, thus enabling the carbon atom to accept a hydride from the reduced cofactor.

Common themes in redox chemistry emerge from the X-ray structure of oilseed rape (Brassica napus) enoyl acyl carrier protein reductase.

BACKGROUND: Enoyl acyl carrier protein reductase (ENR) catalyzes the NAD(P)H-dependent reduction of trans-delta 2-enoyl acyl carrier protein, an essential step in de novo fatty acid biosynthesis. Plants contain both NADH-dependent and separate NADPH-dependent ENR enzymes which form part of the dissociable type II fatty acid synthetase. Highly elevated levels of the NADH-dependent enzyme are found during lipid deposition in maturing seeds of oilseed rape (Brassica napus). RESULTS: The crystal structure of an ENR-NAD binary complex has been determined at 1.9 A resolution and consists of a homotetramer in which each subunit forms a single domain comprising a seven-stranded parallel beta sheet flanked by seven alpha helices. The subunit has a topology highly reminiscent of a dinucleotide-binding fold. The active site has been located by difference Fourier analysis of data from crystals equilibrated in NADH. CONCLUSIONS: The structure of ENR shows a striking similarity with the epimerases and short-chain alcohol dehydrogenases, in particular, 3 alpha,20 beta-hydroxysteroid dehydrogenase (HSD). The similarity with HSD extends to the conservation of a catalytically important lysine that stabilizes the transition state and to the use of a tyrosine as a base--with subtle modifications arising from differing requirements of the reduction chemistry.

Crystallization and preliminary X-ray diffraction studies of a mammalian steroid dehydrogenase.

20 beta-Hydroxysteroid dehydrogenase from the cytosolic fraction of neonatal pig testis is a NADPH-dependent enzyme that catalyzes the reduction of the C-20 ketone of C21-steroids. It is 85% homologous in amino acid sequence to the human enzyme, carbonyl reductase. The enzyme has been crystallized from 36% saturated ammonium sulfate in 10 mM 2-[N-Morpholino]ethanesulfonic acid buffer. The size and the quality of nicely formed square bi-pyramidal crystals were improved by using a "seeding" technique. The crystals diffract X-rays to at least 2.5 A resolution. The space group is P4(1)2(1)2 (or P4(3)2(1)2) and the unit-cell dimensions are a = b = 58.53 A, c = 165.64 A. There is one molecule (M(r) = 30.5 kDa; 289 amino acid residues) in the asymmetric unit. An intensity data set to 2.5 A has been collected with an overall Rmerge of 6.6% for all reflections.

The rat 17 alpha-hydroxylase-17,20-desmolase (CYP17) active site: computerized homology modeling and site directed mutagenesis.

A homology model of the rat 17 alpha-hydroxylase-17,20 desmolase (CYP17) steroid binding domain was derived from the alpha/beta F supersecondary structural element of the 3 alpha/20 beta hydroxysteroid dehydrogenase (HSD) of Streptomyces hydrogenans that constitutes a major segment of the C19 steroid binding cavity. A CYP17 arginine-rich domain, including Arg346, Arg361 and Arg363, that has previously been shown to be important to CYP17 catalytic activity, is conserved in this HSD structural element between two HSD domains known to be important to C19 steroid binding. These two HSD motifs, in addition to a C-terminal domain at the apex of the steroid binding cavity, are also present in similar though not identical forms in the rat CYP17 sequence. The model was evaluated in terms of both hydroxylase/lyase activity and stability of CYP17 mutant proteins (Tyr334Phe, Phe343Ile, Arg357Ala, Arg361Ala, Asp345Ala), and further tested with mutagenesis of Glu353, Glu358, and Tyr431. Those amino acids located at folding junctions in the model steroid binding domain (Glu358, Arg361, and Tyr431) are each individually required to prevent degradation of the nascent protein, as well as for basic hydroxylase/lyase activity. Genomic analysis of the rat CYP17 gene reveals that this domain is contained in exon 6, and a correlation exists between the length of exon 6 and the boundaries of the HSD supersecondary element. These studies demonstrate that exon 6 of the rat CYP17 is essential for CYP17 activity, and may be structurally related to the NAD-linked prokaryote alpha/beta F supersecondary element.

Structure and function of steroid dehydrogenases involved in hypertension, fertility, and cancer.

Short-chain dehydrogenase reductase (SDR) enzymes influence mammalian reproduction, hypertension, neoplasia, and digestion. The three-dimensional structures of two members of the SDR family reveal the position of the conserved catalytic triad, a possible mechanism of keto-hydroxyl interconversion, the molecular mechanism of inhibition, and the basis for selectivity. Glycyrrhizic acid, the active ingredient in licorice, and its metabolite carbenoxolone are potent inhibitors of bacterial 3 alpha, 20 beta-hydroxysteroid dehydrogenase (3 alpha, 20 beta-HSD). The three-dimensional structure of the 3 alpha,20 beta-HSD carbenoxolone complex unequivocally verifies the postulated active site of the enzyme, shows that inhibition is a result of direct competition with the substrate for binding, and provides a plausible model for the mechanism of inhibition of 11 beta-hydroxysteroid dehydrogenase and 15-hydroxyprostaglandin dehydrogenase by carbenoxolone. The structure of human 17 beta-hydroxysteroid dehydrogenase type 1 (17 beta-HSD) suggests the details of binding of estrone and 17 beta-estradiol in the active site of the enzyme and the possible roles of various amino acids in the catalytic cleft. The SDR family includes over 50 proteins from human, mammalian, insect, and bacterial sources. Only five residues are conserved in all members of the family, including the YXXXK sequence. X-ray crystal structures of five members of the family have been completed. When the alpha-carbon backbone of the cofactor binding domains of the five structures are superimposed, the conserved residues are at the core of the structure and in the cofactor binding domain, but not in the substrate binding pocket.

Three-dimensional structure of holo 3 alpha,20 beta-hydroxysteroid dehydrogenase: a member of a short-chain dehydrogenase family.

The x-ray structure of a short-chain dehydrogenase, the bacterial holo 3 alpha,20 beta-hydroxysteroid dehydrogenase (EC 1.1.1.53), is described at 2.6 A resolution. This enzyme is active as a tetramer and crystallizes with four identical subunits in the asymmetric unit. It has the alpha/beta fold characteristic of the dinucleotide binding region. The fold of the rest of the subunit, the quaternary structure, and the nature of the cofactor-enzyme interactions are, however, significantly different from those observed in the long-chain dehydrogenases. The architecture of the postulated active site is consistent with the observed stereospecificity of the enzyme and the fact that the tetramer is the active form. There is only one cofactor and one substrate-binding site per subunit; the specificity for both 3 alpha- and 20 beta-ends of the steroid results from the binding of the steroid in two orientations near the same cofactor at the same catalytic site.

Steroid dehydrogenase structures, mechanism of action, and disease.

Steroid dehydrogenase enzymes influence mammalian reproduction, hypertension, neoplasia, and digestion. The three-dimensional structures of steroid dehydrogenase enzymes reveal the position of the catalytic triad, a possible mechanism of keto-hydroxyl interconversion, a molecular mechanism of inhibition, and the basis for selectivity. Glycyrrhizic acid, the active ingredient in licorice, and its metabolite carbenoxolone are potent inhibitors of human 11 beta-hydroxysteroid dehydrogenase and bacterial 3 alpha, 20 beta-hydroxysteroid dehydrogenase (3 alpha, 20 beta-HSD). The three-dimensional structure of the 3 alpha, 20 beta-HSD carbenoxolone complex unequivocally verifies the postulated active site of the enzyme, shows that inhibition is a result of direct competition with the substrate for binding, and provides a plausible model for the mechanism of inhibition of 11 beta-hydroxysteroid dehydrogenase by carbenoxolone. The structure of the ternary complex of human 17 beta-hydroxysteroid dehydrogenase type 1 (17 beta-HSD) with the cofactor NADP+ and the antiestrogen equilin reveals the details of binding of an inhibitor in the active site of the enzyme and the possible roles of various amino acids in the catalytic cleft. The short-chain dehydrogenase reductase (SDR) family includes these steroid dehydrogenase enzymes and more than 60 other proteins from human, mammalian, insect, and bacterial sources. Most members of the family contain the tyrosine and lysine of the catalytic triad in a YxxxK sequence. X-ray crystal structures of 13 members of the family have been completed. When the alpha-carbon backbone of the cofactor binding domains of the structures are superimposed, the conserved residues are at the core of the structure and in the cofactor binding domain, but not in the substrate binding pocket.

Isoleucine-15 of rainbow trout carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase is critical for coenzyme (NADPH) binding.

Carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase (CR/20beta-HSD) is an enzyme that converts 17alpha-hydroxyprogesterone to 17alpha, 20beta-dihydroxy-4-pregnen-3-one (the maturation-inducing hormone of salmonid fish). We have previously isolated two types of CR/20beta-HSD cDNAs from ovarian follicle of rainbow trout (Oncorhynchus mykiss). Recombinant proteins produced by expression in Escherichia coli in vitro showed that one (type A) had CR and 20beta-HSD activity but that the other (type B) did not. Among the three distinct residues between the protein products encoded by the two cDNAs, two residues (positions 15 and 27) are located in the N-terminal Rossmann fold, the coenzyme binding site. To investigate the structure/function relationships of CR/20beta-HSDs, we generated mutants by site-directed mutagenesis at the following positions: MutA/I15T, MutB/T15I, and MutB/Q27K. Enzyme activity of wild-type A was abolished by substitution of Ile-15 by Thr (MutA/I15T). Conversely, enzyme activity was acquired by the replacement of Thr-15 with Ile in type B (MutB/T15I). MutB/T15I mutant showed properties similar to the wild-type A in every aspect tested. Mutation MutB/Q27K had only partial enzyme activity, indicating that Ile-15 plays an important role in enzyme binding of cofactor NADPH.

Cloning of zebrafish ovarian carbonyl reductase-like 20 beta-hydroxysteroid dehydrogenase and characterization of its spatial and temporal expression.

20 beta-Hydroxysteroid dehydrogenase (20 beta-HSD) is a crucial enzyme that converts 17 alpha-hydroxyprogesterone to 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (DHP), which triggers oocyte maturation in most teleost fish. A full-length cDNA for a carbonyl reductase-like 20 beta-HSD (CR/20 beta-HSD) has been cloned from the zebrafish ovary. Although the zebrafish CR/20 beta-HSD is expressed in all of the tissues tested, it is predominantly expressed in the ovary, testis, kidney, and gill. In the ovary, the enzyme was shown to be expressed in the follicle cells and its expression appeared to be constitutive. No significant difference was noticed in the level of CR/20 beta-HSD expression among follicles of different stages. Furthermore, analysis of the ovarian samples taken at different times before spawning showed no significant change of the enzyme expression. In agreement with these results, treatment of the cultured zebrafish ovarian follicle cells with gonadotropin and activin had little effect on the expression of the enzyme. Taken together, these results point to the possibility that the gonadotropin-induced DHP production and final oocyte maturation in the zebrafish may not involve significant change of CR/20 beta-HSD expression as evidenced in the salmonids, or that there might be other isoforms of 20 beta-HSD whose expression is tightly controlled by endocrine and paracrine factors.

Short-chain dehydrogenases. Proteolysis and chemical modification of prokaryotic 3 alpha/20 beta-hydroxysteroid, insect alcohol and human 15-hydroxyprostaglandin dehydrogenases.

Prokaryotic 3 alpha/20 beta-hydroxysteroid dehydrogenase exhibits one segment sensitive to proteolysis with Glu-C protease and trypsin (cleaving after Glu192 and Arg196, respectively). Cleavage is associated with dehydrogenase inactivation; the presence of NADH offers almost complete protection and substrate (cortisone) gives some protection. Distantly related insect alcohol dehydrogenase is more resistant to proteolysis, but cleavage in a corresponding segment is detectable with Asp-N protease (cleaving before Asp198), while a second site (at Glu243) is sensitive to cleavage with both Glu-C and Asp-N proteases. Combined, the results suggest the presence of limited regions especially sensitive to proteolysis and the possibility of some association between the enzyme active site and the sensitive site(s). Modification of the hydroxysteroid dehydrogenase with tetranitromethane is paralleled by enzyme inactivation. With a 10-fold excess of reagent, labeling corresponds to 1.2 nmol Tyr/nmol protein chain and is recovered largely in Tyr152, with lesser amounts in Tyr251. Tetranitromethane also rapidly inhibits the other two dehydrogenases, but they contain Cys residues, preventing direct correlation with Tyr modification. Together, the proteolysis and chemical modifications highlight three segments of short-chain dehydrogenase subunits, one mid-chain, containing Tyr152 of the steroid dehydrogenase (similar numbers in the other enzymes), strictly conserved and apparently close to the enzyme active site, the other around position 195, sensitive to proteolysis and affected by coenzyme binding, while the third is close to the C-terminus.