Showing papers on "Hydroxysteroid dehydrogenase published in 1995"

Cloning and primary structure of murine 11 beta-hydroxysteroid dehydrogenase/microsomal carbonyl reductase.

Abstract: Screening of a mouse liver λgt 11 cDNA library with a rat liver 11β-hydroxysteroid dehydrogenase cDNA (11β-HSDr1A) and subsequent screening with an isolated mouse probe, resulted in the isolation and structure determination of a mouse cDNA encoding an amino acid sequence which is very similar to human and rat 11β-hydroxysteroid dehydrogenases (78% and 86% similar, respectively), and also to other known vertebrate 11β-hydroxysteroid dehydrogenase structures. Open-reading-frame analysis and the deduced amino acid sequence predict a protein with a molecular mass of 32.3 kDa which belongs to the superfamily of the short-chain dehydrogenase proteins. The amino acid sequence contains two potential glycosylation sites. These data are in agreement with information on the glycoprotein character of the native enzyme. This kind of post-translational modification seems to be a determining factor concerning the equilibrium of the catalyzed 11β-dehydrogenation/11-oxo reduction step [Obeid, J., Curnow, K. M., Aisenberg, J. & White, P. C. (1993) Mol. Endocrinol. 7, 154–160; Agarwal, A. K., Tusie-Luna, M. T., Monder, C. & White, P. C. (1990) Mol. Endocrinol. 4, 1827–18321. After in vitro transcription/translation of the mouse cDNA, immunoprecipitation with anti-(microsomal carbonyl reductase) serum and N-terminal sequence analysis of the purified protein confirms the identity of microsomal 11β-hydroxysteroid dehydrogenase with the previously described, microsomal-bound xenobiotic carbonyl reductase [Maser, E. & Bannenberg, G. (1994) Biochem. Pharmacol., 1805–1812], and points to an involvement of the 11β-HSD1A isofom in the reductive phase-I metabolism of xenobiotic compounds, besides its endocrinological functions. The alignment and comparison to other hydroxysteroid dehydrogenase forms of the same protein superfamily allows the identification of important residues in the 11β-HSD primary structure.

Expression in Escherichia coli and characterization of a bile acid-inducible 3 alpha-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708.

Abstract: We have previously cloned and sequenced three members of a bile acid-inducible gene family from Eubacterium sp. strain VPI 12708 that encode 27,000-Mr polypeptides. Two copies of these genes (baiA1 and baiA3) are identical, while the third copy (baiA2) encodes a polypeptide sharing 92% amino acid identity with the baiA1 and baiA3 gene products. We have overexpressed the baiA1 gene in Escherichia coli and analyzed the expressed activity. Thin-layer chromatography of 14C-labeled bile acid products from reactions using cell-free extracts revealed a 3α-hydroxysteroid dehydrogenase activity for the BaiA1 protein. The BaiA1 protein could utilize both NAD+ and NADP+, and the preferred steroid substrate was the cholyl-coenzyme A conjugate rather than free cholic acid. These results show that the BaiA proteins are novel 3α-hydroxysteroid dehydrogenases.

Expression and kinetic properties of a recombinant 3α-hydroxysteroid/dihydrodiol dehydrogenase isoenzyme of human liver

Abstract: Human liver cytosol contains multiple forms of 3 alpha-hydroxysteroid dehydrogenase and dihydrodiol dehydrogenase with hydroxysteroid dehydrogenase activity, and multiple cDNAs for the enzymes have been cloned from human liver cDNA libraries. To understand the relationship of the multiple enzyme froms to the genes, a cDNA, which has been reported to code for an isoenzyme of human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase, was expressed in Escherichia coli. The recombinant enzyme showed structural and functional properties almost identical to those of the isoenzyme purified from human liver. In addition, the recombinant isoenzyme efficiently reduced 5 alpha-dihydrotestosterone and 5 beta-dihydrocortisone, the known substrates of human liver 3 alpha-hydroxysteroid dehydrogenase and chlordecone reductase previously purified, which suggests that these human liver enzymes are identical. Furthermore, the steady-state kinetic data for NADP(+)-linked (S)-1-indanol oxidation by the recombinant isoenzyme were consistent with a sequential ordered mechanism in which NADP+ binds first. Phenolphthalein inhibited this isoenzyme much more potently than it did the other human liver dihydrodiol dehydrogenases, and was a competitive inhibitor (Ki = 20 nM) that bound to the enzyme-NADP+ complex.

Purification and Characterization of a Novel Dimeric 20α-Hydroxysteroid Dehydrogenase from Tetrahymena Pyriformis.

Abstract: Tetrahymena pyriformis was found to exhibit high NADPH-dependent 20-oxosteroid reductase activity that converted 17 alpha-hydroxyprogesterone into 17 alpha,20 alpha-dihydroxypregn-4-en-3-one. The enzyme was purified 400-fold from the cytosolic fraction. The purified enzyme with a specific activity of 6.4 mumol/min per mg of protein had an isoelectric point of 4.9 and M(r) of 68,000, and was composed of two subunits of equal size. The N-terminal sequence was determined to be LAKTVPLNDGTNFPIFGG. The enzyme reduced pregnanes and pregnanes possessing a 17 alpha-hydroxy group to a greater extent than those without the hydroxy group, and oxidized 20 alpha-hydroxy groups of the steroids in the presence of NADP+. The Km values for 17 alpha-hydroxyprogesterone and 17 alpha-hydroxypregnenolone were 2.9 and 3.4 microM respectively. Although the enzyme was inactive towards androgens and oestrogens with 3- or 17-oxo groups, it reduced several nonsteroidal carbonyl compounds and oxidized trans-benzene dihydrodiol. The enzyme activity was inhibited by synthetic oestrogens, barbiturates, aldose reductase inhibitors and quercitrin. Thus, this enzyme is a novel form of 20 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.149) which structurally and functionally differs from the mammalian and bacterial enzymes.

Production and use of isolated type 5 17β-hydroxysteroid dehydrogenase

Abstract: A novel type 5 17β-hydroxysteroid dehydrogenase is provided. Methods of producing the enzyme and using the enzyme to identify potential compounds which inhibit or alter the activity of the enzyme are described. In addition, methods of using the gene sequence or portions thereof for probes or to produce expression-disrupting sense or antisense DNA fragments thereof, or antisense RNA, are provided.

Molecular Cloning and Sequencing of Mouse Hepatic 11ß-Hydroxysteroid Dehydrogenase/Carbonyl Reductase

Abstract: Extensive studies on carbonyl reducing enzymes in recent years have led to the establishment of the aldo-keto reductase family, members of which are aldehyde reductase (EC 1.1.1.2) and aldose reductase (EC 1.1.1.21) (Bohren et al, 1989). In addition to these “classic” aldo-keto reductases, enzymes like carbonyl reductase (EC 1.1.1.184), dihydrodiol dehydrogenase (EC 1.3.1.20), NAD(P)H:quinone-oxidoreductase (EC 1.6.99.2) and several hydroxysteroid dehydrogenases (3α-, 17s-, 20α-, 3α/20s-) were also shown to be involved in the reductive metabolism of xenobiotic carbonyl compounds (for Review see Maser, 1994). The involvement of hydroxysteroid dehydrogenases in the reductive metabolism of xenobiotic carbonyl compounds and in the inactivation of proximal carcinogens, derived from polycyclic aromatic hydrocarbons, points to other important roles of these proteins besides their normal endocrinological function.

[Cloning of genes coding for 3-alpha-hydroxysteroid dehydrogenase and for (3-17)-beta-hydroxysteroid dehydrogenase from Pseudomonas testosteroni].

Abstract: A genomic library of Pseudomonas testosteroi total DNA constructed from SauIIIA digests ligated to a lambda gt11 vector was probed with different polyclonal antibodies raised against purified 3 alpha-HSD and (3 beta-17 beta)-HSD. Two different clones reacting with one antibody were selected. The clone reacting with (3-17)beta-HSD antibody contained a 2,661-base pair insert and was found to contained an open reading frame of 765 base pair that corresponds to a protein of 254 amino-acid residues. A 1,492-base pair was inserte in pBR 322 plasmid vector; the recombinant bacterie over expressed the (3-17)beta-HSD gene. The clone reacting with 3 alpha-HSD antibody contained a 1746 base pair insert which contained an open reading frame of 696 base pairs that corresponds to a protein of 231 amino-acid residues. A search for homologous proteins was performed. Distant similarities were found between (3-17)beta-HSD and members of the short-chain alcool dehydrogenase (SCAD) family but no similarity was observed between 3 alpha-HSD and proteins of this family.