Publications

• Human glutathione transferase A3-3, a highly efficient catalyst of double-bond isomerization in the biosynthetic pathway of steroid hormones.

  Johansson A.-S., Mannervik B.

  The cDNA of a novel human glutathione transferase (GST) of the Alpha class was cloned, and the corresponding protein, denoted GST A3-3, was heterologously expressed and characterized. GST A3-3 was found to efficiently catalyze obligatory double-bond isomerizations of Delta(5)-androstene-3,17-dione ... >> More

  The cDNA of a novel human glutathione transferase (GST) of the Alpha class was cloned, and the corresponding protein, denoted GST A3-3, was heterologously expressed and characterized. GST A3-3 was found to efficiently catalyze obligatory double-bond isomerizations of Delta(5)-androstene-3,17-dione and Delta(5)-pregnene-3,20-dione, precursors to testosterone and progesterone, respectively, in steroid hormone biosynthesis. The catalytic efficiency (k(cat)/K(m)) with Delta(5)-androstene-3,17-dione was determined as 5 x 10(6) m(-1) s(-1), which is considerably higher than with any other GST substrate tested. The rate of acceleration afforded by GST A3-3 is 6 x 10(8) based on the ratio between k(cat) and the rate constant for the nonenzymatic isomerization of Delta(5)-androstene-3,17-dione. Besides being high in absolute numbers, the k(cat)/K(m) value of GST A3-3 exceeds by a factor of approximately 230 that of 3beta-hydroxysteroid dehydrogenase/isomerase, the enzyme generally considered to catalyze the Delta(5)-Delta(4) double-bond isomerization. Furthermore, GSTA3-specific polymerase chain reaction analysis of cDNA libraries from various tissues showed a message only in those characterized by active steroid hormone biosynthesis, indicating a selective expression of GST A3-3 in these tissues. Based on this finding and the high activity with steroid substrates, we propose that GST A3-3 has evolved to catalyze isomerization reactions that contribute to the biosynthesis of steroid hormones. << Less

  J. Biol. Chem. 276:33061-33065(2001) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• A novel missense mutation in the HSD3B2 gene, underlying nonsalt-wasting congenital adrenal hyperplasia. new insight into the structure-function relationships of 3beta-hydroxysteroid dehidrogenase type II.

  Baquedano M.S., Ciaccio M., Marino R., Perez Garrido N., Ramirez P., Maceiras M., Turjanski A., Defelipe L.A., Rivarola M.A., Belgorosky A.

  <h4>Context</h4>3βHSD2 is a bifunctional microsomal NAD+-dependent enzyme crucial for adrenal and gonad steroid biosynthesis, converting Δ5-steroids to Δ4-steroids. 3βHSD2 deficiency is a rare cause of congenital adrenal hyperplasia caused by recessive loss-of-function HSD3B2 mutations.<h4>Objecti ... >> More

  <h4>Context</h4>3βHSD2 is a bifunctional microsomal NAD+-dependent enzyme crucial for adrenal and gonad steroid biosynthesis, converting Δ5-steroids to Δ4-steroids. 3βHSD2 deficiency is a rare cause of congenital adrenal hyperplasia caused by recessive loss-of-function HSD3B2 mutations.<h4>Objective</h4>The aim was to define the pathogenic consequences of a novel missense mutation in the HSD3B2 gene.<h4>Patient</h4>We report a 7-month-old 46,XX girl referred because of precocious pubarche and postnatal clitoromegaly. Hormonal profile showed inadequate glucocorticoid levels, increased 17OHP and renin levels, and very high DHEAS levels, suggestive of compensated nonsalt-losing 3βHSD2 deficiency.<h4>Design and results</h4>Direct sequencing revealed a novel, homozygous, pG250V HSD3B2 mutation. In vitro analysis in intact COS-7 cells showed impaired enzymatic activity for the conversion of pregnenolone to progesterone and dehydroepiandrosterone to androstenedione (20% and 27% of WT at 6 h, respectively). G250V-3βHSD2 decreased the Vmax for progesterone synthesis without affecting the Km for pregnenolone. Western blot and immunofluorescence suggested that p.G250V mutation has no effect on the expression and intracellular localization of the mutant protein. Molecular homology modeling predicted that mutant V250 affected an L239-Q251 loop next to a β-sheet structure in the NAD+-binding domain.<h4>Conclusions</h4>We identified a novel p.G250V mutation of HSD3B2 which causes an incomplete loss of enzymatic activity, explaining the compensated nonsalt loss phenotype. In vitro and in silico experiments provided insight into the structure-function relationship of the 3βHSD2 protein suggesting the importance of the L239-Q251 loop for the catalytic activity of the otherwise stable 3βHSD2 enzyme. << Less

  J. Clin. Endocrinol. Metab. 100:E191-E196(2015) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• The role of glutathione in the isomerization of delta 5-androstene-3,17-dione catalyzed by human glutathione transferase A1-1.

  Pettersson P.L., Mannervik B.

  Human glutathione transferase (GST) A1-1 efficiently catalyzes the isomerization of Delta(5)-androstene-3,17-dione (AD) into Delta(4)-androstene-3,17-dione. High activity requires glutathione, but enzymatic catalysis occurs also in the absence of this cofactor. Glutathione alone shows a limited ca ... >> More

  Human glutathione transferase (GST) A1-1 efficiently catalyzes the isomerization of Delta(5)-androstene-3,17-dione (AD) into Delta(4)-androstene-3,17-dione. High activity requires glutathione, but enzymatic catalysis occurs also in the absence of this cofactor. Glutathione alone shows a limited catalytic effect. S-Alkylglutathione derivatives do not promote the reaction, and the pH dependence of the isomerization indicates that the glutathione thiolate serves as a base in the catalytic mechanism. Mutation of the active-site Tyr(9) into Phe significantly decreases the steady-state kinetic parameters, alters their pH dependence, and increases the pK(a) value of the enzyme-bound glutathione thiol. Thus, Tyr(9) promotes the reaction via its phenolic hydroxyl group in protonated form. GST A2-2 has a catalytic efficiency with AD 100-fold lower than the homologous GST A1-1. Another Alpha class enzyme, GST A4-4, is 1000-fold less active than GST A1-1. The Y9F mutant of GST A1-1 is more efficient than GST A2-2 and GST A4-4, both having a glutathione cofactor and an active-site Tyr(9) residue. The active sites of GST A2-2 and GST A1-1 differ by only four amino acid residues, suggesting that proper orientation of AD in relation to the thiolate of glutathione is crucial for high catalytic efficiency in the isomerization reaction. The GST A1-1-catalyzed steroid isomerization provides a complement to the previously described isomerase activity of 3beta-hydroxysteroid dehydrogenase. << Less

  J. Biol. Chem. 276:11698-11704(2001) [PubMed] [EuropePMC]

• Crystal structure of human glutathione S-transferase A3-3 and mechanistic implications for its high steroid isomerase activity.

  Gu Y., Guo J., Pal A., Pan S.S., Zimniak P., Singh S.V., Ji X.

  The crystal structure of human class alpha glutathione (GSH) S-transferase A3-3 (hGSTA3-3) in complex with GSH was determined at 2.4 A. Despite considerable amino acid sequence identity with other human class alpha GSTs (e.g., hGSTA1-1), hGSTA3-3 is unique due to its exceptionally high steroid dou ... >> More

  The crystal structure of human class alpha glutathione (GSH) S-transferase A3-3 (hGSTA3-3) in complex with GSH was determined at 2.4 A. Despite considerable amino acid sequence identity with other human class alpha GSTs (e.g., hGSTA1-1), hGSTA3-3 is unique due to its exceptionally high steroid double bond isomerase activity for the transformation of Delta(5)-androstene-3,17-dione (Delta(5)-AD) to Delta(4)-androstene-3,17-dione. A comparative analysis of the active centers of hGSTA1-1 and hGSTA3-3 reveals that residues in positions 12 and 208 may contribute to their disparate isomerase activity toward Delta(5)-AD. Substitution of these two residues of hGSTA3-3 with the corresponding residues in hGSTA1-1 followed by kinetic characterization of the wild-type and the mutant enzymes supported this prediction. On the basis of our model of the hGSTA3-3.GSH.Delta(5)-AD ternary complex and available biochemical data, we propose that the thiolate group of deprotonated GSH (GS(-)) serves as a base to initiate the reaction by accepting a proton from the steroid and the nonionized hydroxyl group of catalytic residue Y9 (HO-Y9) functions as part of a proton-conducting wire to transfer a proton back to the steroid. Residue R15 may function to stabilize the deprotonated thiolate group of GSH (GS(-)), and a GSH-bound water molecule may donate a hydrogen bond to the 3-keto group of Delta(5)-AD and thus help the thiolate of GS(-) to initiate the proton transfer and the subsequent stabilization of the reaction intermediate. << Less

  Biochemistry 43:15673-15679(2004) [PubMed] [EuropePMC]

• Rv1106c from Mycobacterium tuberculosis is a 3beta-hydroxysteroid dehydrogenase.

  Yang X., Dubnau E., Smith I., Sampson N.S.

  New approaches are required to combat Mycobacterium tuberculosis (Mtb), especially the multi-drug resistant and extremely drug resistant organisms (MDR-TB and XDR-TB). There are many reports that mycobacteria oxidize 3beta-hydroxysterols to 3-ketosteroids, but the enzymes responsible for this acti ... >> More

  New approaches are required to combat Mycobacterium tuberculosis (Mtb), especially the multi-drug resistant and extremely drug resistant organisms (MDR-TB and XDR-TB). There are many reports that mycobacteria oxidize 3beta-hydroxysterols to 3-ketosteroids, but the enzymes responsible for this activity have not been identified in mycobacterial species. In this work, the Rv1106c gene that is annotated as a 3beta-hydroxysteroid dehydrogenase in Mtb has been cloned and heterologously expressed. The purified enzyme was kinetically characterized and found to have a pH optimum between 8.5 and 9.5. The enzyme, which is a member of the short chain dehydrogenase superfamily, uses NAD+ as a cofactor and oxidizes cholesterol, pregnenolone, and dehydroepiandrosterone to their respective 3-keto-4-ene products. The enzyme forms a ternary complex with NAD+ binding before the sterol. The enzyme shows no substrate preference for dehydroepiandrosterone versus pregnenolone with second-order rate constants (kcat/Km) of 3.2 +/- 0.4 and 3.9 +/-0.9 microM-1 min-1, respectively, at pH 8.5, 150 mM NaCl, 30 mM MgCl2, and saturating NAD+. Trilostane is a competitive inhibitor of dehydroepiandrosterone with a Ki of 197 +/-8 microM. The expression of the 3beta-hydroxysteroid dehydrogenase in Mtb is intracellular. Disruption of the 3beta-hydroxysteroid dehydrogenase gene in Mtb abrogates mycobacterial cholesterol oxidation activity. These data are consistent with the Rv1106c gene being the one responsible for 3beta-hydroxysterol oxidation in Mtb. << Less

  Biochemistry 46:9058-9067(2007) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

• Characterization, expression, and immunohistochemical localization of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase in human skin.

  Dumont M., Van L.T., Dupont E., Pelletier G., Labrie F.

  Three beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) catalyses an obligatory step in the biosynthesis of all classes of hormonal steroids, namely, the oxidation/isomerization of 3 beta-hydroxy-5-ene steroids into the corresponding 3-keto-4-ene steroids in gonadal as well ... >> More

  Three beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) catalyses an obligatory step in the biosynthesis of all classes of hormonal steroids, namely, the oxidation/isomerization of 3 beta-hydroxy-5-ene steroids into the corresponding 3-keto-4-ene steroids in gonadal as well as in peripheral tissues. Because humans are unique with some primates in having adrenals that secrete large amounts of the steroid precursors dehydropiandrosterone (DHEA) and its sulfate (DHEA-S) and its exceptionally large volume makes the skin an important site of steroid biosynthesis, we have isolated and characterized cDNA clones encoding 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase from a human skin lambda gt11 library. The longest clone obtained contains the entire coding sequence for type I 3 beta-HSD (372 amino acids) as well as an additional 131 nucleotides in the 5'-untranslated region. The insert of 1647 bp containing the entire coding region has been inserted in a pCMV expression vector and transfected into human cervical carcinoma cells (HeLa). The expressed enzyme efficiently catalyzes the transformation of pregnenolone, DHEA, and dihydrotestosterone into progesterone, 4-androstenedione, and 5 alpha-androstane-3 beta, 17 beta-diol, respectively. Using the enzyme expressed in HeLa cells, we have shown cyproterone acetate, a progestin used in the treatment of acne and hirsutism, as well as norgestrel and norethindrone, two steroids widely used as oral contraceptives, to be relatively potent inhibitors, with Ki values of 0.38 microM, 1.3 microM, and 1.2 microM, respectively. Immunohistochemical localization of 3 beta-HSD, illustrated by using an antibody raised against human placental 3 beta-HSD, shows that the enzyme is localized in sebaceous glands. << Less

  J. Invest. Dermatol. 99:415-421(1992) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.