Structure/function relationships responsible for coenzyme specificity and the isomerase activity of human type 1 3 beta-hydroxysteroid dehydrogenase/isomerase

Human type 1 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD/isomerase) catalyzes the two sequential enzyme reactions on a single protein that converts dehydroepiandrosterone or pregnenolone to androstenedione or progesterone, respectively, in placenta, mammary gland, breast tumors, prostate, prostate tumors, and other peripheral tissues. Our earlier studies show that the two enzyme reactions are linked by the coenzyme product, NADH, of the 3 beta-HSD activity. NADH activates the isomerase activity by inducing a time-dependent conformational change in the enzyme protein. The current study tested the hypothesis that the 3 beta-HSD and isomerase activities shared a common coenzyme domain, and it characterized key amino acids that participated in coenzyme binding and the isomerase reaction. Homology modeling with UDP-galactose-4-epimerase predicts that Asp36 is responsible for the NAD(H) specificity of human 3 beta-HSD/isomerase and identifies the Rossmann-fold coenzyme domain at the amino terminus. The D36A/K37R mutant in the potential coenzyme domain and the D241N, D257L, D258L, and D265N mutants in the potential isomerase domain (previously identified by affinity labeling) were created, expressed, and purified. The D36A/K37R mutant shifts the cofactor preference of both 3 beta-HSD and isomerase from NAD(H) to NADP(H), which shows that the two activities utilize a common coenzyme domain. The D257L and D258L mutations eliminate isomerase activity, whereas the D241N and D265N mutants have nearly full isomerase activity. Kinetic analyses and pH dependence studies showed that either Asp257 or Asp258 plays a catalytic role in the isomerization reaction. These observations further characterize the structure/function relationships of human 3 beta-HSD/isomerase and bring us closer to the goal of selectively inhibiting the type 1 enzyme in placenta (to control the timing of labor) or in hormone-sensitive breast tumors (to slow their growth).     

Structure/function of the inhibition of human 3beta-hydroxysteroid dehydrogenase type 1 and type 2 by trilostane

The human type 1 (placenta, breast tumors) and type 2 (gonads, adrenals) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) are key enzymes in biosynthesis of all active steroid hormones. Human 3beta-HSD1 is a critical enzyme in the conversion of DHEA to estradiol in breast tumors and may be a major target enzyme for the treatment of breast cancer. 3beta-HSD2 participates in the production of cortisol and aldosterone in the human adrenal gland. The goals of this project are to evaluate the role of the 2alpha-cyano group on trilostane (2alpha-cyano-4alpha,5alpha-epoxy-17beta-ol-androstane-3-one) and determine which amino acids may be critical for 3beta-HSD1 specificity. Trilostane without the 2alpha-cyano group, 4alpha,5alpha-epoxy-testosterone, was synthesized. Using our structural model of 3beta-HSD1, trilostane or 4alpha,5alpha-epoxy-testosterone was docked in the active site using Autodock 3.0, and the potentially critical residues (Met187 and Ser124) were identified. The M187T and S124T mutants of 3beta-HSD1 were created, expressed and purified. Dixon analyses of the inhibition of wild-type 3beta-HSD1, 3beta-HSD2, M187T and S124T by trilostane and 4alpha,5alpha-epoxy-testosterone suggest that the 2alpha-cyano group of trilostane is anchored by Ser124 in both isoenzymes. Kinetic analyses of cofactor and substrate utilization as well as the inhibition kinetics of M187T and the wild-type enzymes suggest that the 16-fold higher-affinity inhibition of 3beta-HSD1 by trilostane may be related to the presence of Met187 in 3beta-HSD1 and Thr187 in 3beta-HSD2. This structure/function information may lead to the production of more highly specific inhibitors of 3beta-HSD1 to block the hormone-dependent growth of breast tumors.     

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

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.     

Oxidative 3alpha-hydroxysteroid dehydrogenase activity of human type 10 17beta-hydroxysteroid dehydrogenase

In vitro enzyme assays have demonstrated that human type 10 17beta-hydroxysteroid dehydrogenase (17beta-HSD10) catalyzes the oxidation of 5alpha-androstane-3alpha,17beta-diol (adiol), an almost inactive androgen, to dihydrotestosterone (DHT) rather than androsterone or androstanedione. To further investigate the role of this steroid-metabolizing enzyme in intact cells, we produced stable transfectants expressing 17beta-HSD10 or its catalytically inactive Y168F mutant in human embryonic kidney (HEK) 293 cells. It was found that DHT levels in HEK 293 cells expressing 17beta-HSD10, but not its catalytically inactive mutant, will dramatically increase if adiol is added to culture media. Moreover, certain malignant prostatic epithelial cells have more 17beta-HSD10 than normal controls, and can generate DHT, the most potent androgen, from adiol. This event might promote prostate cancer growth. Analysis of the 17beta-HSD10 sequence shows that this enzyme does not have any ER retention signal or transmembrane segments and has not originated by divergence from a retinol dehydrogenase. The data suggest that the unique mitochondrial location of this HSD [Eur. J. Biochem. 268 (2001) 4899] does not prevent it from oxidizing the 3alpha-hydroxyl group of a C19 sterol in living cells. The experimental results lead to the conclusion that mitochondrial 17beta-HSD10 plays a significant part in a non-classical androgen synthesis pathway along with microsomal retinol dehydrogenases.     

Calcium inhibits human placental 11beta-hydroxysteroid dehydrogenase type 2 activity

The effect of Ca2+ on the conversion of cortisol to its inert metabolite cortisone, the reaction catalyzed by the microsomal enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), was investigated in human placental microsomes. Placental microsomal 11beta-HSD2 activity, as determined by the rate of conversion of cortisol to cortisone, was inhibited up to 50% by increasing free Ca2+ concentrations from 22 to 268 nM. The Ca2+-induced inhibition was reversible since chelation of endogenous Ca2+ with EGTA increased 11beta-HSD2 activity up to 200%. Ca2+ decreased the maximal velocity (Vmax) of the 11beta-HSD2 catalyzed conversion of cortisol to cortisone without altering the Km of 11beta-HSD2 for cortisol, indicating that Ca2+ modulates the catalytic efficiency rather than the substrate binding of 11beta-HSD2. Moreover, the Ca2+-induced inhibition does not appear to involve altered cofactor (NAD+) binding since the inhibition of microsomal 11beta-HSD2 activity by a sub-maximal concentration of free Ca2+ was not overcome by increasing the concentration of NAD+. These findings in the microsomes were then extended to an intact cell system, JEG-3 cells, an established model for human placental trophoblasts. In these cells, an increase in cytosolic free Ca2+ concentration ([Ca2+]i) elicited by a known physiological stimulus, PGF(2alpha), was accompanied by a 40% decrease in the level of 11beta-HSD2 activity. Furthermore, the PGF(2alpha)-induced inhibition of 11beta-HSD2 activity was abrogated when increases in [Ca2+]i were blocked with the intracellular Ca2+ chelator, BAPTA. Collectively, these results demonstrate for the first time that Ca2+ inhibits human placental 11beta-HSD2 activity by a post-translational mechanism not involving substrate or cofactor binding.     

Characterization of type 12 17beta-hydroxysteroid dehydrogenase, an isoform of type 3 17beta-hydroxysteroid dehydrogenase responsible for estradiol formation in women

A novel 17beta-hydroxysteroid dehydrogenase (17beta-HSD) chronologically named type 12 17beta-HSD (17beta-HSD12), that transforms estrone (E1) into estradiol (E2) was identified by sequence similarity with type 3 17beta-HSD (17beta-HSD3) that catalyzes the formation of testosterone from androstenedione in the testis. Both are encoded by large genes spanning 11 exons, most of them showing identical size. Using human embryonic kidney-293 cells stably expressing 17beta-HSD12, we have found that the enzyme catalyzes selectively and efficiently the transformation of E1 into E2, thus identifying its role in estrogen formation, in contrast with 17beta-HSD3, the enzyme involved in the biosynthesis of the androgen testosterone in the testis. Using real-time PCR to quantify mRNA in a series of human tissues, the expression levels of 17beta-HSD12 as well as two other enzymes that perform the same transformation of E1 into E2, namely type 1 17beta-HSD and type 7 17beta-HSD, it was found that 17beta-HSD12 mRNA is the most highly expressed in the ovary and mammary gland. To obtain a better understanding of the structural basis of the difference in substrate specificity between 17beta-HSD3 and 17beta-HSD12, we have performed tridimensional structure modelization using the coordinates of type 1 17beta-HSD and site-directed mutagenesis. The results show the potential role of bulky amino acid F234 in 17beta-HSD12 that blocks the entrance of androstenedione. Overall, our results strongly suggest that 17beta-HSD12 is the major estrogenic 17beta-HSD responsible for the conversion of E1 to E2 in women, especially in the ovary, the predominant source of estrogens before menopause.     

Identification of key amino acids responsible for the substantially higher affinities of human type 1 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD1) for substrates, coenzymes, and inhibitors relative to human 3beta-HSD2

The human type 1 (placenta, breast tumors, and prostate tumors) and type 2 (adrenals and gonads) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD1 and 3beta-HSD2) are encoded by two distinct genes that are expressed in a tissue-specific pattern. Our recent studies have shown that His156 contributes to the 14-fold higher affinity that 3beta-HSD1 exhibits for substrate and inhibitor steroids compared with human 3beta-HSD2 containing Tyr156 in the otherwise identical catalytic domain. Our structural model of human 3beta-HSD localizes His156 or Tyr156 in the subunit interface of the enzyme homodimer. The model predicts that Gln105 on one enzyme subunit has a higher probability of interacting with His156 on the other subunit in 3beta-HSD1 than with Tyr156 in 3beta-HSD2. The Q105M mutant of 3beta-HSD1 (Q105M1) shifts the Michaelis-Menten constant (Km) for 3beta-HSD substrate and inhibition constants (Ki) for epostane and trilostane to the much lower affinity profiles measured for wild-type 3beta-HSD2 and H156Y1. However, the Q105M2 mutant retains substrate and inhibitor kinetic profiles similar to those of 3beta-HSD2. Our model also predicts that Gln240 in 3beta-HSD1 and Arg240 in 3beta-HSD2 may be responsible for the 3-fold higher affinity of the type 1 isomerase activity for substrate steroid and cofactors. The Q240R1 mutation increases the isomerase substrate Km by 2.2-fold to a value similar to that of 3beta-HSD2 isomerase and abolishes the allosteric activation of isomerase by NADH. The R240Q2 mutation converts the isomerase substrate, cofactor, and inhibitor kinetic profiles to the 4-14-fold higher affinity profiles of 3beta-HSD1. Thus, key structural reasons for the substantially higher affinities of 3beta-HSD1 for substrates, coenzymes, and inhibitors have been identified. These structure and function relationships can be used in future docking studies to design better inhibitors of the 3beta-HSD1 that may be useful in the treatment of hormone-sensitive cancers and preterm labor.     

Flavonoid inhibition of overexpressed human 3beta-hydroxysteroid dehydrogenase type II

The inhibitory effects of various flavonoids on human 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase type II (3beta-HSD type II), overexpressed in baculovirus, were investigated, and the structure-inhibition relationship was examined. The isoflavone derivatives daidzein, genistein, formononetin and biochanin A inhibited 3beta-HSD type II activity at a concentration of 10 microM and of these, genistein was the most potent inhibitor. 6-Hydroxyflavone (6-HF), a synthetic flavone, also strongly inhibited 3beta-HSD activity but 5-HF, 7-HF and other natural flavones were less potent. Energy minimization structures of the flavonoids, as produced using MOE software, showed that isoflavones and flavones have an almost flat A-C ring structure, and that flavonoids that acted as inhibitors had similar steric structures to DHEA. Genistein, 6-HF and cyanoketone, which is known as a typical 3beta-HSD inhibitor, were found to act as competitive inhibitors with K(i) values of 0.12 microM, 0.19 microM and 0.67 nM, respectively. Furthermore, the LUMO (lowest unoccupied molecular orbital (LUMO)) values, as calculated using WinMOPAC (Fujitsu, Japan), of the inhibitors were correlated with the IC(50) values (r2 = 0.84). From these results, it appears that inhibitory effects of flavonoids are due to the combination of steric structure and electron affinity between the active center of 3beta-HSD type II and the flavonoid molecule.     

Novel enzymological profiles of human 11beta-hydroxysteroid dehydrogenase type 1

The human enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD) catalyzes the reversible oxidoreduction of 11beta-OH/11-oxo groups of glucocorticoid hormones. Besides this important endocrinological property, the type 1 isozyme (11beta-HSD1) mediates reductive phase I reactions of several carbonyl group bearing xenobiotics, including drugs, insecticides and carcinogens. The aim of this study was to explore novel substrate specificities of human 11beta-HSD1, using heterologously expressed protein in the yeast system Pichia pastoris. In addition to established phase I xenobiotic substrates, it is now demonstrated that transformed yeast strains catalyze the reduction of ketoprofen to its hydroxy metabolite, and the oxidation of the prodrug DFU-lactol to the pharmacologically active lactone compound. Purified recombinant 11beta-HSD1 mediated oxidative reactions, however, the labile reductive activity component could not be maintained. In conclusion, evidence is provided that human 11beta-HSD1 in vitro is involved in phase I reactions of anti-inflammatory non-steroidal drugs like ketoprofen and DFU-lactol.     

Novel non-steroidal inhibitors of human 11beta-hydroxysteroid dehydrogenase type 1

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) regulates glucocorticoid action at the pre-receptor stage by converting cortisone to cortisol. 11β-HSD1 is selectively expressed in many tissues including the liver and adipose tissue where metabolic events are important. Metabolic syndrome relates to a number of metabolic abnormalities and currently has a prevalence of >20% in adult Americans. 11β-HSD1 inhibitors are being investigated by many major pharmaceutical companies for type 2 diabetes and other abnormalities associated with metabolic syndrome. In this area of intense interest a number of structural types of 11β-HSD1 inhibitor have been identified. It is important to have an array of structural types as the physicochemical properties of the compounds will determine tissue distribution, HPA effects, and ultimately clinical utility. Here we report the discovery and synthesis of three structurally different series of novel 11β-HSD1 inhibitors that inhibit human 11β-HSD1 in the low micromolar range. Docking studies with 1–3 into the crystal structure of human 11β-HSD1 reveal how the molecules may interact with the enzyme and cofactor and give further scope for structure based drug design in the optimisation of these series.     

Characterization of a monoclonal antibody for human aldo-keto reductase AKR1C3 (type 2 3alpha-hydroxysteroid dehydrogenase/type 5 17beta-hydroxysteroid dehydrogenase); immunohistochemical detection in breast and prostate

Human aldo-keto reductase AKR1C3 (type 2 3alpha-hydroxysteroid dehydrogenase/type 5 17beta-hydroxysteroid dehydrogenase) catalyzes the reduction of Delta(4)-androstene-3,17-dione to yield testosterone, the reduction of 5alpha-dihydrotestosterone to yield 3alpha- and 3beta-androstanediol, and the reduction of estrone to yield 17beta-estradiol. Relatively, high mRNA expression of AKR1C3 was found in human prostate and mammary gland where it is implicated in regulating ligand access to the androgen and estrogen receptor, respectively. AKR1C3 shares high sequence identity >86% with related plastic human 20alpha-hydroxysteroid dehydrogenases (AKR1C1), type 3 3alpha-hydroxysteroid dehydrogenase (AKR1C2) and type 1 3alpha-hydroxysteroid dehydrogenase (AKR1C4), and reagents are urgently needed to discriminate between these enzymes at the mRNA, protein and functional level. We describe the characterization of a high-titer isoform specific monoclonal antibody (Ab) for AKR1C3. It does not cross react with human AKR1C1, AKR1C2 or AKR1C4, human aldehyde reductase AKR1A1 or rat 3alpha-hydroxysteroid dehydrogenase (AKR1C9) on immunoblot analysis. The monoclonal Ab can be used to detect AKR1C3 expression by immunohistochemistry in sections of paraffin-embedded mammary gland and prostate. In the breast enzyme staining was detected in ductal carcinoma in situ where the cancerous cells were strongly immunoreactive. In normal prostate immunoreactivity was limited to stromal cells with only faint staining in the epithelial cells. In adenocarcinoma of the prostate elevated staining was observed in the endothelial cells and carcinoma cells. The reagent thus has utility to access the localized expression of AKR1C3 in hormonal dependent malignancies of the breast and prostate.     

Rapid hepatic metabolism of 7-ketocholesterol by 11beta-hydroxysteroid dehydrogenase type 1: species-specific differences between the rat, human, and hamster enzyme

The role of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in the local activation of the glucocorticoid receptor by converting inactive 11-ketoglucocorticoids to active 11beta-hydroxyglucocorticoids is well established. Currently, 11beta-HSD1 is considered a promising target for treatment of obese and diabetic patients. Here, we demonstrate a role of 11beta-HSD1 in the metabolism of 7-ketocholesterol (7KC), the major dietary oxysterol. Comparison of recombinant 11beta-HSD1, transiently expressed in human embryonic kidney 293 cells, revealed the stereo-specific interconversion of 7KC and 7beta-hydroxycholesterol by rat and human 11beta-HSD1, whereas the hamster enzyme interconverted 7alpha-hydroxycholesterol, 7beta-hydroxycholesterol, and 7KC. In contrast to lysates, which efficiently catalyzed both oxidation and reduction, intact cells exclusively reduced 7KC. These findings were confirmed using rat and hamster liver homogenates, intact rat hepatocytes, and intact hamster liver tissue slices. Reduction of 7KC was abolished upon inhibition of 11beta-HSD1 by carbenoxolone (CBX) or 2'-hydroxyflavanone. In vivo, after gavage feeding rats, 7KC rapidly appeared in the liver and was converted to 7beta-hydroxycholesterol. CBX significantly decreased the ratio of 7beta-hydroxycholesterol to 7KC, supporting the evidence from cell culture experiments for 11beta-HSD1-dependent reduction of 7KC to 7beta-hydroxycholesterol. Upon inhibition of 11beta-HSD1 by CBX, 7KC tended to accumulate in the liver, and plasma 7KC concentration increased. Together, our results suggest that 11beta-HSD1 efficiently catalyzes the first step in the rapid hepatic metabolism of dietary 7KC, which may explain why dietary 7KC has little or no effect on the development of atherosclerosis.     

Proinflammatory cytokines inhibit human placental 11beta-hydroxysteroid dehydrogenase type 2 activity through Ca2+ and cAMP pathways

Excessive fetal exposure to glucocorticoids has been implicated in the etiology of adult metabolic and cardiovascular disease. Placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) may protect the fetus from excessive glucocorticoid exposure. Maternal stress may be accompanied by elevated levels of cortisol and increased proinflammatory cytokines [interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha (TNF-alpha)]. We hypothesize that proinflammatory cytokines inhibit human placental 11beta-HSD activity. We incubated explant cultures of term human placental villi in the presence or absence of 10 ng/ml IL-1beta, IL-6, or TNF-alpha, with or without agonists or antagonists of intracellular Ca2+ and adenylyl cyclase. Activity for 11beta-HSD2 was estimated using a radioisotope assay, and mRNA was measured using quantitative RT-PCR. All cytokines significantly (P < or = 0.05) reduced 11beta-HSD2 activity (>75% suppression); maximal inhibition occurred within 2 h and was maintained for at least 24 h. The IL-1beta-induced inhibitory activity was attenuated using a Ca2+ channel blocker (nifedipine), an intracellular Ca2+ antagonist [8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate], or the adenylyl cyclase stimulant forskolin. Conversely, 11beta-HSD2 activity was diminished in the presence of the Ca2+ ionophore A-23187 or the adenylyl cyclase inhibitor SQ-22536. mRNA levels for 11beta-HSD2 were not changed by any of the treatments. Proinflammatory cytokines inhibit human placental 11beta-HSD2 activity through a mechanism that involves increased intracellular Ca2+ and inhibition of adenylyl cyclase. This could result in excessive fetal exposure to maternal cortisol. This mechanism might mediate part of the increased risk of metabolic and cardiovascular disease in adult offspring.     

Structure/function of human type 1 3beta-hydroxysteroid dehydrogenase: An intrasubunit disulfide bond in the Rossmann-fold domain and a Cys residue in the active site are critical for substrate and coenzyme utilization

The human type 1 (placenta, breast tumors) and type 2 (gonads, adrenals) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) are key enzymes in steroidogenic pathways leading to the production of all active steroid hormones. Kinetic analyses of purified 3beta-HSD1 show that the Michaelis-Menten constants (Km) for substrates and cofactor are decreased dramatically (three- to eight-fold) by the addition of beta-mercaptoethanol (BME), which suggest that a disulfide bond may be critical to ligand utilization. Western immunoblots and SDS-PAGE of purified 3beta-HSD1 in the presence or absence of BME showed a lack of intersubunit disulfide bonds in the dimeric enzyme. The Rossmann-fold domain of 3beta-HSD1 contains two Cys residues, Cys72 and Cys111, which are capable of forming an intrasubunit disulfide bond based on their proximity in our structural model. Our structural model also predicts that Cys83 may affect the orientation of substrate and cofactor. To test these predictions, the C72S, C72F, C111S, C111A, C83S and C83A mutants of 3beta-HSD1 were produced, expressed, and purified. BME failed to diminish the Km values of substrate and cofactor for C72S, C72F, C111S and C111A but produced a 2.5 decrease in Km values for C83A ligands similar to wild-type 3beta-HSD. Thus, our results support the presence of an intrasubunit disulfide bond between Cys72 and Cys111 that participates in the tertiary structure of the Rossmann-fold domain. Although C83S had no enzyme activity, the C83A mutant enzyme exhibited two- to five-fold higher Km values for substrate and cofactor but had similar K(cat) values compared to wild-type 3beta-HSD. These data characterize the roles of Cys residues in 3beta-HSD and validate the predictions of our structural model.     

Characterization of human DHRS4: an inducible short-chain dehydrogenase/reductase enzyme with 3beta-hydroxysteroid dehydrogenase activity

Human DHRS4 is a peroxisomal member of the short-chain dehydrogenase/reductase superfamily, but its enzymatic properties, except for displaying NADP(H)-dependent retinol dehydrogenase/reductase activity, are unknown. We show that the human enzyme, a tetramer composed of 27 kDa subunits, is inactivated at low temperature without dissociation into subunits. The cold inactivation was prevented by a mutation of Thr177 with the corresponding residue, Asn, in cold-stable pig DHRS4, where this residue is hydrogen-bonded to Asn165 in a substrate-binding loop of other subunit. Human DHRS4 reduced various aromatic ketones and α-dicarbonyl compounds including cytotoxic 9,10-phenanthrenequinone. The overexpression of the peroxisomal enzyme in cultured cells did not increase the cytotoxicity of 9,10-phenanthrenequinone. While its activity towards all-trans-retinal was low, human DHRS4 efficiently reduced 3-keto-C₁₉/C₂₁-steroids into 3β-hydroxysteroids. The stereospecific conversion to 3β-hydroxysteroids was observed in endothelial cells transfected with vectors expressing the enzyme. The mRNA for the enzyme was ubiquitously expressed in human tissues and several cancer cells, and the enzyme in HepG2 cells was induced by peroxisome-proliferator-activated receptor α ligands. The results suggest a novel mechanism of cold inactivation and role of the inducible human DHRS4 in 3β-hydroxysteroid synthesis and xenobiotic carbonyl metabolism.     

Distinctive molecular inhibition mechanisms for selective inhibitors of human 11beta-hydroxysteroid dehydrogenase type 1

11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the NADPH dependent interconversion of inactive cortisone to active cortisol. Excess 11beta-HSD1 or cortisol leads to insulin resistance and metabolic syndrome in animal models and in humans. Inhibiting 11beta-HSD1 activity signifies a promising therapeutic strategy in the treatment of Type 2 diabetes and related diseases. Herein, we report two highly potent and selective small molecule inhibitors of human 11beta-HSD1. While compound 1, a sulfonamide, functions as a simple substrate competitive inhibitor, compound 2, a triazole, shows the kinetic profile of a mixed inhibitor. Co-crystal structures reveal that both compounds occupy the 11beta-HSD1 catalytic site, but present distinct molecular interactions with the protein. Strikingly, compound 2 interacts much closer to the cofactor NADP+ and likely modifies its binding. Together, the structural and kinetic analyses demonstrate two distinctive molecular inhibition mechanisms, providing valuable information for future inhibitor design.