Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.1.1.3 (HSD)
3,464 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

3 beta-Hydroxy-5-ene-steroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD) is a NAD(+)-dependent membrane-bound enzyme that catalyzes the oxidation of delta 5-3 beta-hydroxysteroids to delta 4-3-keto structures during adrenal, gonadal, and placental steroidogenesis. Enzyme activity is located in both microsomes and mitochondria. In these experiments we examined the membrane topologies of 3 beta HSD in rat and calf adrenal microsomes and mitochondria by comparing access to the active sites of coenzyme and the inhibitor mersalyl, a nonpenetrant organic mercurial anion. Microsomal activity required exogenous NAD+ and was inhibited by mersalyl, indicating that the active site faced the medium in vitro and the cytoplasm in vivo. In contrast, mitochondrial 3 beta HSD used matrix space NAD+, was inhibited by reduction of intramitochondrial NAD(P)+, and was insensitive to mersalyl. Mitochondrial activity was decreased by exogenous NADH (apparent Ki, 2.8 microM) and increased by added NAD+ (apparent Ka, 2.4 microM). However, mersalyl blocked the effects of exogenous NADH and NAD+ and returned the activity to that observed before coenzyme addition. The membrane-sidedness of the NAD+ activation was examined further in submitochondrial particles prepared by sonication of pyridine nucleotide-depleted calf adrenal cortex mitochondria. Particles were prepared in the absence or presence of 10 mM NAD+ and contained none or 2.9-7.3 nmol NAD+/mg protein, respectively. Both groups of submitochondrial particles required exogenous NAD+ for 3 beta HSD activity, indicating that the active site faced the medium (the particles were everted), and the contained NAD+ was inside the particles. However, 3 beta HSD activity was increased 12-140% in particles that contained NAD+. The results suggest that mitochondrial 3 beta HSD is an integral inner membrane protein, that the active site faces the matrix space and is influenced by coenzyme availability, and that a regulatory site(s) faces the intermembrane space. Binding of NAD+ or NADH to this external site increases or decreases, respectively, the rate of catalysis at the active site. Mitochondrial 3 beta HSD activity may be enhanced by oxidation of intermembrane space NADH via an active rotenone- and antimycin-a-insensitive NADH oxidase.
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PMID:Topology of 3 beta-hydroxy-5-ene-steroid dehydrogenase/delta 5-delta 4-isomerase in adrenal cortex mitochondria and microsomes. 829 70

The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-Isomerase (3 beta HSD) catalyzes the conversion of delta 5-3 beta-hydroxysteroids to delta 4-3-ketosteroids, an essential step in the biosynthesis of all biologically active steroid hormones. We previously reported the isolation of three distinct mouse cDNAs for 3 beta HSD (3 beta HSD I, II, and III) and tissue-specific expression of their mRNAs. 3 beta HSD I is expressed only in gonads and adrenal glands, and 3 beta HSD II and III are expressed in both liver and kidneys. In the current study, we present data which demonstrate that transiently expressed 3 beta HSD I and 3 beta HSD III proteins can catalyze the conversion of the delta 5-steroids, pregnenolone and dehydroepiandrosterone, to their respective delta 4-3-ketosteroids, progesterone and androstenedione. They also can dehydrogenate the 3 beta-hydroxy group of the 5 alpha-reduced steroid 5 alpha-androstanediol to yield dihydrotestosterone in the presence of the cofactor NAD+. The Km values of the expressed 3 beta HSD I (for each of these substrates) were all below 0.2 microM. Km values of 3 beta HSD III were greater for all substrates, with the greatest increase observed for pregnenolone, which was over 10-fold greater. Both forms of expressed protein can catalyze the reduction of dihydrotestosterone to 5 alpha-androstanediol in the presence of the cofactor NADH, but with considerably higher Km values (5.5 microM for form I and 6.8 microM for form III). The observed maximum velocity of form I was much higher for all substrates examined. RNase protection and immunoblot analysis of expressed 3 beta HSD I and III indicate that the difference in maximum velocity reflect differences in the steady state levels of mRNA and amounts of protein. In addition, the expressed 3 beta HSD III protein analyzed by Western blot has a lower mobility than the 3 beta HSD I protein, both similar in mol wt to the 3 beta HSD proteins detected in mouse liver and adrenal glands, respectively. These data demonstrate that an isoform of 3 beta HSD expressed in liver and kidney has the capacity to convert delta 5-3 beta-hydroxysteroids to delta 4-3-ketosteroids. The data suggest that a homologous human 3 beta HSD isoform could play an important role in cases of genetic deficiency of the gonadal and adrenal isoform.
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PMID:Enzyme characteristics of two distinct forms of mouse 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase complementary deoxyribonucleic acids expressed in COS-1 cells. 847 48

The prostaglandin-E2 9-reductase (PGE2 9-reductase) activity in the corpus luteum of rabbits corresponds to a cytosolic, NADPH-dependent enzyme with a molecular mass of 36 kDa. This enzyme was purified from corpora lutea on day 12 of pseudopregnancy with a 266-fold enrichment. The main purification step was affinity chromatography using Red Sepharose CL-6B. The efficiency of this column was improved by elution with 1 mM NADH prior to elution of the active fractions with 1 mM NADPH. Amino acid sequence data demonstrate that the rabbit luteal PGE2 9-reductase has to be classified as a member of the aldo-keto reductase superfamily. The enzyme revealed a wide substrate specificity comprising the reduction of aldehydes, ketones, and quinones. Apparent kinetic constants were determined using methylglyoxal, DL-glyceraldehyde, and 9,10-phenanthrenquinone as substrates. The fully purified enzyme showed two catalytic activities of particular interest: PGE2 9-reductase and 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activities. The competitive inhibition of 20 alpha-HSD activity by PGE2 indicates that progesterone and PGE2 are substrates for the same enzyme. From these results, we conclude that prostaglandin and steroid metabolism are tightly linked to each other. For this reason the aldo-keto reductase could be a key enzyme in the cascade of events leading to the regression of the corpus luteum in the rabbit.
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PMID:Prostaglandin-E2 9-reductase from corpus luteum of pseudopregnant rabbit is a member of the aldo-keto reductase superfamily featuring 20 alpha-hydroxysteroid dehydrogenase activity. 852 51

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) modulates glucocorticoid interactions with mineralocorticoid and glucocorticoid receptors in vivo, by converting 11 beta-hydroxyglucocorticoids to their inactive 11-ketone derivatives. Defective 11 beta-oxidation of glucocorticoids has been associated with hypertension. The objective of this study was to investigate whether 11 beta-HSD contributes to the occurrence of hypertension in spontaneously hypertensive rats (SHRs). The liver and kidney microsomal oxidations of corticosterone (the physiological glucocorticoid in rats) in organs from juvenile (3 weeks old) and adult (3 months old) SHR and Wistar-Kyoto (WKY) rats, with NAD and NADP, show no differences between rat strains. For cortisol, with NADP, adult SHRs show (1.3-3 times; P < 0.05) lower kidney microsomal oxidation rates. The liver microsomal reduction of cortisone shows remarkable interstrain differences; with NADH, reduction is conducted only by adult WKY rats, whereas with NADPH, juvenile animals show similar reduction rates, but at adulthood, only WKYs reduce cortisone. Using Western blot analysis with antibodies against 11 beta-HSD1, positive signals are obtained only for liver microsomes, appearing somewhat lower in SHRs for juvenile but not adult animals. Urinary corticosterone/11-dehydrocorticosterone ratios (measured in adult animals) are not different between rat strains, but are elevated after administration of corticosterone in both strains (although significant only in SHRs). The data provide no indications for exaggerated stimulation of renal corticosteroid receptors, due to modified 11 beta-HSD, in SHRs. However, the experiments suggest the existence of multiple 11 beta-HSDs, in addition to 11 beta-HSD1 and 11 beta-HSD2, some of which may be modified in SHR, but the nature and physiological role of these 11 beta-HSDs is unclear.
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PMID:Comparison of 11 beta-hydroxysteroid dehydrogenase in spontaneously hypertensive and Wistar-Kyoto rats. 858 2

7 alpha-Hydroxysteroid dehydrogenase (7 alpha-HSDH;1 EC 1.1.1.159) is an NAD+-dependent oxidoreductase belonging to the short-chain dehydrogenase/reductase (SDR) 1 family. It catalyzes the dehydrogenation of a hydroxyl group at position 7 of the steroid skeleton of bile acids. The crystal structure of the binary (complexed with NAD+) complex of 7 alpha-HSDH has been solved at 2.3 A resolution by the multiple isomorphous replacement method. The structure of the ternary complex [the enzyme complexed with NADH, 7-oxoglycochenodeoxycholic acid (as a reaction product), and possibly partially glycochenodeoxycholic acid (as a substrate)] has been determined by a difference Fourier method at 1.8 A resolution. The enzyme 7 alpha-HSDH is an alpha/beta doubly wound protein having a Rossmann-fold domain for NAD (H) binding. Upon substrate binding, large conformation changes occur at the substrate binding loop (between the beta F strand and alpha G helix) and the C-terminal segment (residues 250-255). The variable amino acid sequences of the substrate-binding loop appear to be responsible for the wide variety of substrate specificities observed among the enzymes of the SDR family. The crystal structure of the ternary complex of 7 alpha-HSDH, which is the only structure available as the ternary complex among the enzymes of the SDR family, indicates that the highly conserved Tyr159 and Ser146 residues most probably directly interact with the hydroxyl group of the substrates although this observation cannot be definite due to an insufficiently characterized nature of the ternary complex. The strictly conserved Lys163 is hydrogen-bonded to both the 2'- and 3'-hydroxyl groups of the nicotinamide ribose of NAD(H). We propose a new catalytic mechanism possibly common to all the enzymes belonging to the SDR family in which a tyrosine residue (Tyr159) acts as a catalytic base and a serine residue (Ser146) plays a subsidiary role of stabilizing substrate binding.
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PMID:Crystal structures of the binary and ternary complexes of 7 alpha-hydroxysteroid dehydrogenase from Escherichia coli. 867 72

Benign meningioma tumors possess significant levels of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity. Two different 17 beta-HSDs were discovered in human placenta: one highly estrogen specific and using NADP+/NADPH as cofactors (type-1 17 beta-HSD), and a second one that utilizes both androgens and estrogens as substrates with NAD+/NADH (type-2 17 beta-HSD). Recently, two further human 17 beta-HSDs were isolated. A testis-specific 17 beta-HSD (type-3 17 beta-HSD) favors the reduction of delta 4-androstenedione to testosterone, and a ubiquitously expressed type-4 17 beta-HSD preferentially catalyzes the oxidation of estradiol and delta 5-androstenediol. In this study we characterize the expression levels of different types of 17 beta-HSD in a wide series of tumors. Using the Northern blotting method we show that type-1, -3, and -4 17 beta-HSDs are not detectable in meningiomas. In contrast, the type-2 17 beta-HSD RNA is present in 6 of 17 meningiomas and its abundance is directly correlated with estrogenic 17 beta-HSD activity (r2 = 0.74). The presence of type-2 17 beta-HSD is also demonstrated by in situ hybridization. RT-PCR and Western blots show that type-4 17 beta-HSD is also present, though at much lower levels. The progesterone receptor level, the epidermal growth factor receptor level, and the age of the patients are not correlated with the estrogenic 17 beta-HSD activity or type-2 17 beta-HSD mRNA expression level.
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PMID:17 beta-Hydroxysteroid dehydrogenase activity correlates with the type-2 17 beta-hydroxysteroid dehydrogenase mRNA abundance in human meningioma tumors. 881 69

Mineralocorticoid receptor (MR) selectivity for aldosterone is thought to be exerted by enzymes which inactivate competing glucocorticoids before they bind the receptor. Two different 11 beta-hydroxysteroid dehydrogenases (11 beta-HSD) have been described. 11 beta-HSD-1 is NADP(+)-dependent and has a Km in the micromolar range and bidirectional activity. 11 beta-HSD-2 is NAD(+)-dependent, has a Km in the nanomolar range, exhibits only oxidase activity, and colocalizes with the MR in the kidney, so is likely to serve as the gatekeeper for the MR. We have further characterized 11 beta-HSD activity in JEG-3 cells, a cell line derived from a human choriocarcinoma which was reported to have only the high affinity, NAD(+)-dependent 11 beta-HSD-2. We found that the Km for the conversion of corticosterone to 11-dehydrocorticosterone in intact cells and homogenates was about 16 nM. NAD(+)-dependent corticosterone conversion was equal in the nuclear and mitochondrial fractions and less, but significant, in the microsomal fraction. A high affinity, Km = 40 nM, NADP(+)-dependent enzyme was also found in homogenates. The subcellular distribution of this high affinity activity was greatest in the mitochondria, less in the nuclei, and even less, but still significant, in microsomes. Because of its cofactor dependency, high affinity, and different subcellular distribution, we suggest that this enzyme is neither the 11 beta-HSD-1 nor the 11 beta-HSD-2 and have named it 11 beta-HSD-3. Conversion of 11-dehydrocorticosterone to corticosterone did not occur in intact cells or in homogenates incubated with NADH or NADPH. Enzyme activity in intact cells was inhibited by glycyrrhetinic acid, carbenoxolone, progesterone, 5 beta-dihydroprogesterone, and 5 alpha-dihydroprogesterone, but not bile acids.
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PMID:11 beta-hydroxysteroid dehydrogenases of the choriocarcinoma cell line JEG-3 and their inhibition by glycyrrhetinic acid and other natural substances. 885 27

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

Recently, two distinct isoenzymes of 11beta-hydroxysteroid-dehydrogenase (11beta-HSD) have been cloned and characterized in several species: The isoenzyme 11beta-HSD-I is widely distributed, bidirectional, prefers NADP(H) and has a low substrate affinity. The isoenzyme 11beta-HSD-II seems to exclusively oxidize physiological glucocorticoids, uses NAD as cosubstrate, has high substrate affinity, and is only found in mineralocorticoid target tissues and the placenta. Synthetic steroids fluorinated in position 9, however, are rapidly reduced by human kidney cortex slices. We attempted to find out which isoenzyme is responsible for this unexpected reductase activity. We studied the 11beta-HSD activity towards cortisol (F)/cortisone (E) and dexamethasone (D)/11-dehydro-dexamethasone (DH-D) in microsomes prepared from human kidney cortex. For the reaction E to F (not for DH-D to D!), glucose-6-phosphate and glucose-6-phosphate-dehydrogenase had to be added as a NADH/NADPH-regenerating system. Oxidation of F to E: NAD was the exclusively used cosubstrate; the affinity [Michael's constant (Km) for F = 25.5 nmol/L] and the maximum velocity (Vmax = 22.9 nmol/mg/min) were high. Reduction of E to F: Without the NADH/NADPH-regenerating system, this reaction was very slow. With this system, the Km value for E was in the nanomolar range (80.6 nmol/L) and the Vmax value was very low (0.88 nmol/mg/min). The reaction was clearly NADH-preferring. For the steroid pair F/E, the quotient Vmax(oxidation)/Vmax(reduction) (=26) demonstrates an equilibrium far on the 11-keto side. Oxidation of D to DH-D: With NAD as the only used cosubstrate, the kinetic analysis is compatible with the existence of two different NAD-dependent isoenzymes: Km for D = 327 nmol/L, Vmax = 53.5 nmol/mg/min and Km for D = 81.2 nmol/L; Vmax = 20.4 nmol/mg/min. Reduction of DH-D to D: The maximum velocity was higher than that of all other reactions tested: Vmax = 226.0 nmol/mg/min. The reaction was exclusively NADH-dependent; the Km value for DH-D was 68.4 nmol/L. For D/DH-D, the ratio Vmax(oxidation)/Vmax(reduction) was 0.24, demonstrating a shift to reductase activity with the reaction equilibrium far on the 11-hydroxy side. The reaction F to E was inhibited by E, DH-D, and D in a concentration-dependent manner. In conclusion, the cosubstrate dependence, the Km value of the oxidation of F and the product inhibition are in good correspondence with data for the cloned human 11beta-HSD-II. The NADH-dependent 11beta-reduction of E and especially of DH-D are inconsistent with the dogma of an unidirectional 11beta-HSD-II. The preference of D for the reductase reaction in human kidney slices is probably caused by the fluor atom in position 9, is catalyzed by 11beta-HSD-II, and leads to an activation of 11-DH-D to D in the human kidney.
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PMID:Metabolism of dexamethasone in the human kidney: nicotinamide adenine dinucleotide-dependent 11beta-reduction. 914 56

In the human and in rodents like the rat and mouse, the liver enzyme 11 beta-hydroxysteroid dehydrogenase type I (11 beta-HSD-I) is a functional oxidoreductase preferring NADP+/NADPH as cosubstrate, while the renal isoenzyme (11 beta-HSD-II) prefers NAD+ as cosubstrate, and seems to be a pure oxidase and protects the tubular, mineralocorticoid (MC) receptor from occupancy by cortisol and corticosterone. We studied the enzyme kinetics of 11 beta-HSDs in kidney and liver microsomes of the guinea pig, a species whose zoological classification is still a matter of debate. With a fixed concentration of 10(-6) mol/l cortisol, liver and kidney microsomes preferred NAD+ to NADP+ (10(-3) mol/l) for the conversion to cortisone. Kidney microsomes converted cortisol to cortisone with K(m) values of 0.64 mumol/l and 9.8 mumol/l with NAD+ and NADP+ as cosubstrates respectively. The reduction of cortisone to cortisol was slow with kidney microsomes, but could be markedly enhanced by adding an NADH/NADPH regenerating system: with NADPH as preferred cosubstrate, the approximate K(m) was 7.2 mumol/l. This indicated the existence of both isoenzymes in the guinea pig kidney. Liver microsomes oxidized cortisol to cortisone with similar K(m) and Vmax values for NAD+ to NADP+ as cosubstrates (K(m) of 4.3 mumol/l and 5.0 mumol/l respectively). The NAD+ preference for the oxidation of 10(-6) mol/l cortisol described above may be due to a second, NAD(+)-preferring 11 beta-HSD with a K(m) of 1.4 mumol/l. In contrast to the kidney, liver microsomes actively converted cortisone to cortisol with a preference for NADPH (K(m): 1.2 mumol/l; Vmax: 467 nmol/min per mg protein). Thus, the main liver enzyme is similar to the oxidoreductase of other species (11 beta-HSD-I) and is also present in the kidney, while the main kidney enzyme is clearly NAD(+)-preferring. This kidney enzyme (analogous to 11 beta-HSD-II of other species) seems to be suitable for the protection of the MC receptor from the high free plasma cortisol levels of the guinea pig.
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PMID:Evidence for isoforms of 11 beta-hydroxysteroid dehydrogenase in the liver and kidney of the guinea pig. 916 19


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