UNIPROT:Q7LGC8 - FACTA Search

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Query: UNIPROT:Q7LGC8 (HSD)
3,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Within potential target cells, the actions of physiological glucocorticoids (cortisol and corticosterone) are modulated by isoforms of the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD). To date, two isoforms of 11 beta HSD have been cloned: 11 beta HSD1 acts predominantly as an NADP(H)-dependent reductase to generate active cortisol or corticosterone, and 11 beta HSD2 is a high affinity NAD(+)-dependent enzyme that catalyses the enzymatic inactivation of glucocorticoids. Whereas the regeneration of active glucocorticoids by 11 beta HSD1 has been implicated in the cellular mechanisms of pituitary function, ovulation and parturition, the enzymatic inactivation of cortisol and corticosterone by 11 beta HSD enzymes appears to be central to the protection of gonadal steroidogenesis, prevention of intra-uterine growth retardation, and lactation. Recent evidence indicates that follicular fluid contains endogenous modulators of cortisol metabolism by 11 beta HSD1, the concentrations of which are associated with the clinical outcome of assisted conception cycles and are altered in cystic ovarian disease. In conclusion, the two cloned isoforms of 11 beta HSD fulfil diverse roles in a wide range of reproductive processes from conception to lactation.
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PMID:Glucocorticoid metabolism and reproduction: a tale of two enzymes. 1452 25

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) behaves predominantly as an oxoreductase converting the receptor-inactive glucocorticoids to their active forms in vivo, while the type 2 isoform (11beta-HSD2) possesses only dehydrogenase activity and inactivates cortisol in human or corticosterone in rat. We determined enzyme activity of 11beta-HSD in rat lungs from fetus to adult, and examined whether 11beta-HSD1 exists in alveolar type II cells, the most important site for the synthesis of pulmonary surfactant in mature lungs, by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Enzyme activity of 11beta-HSD1 and 2 in lung tissue homogenate were determined as NADP(+)- and NAD(+)-dependent conversion of corticosterone to 11-dehydrocorticosterone, respectively. We found that 11beta-HSD1 activity was increased progressively from 21 days gestation to 7 weeks after birth. 11beta-HSD2 activity was significantly lower than that of 11beta-HSD1 throughout gestation and after birth. Immunoreactivity for 11beta-HSD1 was detected in the cytoplasm of the cells in the alveolar region of adult rats. Some of these expressing 11beta-HSD1 were considered to be alveolar type II cells, because of their cuboid shape and localization at the corner of the alveoli. RT-PCR demonstrated 11beta-HSD1 mRNA in isolated alveolar type II cells. Our results suggest that alveolar type II cells enhance intracellular glucocorticoid availability via 11beta-HSD1. 11beta-HSD1 in alveolar type II cells is thought of as an autocrine amplifier of glucocorticoid action in the lung.
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PMID:Expression of 11 beta-hydroxysteroid dehydrogenase type 1 in alveolar epithelial cells in rats. 1459 19

Prostaglandin H(2) (PGH(2)) formed from arachidonic acid is an unstable intermediate and is efficiently converted into more stable arachidonate metabolites (PGD(2), PGE(2), and PGF(2)) by the action of three groups of enzymes. Prostaglandin F synthase (PGFS) was first purified from bovine lung and catalyzes the formation of 9 alpha,11 beta-PGF(2) from PGD(2) and PGF(2)(alpha) from PGH(2) in the presence of NADPH. Human PGFS is 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) type II and has PGFS activity and 3 alpha-HSD activity. Human lung PGFS has been crystallized with the cofactor NADP(+) and the substrate PGD(2), and with the cofactor NADPH and the inhibitor rutin. These complex structures have been determined at 1.69 A resolution. PGFS has an (alpha/beta)(8) barrel structure. The cofactor and substrate or inhibitor bind in a cavity at the C-terminal end of the barrel. The cofactor binds deeply in the cavity and has extensive interactions with PGFS through hydrogen bonds, whereas the substrate (PGD(2)) is located above the bound cofactor and has little interaction with PGFS. Despite being largely structurally different from PGD(2), rutin is located at the same site of PGD(2), and its catechol and rhamnose moieties are involved in hydrogen bonds with PGFS. The catalytic site of PGFS contains the conserved Y55 and H117 residues. The carbonyl O(11) of PGD(2) and the hydroxyl O(13) of rutin are involved in hydrogen bonds with Y55 and H117. The cyclopentane ring of PGD(2) and the phenyl ring of rutin face the re-side of the nicotinamide ring of the cofactor. On the basis of the catalytic geometry, a direct hydride transfer from NADPH to PGD(2) would be a reasonable catalytic mechanism. The hydride transfer is facilitated by protonation of carbonyl O(11) of PGD(2) from either H117 (at low pH) or Y55 (at high pH). Since the substrate binding cavity of PGFS is relatively large in comparison with those of AKR1C1 and AKR1C2, PGFS (AKR1C3) could catalyze the reduction and/or oxidation reactions of various compounds over a relatively wide pH range.
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PMID:Crystal structure of human prostaglandin F synthase (AKR1C3). 1497 15

The aldo-keto reductase rabbit 20alpha-hydroxysteroid dehydrogenase (rb20alpha-HSD; AKR1C5) is less selective than other HSDs, since it exerts its activity both on androgens (C19 steroids) and progestins (C21 steroids). In order to identify the molecular determinants responsible for this reduced selectivity, binary (NADPH) and ternary (NADP(+)/testosterone) complex structures were solved to 1.32A and 2.08A resolution, respectively. Inspection of the cofactor-binding cavity led to the identification of a new interaction between side-chains of residues His222 and Lys270, which cover the central phosphate chain of the cofactor, reminiscent of the "safety-belt" found in other aldo-keto reductases. Testosterone is stabilized by a phenol/benzene tunnel composed of side-chains of numerous residues, among which Phe54, which forces the steroid to take up an orientation markedly contrasting with that found in HSD ternary complexes reported. Combining structural, site-directed mutagenesis, kinetic and fluorescence titration studies, we found that the selectivity of rb20alpha-HSD is mediated by (i) the relaxation of loop B (residues 223-230), partly controlled by the nature of residue 230, (ii) the nature of the residue found at position 54, and (iii) the residues found in the C-terminal tail of the protein especially the side-chain of the amino acid 306.
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PMID:Loop relaxation, a mechanism that explains the reduced specificity of rabbit 20alpha-hydroxysteroid dehydrogenase, a member of the aldo-keto reductase superfamily. 1512 23

3Alpha-hydroxysteroid dehydrogenases (3alpha-HSDs) catalyze the interconversion between 5alpha-dihydrotestosterone (5alpha-DHT), the most potent androgen, and 3alpha-androstanediol (3alpha-diol), a weak androgen metabolite. To identify the rate-determining step in this physiologically important reaction, rat liver 3alpha-HSD (AKR1C9) was used as the protein model for the human homologues in fluorescence stopped-flow transient kinetic and kinetic isotope effect studies. Using single and multiple turnover experiments to monitor the NADPH-dependent reduction of 5alpha-DHT, it was found that k(lim) and k(max) values were identical to k(cat), indicating that chemistry is rate-limiting overall. Kinetic isotope effect measurements, which gave (D)k(cat) = 2.4 and (D)2(O)k(cat) = 3.0 at pL 6.0, suggest that the slow chemical transformation is significantly rate-limiting. When the NADP(+)-dependent oxidation of 3alpha-diol was monitored, single and multiple turnover experiments showed a k(lim) and burst kinetics consistent with product release as being rate-limiting overall. When NAD(+) was substituted for NADP(+), burst phase kinetics was eliminated, and k(max) was identical to k(cat). Thus with the physiologically relevant substrates 5alpha-DHT plus NADPH and 3alpha-diol plus NAD(+), the slowest event is chemistry. R276 forms a salt-linkage with the phosphate of 2'-AMP, and when it is mutated, tight binding of NAD(P)H is no longer observed [Ratnam, K., et al. (1999) Biochemistry 38, 7856-7864]. The R276M mutant also eliminated the burst phase kinetics observed for the NADP(+)-dependent oxidation of 3alpha-diol. The data with the R276M mutant confirms that the release of the NADPH product is the slow event; and in its absence, chemistry becomes rate-limiting. W227 is a critical hydrophobic residue at the steroid binding site, and when it is mutated to alanine, k(cat)/K(m) for oxidation is significantly depressed. Burst phase kinetics for the NADP(+)-dependent turnover of 3alpha-diol by W227A was also abolished. In the W227A mutant, the slow release of NADPH is no longer observed since the chemical transformation is now even slower. Thus, residues in the cofactor and steroid-binding site can alter the rate-determining step in the NADP(+)-dependent oxidation of 3alpha-diol to make chemistry rate-limiting overall.
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PMID:Dissection of the physiological interconversion of 5alpha-DHT and 3alpha-diol by rat 3alpha-HSD via transient kinetics shows that the chemical step is rate-determining: effect of mutating cofactor and substrate-binding pocket residues on catalysis. 1537 43

During the peri-implantation period, molecular signaling between embryo and endometrium (layer of tissue lining the uterus lumen) is supposed to be crucial for the maintenance of pregnancy. To investigate embryo-induced alterations in the proteome of bovine endometrium in the preattachment period (day 18), we used monozygotic cattle twins (generated by embryo splitting) as a model eliminating genetic variability as a source for proteome differences. One of the twins was pregnant after the transfer of two in vitro produced blastocysts, while the corresponding twin received a sham-transfer and served as a nonpregnant control. The two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) analysis of the endometrium samples of three twin pairs (pregnant/nonpregnant) revealed four proteins with significantly higher abundance (p < 10(-9)) in each sample derived from the pregnant animals: Rho GDP dissociation inhibitor beta; 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD); soluble NADP(+)-dependent isocitrate dehydrogenase 1; and acyl-CoA-binding protein. To verify the accuracy of the 2-D DIGE quantification, the abundances of 20 alpha-HSD were quantified by a targeted cleavable isotope-coded affinity tag (ICAT) approach. The mass spectrometry-based ICAT quantification matched perfectly the results obtained by 2-D DIGE quantification, demonstrating the accuracy of our data. These results demonstrate that our model (monozygotic twins) in combination with the appropriate analytical tools is particularly suitable for the detection of the proteins involved in the embryo-maternal interactions.
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PMID:Holistic differential analysis of embryo-induced alterations in the proteome of bovine endometrium in the preattachment period. 1591 10

Rat liver AKR1C9 is the best-studied 3alpha-hydroxysteroid dehydrogenase (3alphaHSD) of the aldo-keto reductase superfamily. The physiologic function of AKR1C9 is to catalyze the reduction of 5alpha-androstane-17beta-ol-3-one (dihydrotestosterone) to 5alpha-androstane-3alpha,17beta-diol (androstanediol) rather than the reverse reaction, and all of the known AKR1C enzymes with 3alphaHSD activity also preferentially catalyze dihydrotestosterone reduction in intact cells. Because the utilization of pyridine-nucleotide cofactors NAD(P)(H) primarily governs the directional preference of HSD enzymes in intact cells, and because R276 participates in NADP(H) binding, we hypothesized that mutation of R276 would alter directional preference in intact cells. To test this model, we constructed stable lines of human embryonic kidney 293 cells expressing wild-type AKR1C9 and mutations R276M, R276G, and R276E. Mutations R276M and R276G retained reductive preference with slightly reduced magnitude compared with wild-type AKR1C9. NADPH depletion by glucose deprivation minimally altered the equilibrium steroid distribution for wild-type AKR1C9 but further reduced the reductive preference of mutations R276M and R276G. Mutation R276E, in contrast, showed an oxidative preference under all conditions. The intrinsic rates of the reductive and oxidative reactions for all four enzymes were similar at the functional equilibrium states. We conclude the R276 maximizes the reductive preference of AKR1C9 in intact cells and maintains this strong preference despite NADPH depletion; mutation R276E reverses the directional preference.
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PMID:Arginine 276 controls the directional preference of AKR1C9 (rat liver 3alpha-hydroxysteroid dehydrogenase) in human embryonic kidney 293 cells. 1654 77

Mammalian 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) is a member of the short chain dehydrogenase/reductase. It is a key steroidogenic enzyme that catalyzes the first step of the multienzyme pathway conversion of circulating dehydroepiandrosterone and pregnenolone to active steroid hormones. A three dimensional model of a ternary complex of human 3beta-HSD type 1 (3beta-HSD_1) with an NAD cofactor and androstenedione product has been developed based upon X-ray structures of the ternary complex of E. coli UDP-galactose 4-epimerase (UDPGE) with an NAD cofactor and substrate (PDB_AC: 1NAH) and the ternary complex of human type 1 17beta-hydroxysteroid dehydrogenase (17beta-HSD_1) with an NADP cofactor and androstenedione (PDB_AC: 1QYX). The dimeric structure of the enzyme was built from two monomer models of 3beta-HSD_1 by respective 3D superposition with A and B subunits of the dimeric structure of Streptococcus suis DTDP-D-glucose 4,6-dehydratase (PDB_AC: 1KEP). The 3D model structure of 3beta-HSD_1 has been successfully used for the rational design of mutagenic experiments to further elucidate the key substrate binding residues in the active site as well as the basis for dual function of the 3beta-HSD_1 enzyme. The structure based mutant enzymes, Asn100Ser, Asn100Ala, Glu126Leu, His232Ala, Ser322Ala and Asn323Leu, have been constructed and functionally characterized. The mutagenic experiments have confirmed the predicted roles of the His232 and Asn323 residues in recognition of the 17-keto group of the substrate and identified Asn100 and Glu126 residues as key residues that participate for the dehydrogenase and isomerization reactions, respectively.
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PMID:Rational proteomics V: structure-based mutagenesis has revealed key residues responsible for substrate recognition and catalysis by the dehydrogenase and isomerase activities in human 3beta-hydroxysteroid dehydrogenase/isomerase type 1. 1688 58

Very recently, the mouse 17alpha-hydroxysteroid dehydrogenase (m17alpha-HSD), a member of the aldo-keto reductase (AKR) superfamily, has been characterized and identified as the unique enzyme able to catalyze efficiently and in a stereospecific manner the conversion of androstenedione (Delta4) into epitestosterone (epi-T), the 17alpha-epimer of testosterone. Indeed, the other AKR enzymes that significantly reduce keto groups situated at position C17 of the steroid nucleus, the human type 3 3alpha-HSD (h3alpha-HSD3), the human and mouse type 5 17beta-HSD, and the rabbit 20alpha-HSD, produce only 17beta-hydroxy derivatives, although they possess more than 70% amino acid identity with m17alpha-HSD. Structural comparisons of these highly homologous enzymes thus offer an excellent opportunity of identifying the molecular determinants responsible for their 17alpha/17beta-stereospecificity. Here, we report the crystal structure of the m17alpha-HSD enzyme in its apo-form (1.9 A resolution) as well as those of two different forms of this enzyme in binary complex with NADP(H) (2.9 A and 1.35 A resolution). Interestingly, one of these binary complex structures could represent a conformational intermediate between the apoenzyme and the active binary complex. These structures provide a complete picture of the NADP(H)-enzyme interactions involving the flexible loop B, which can adopt two different conformations upon cofactor binding. Structural comparison with binary complexes of other AKR1C enzymes has also revealed particularities of the interaction between m17alpha-HSD and NADP(H), which explain why it has been possible to crystallize this enzyme in its apo form. Close inspection of the m17alpha-HSD steroid-binding cavity formed upon cofactor binding leads us to hypothesize that the residue at position 24 is of paramount importance for the stereospecificity of the reduction reaction. Mutagenic studies have showed that the m17alpha-HSD(A24Y) mutant exhibited a completely reversed stereospecificity, producing testosterone only from Delta4, whereas the h3alpha-HSD3(Y24A) mutant acquires the capacity to metabolize Delta4 into epi-T.
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PMID:Crystal structures of mouse 17alpha-hydroxysteroid dehydrogenase (apoenzyme and enzyme-NADP(H) binary complex): identification of molecular determinants responsible for the unique 17alpha-reductive activity of this enzyme. 1703 17

Reduction of 17-ketosteroids is a biocatalytic process of economic significance for the production of steroid drugs. This reaction can be catalyzed by different microbial 17beta-hydroxysteroid dehydrogenases (17beta-HSD), like the 17beta-HSD activity of Saccharomyces cerevisiae, Pichia faranosa and Mycobacterium sp., and by purified 3beta,17beta-HSD from Pseudomonas testosteroni. In addition to the bacterial 3beta,17beta-HSD the 17beta-HSD of the filamentous fungus Cochliobolus lunatus is the only microbial 17beta-HSD that has been expressed as a recombinant protein and fully characterized. On the basis of its modeled 3D structure, we selected several positions for the replacement of amino acids by site-directed mutagenesis to change substrate specificity, alter coenzyme requirements, and improve overall catalytic activity. Replacement of Val161 and Tyr212 in the substrate-binding region by Gly and Ala, respectively, increased the initial rates for the conversion of androstenedione to testosterone. Replacement of Tyr49 within the coenzyme binding site by Asp changed the coenzyme specificity of the enzyme. This latter mutant can convert the steroids not only in the presence of NADP(+) and NADPH, but also in the presence of NADH and NAD(+). The replacement of His164, located in the non-flexible part of the 'lid' covering the active center resulted in a conformation of the enzyme that possessed a higher catalytic activity.
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PMID:Rational design of novel mutants of fungal 17beta-hydroxysteroid dehydrogenase. 1719 85

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