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.

• 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.