Publications

• In search of sustainable chemical processes: cloning, recombinant expression, and functional characterization of the 7alpha- and 7beta-hydroxysteroid dehydrogenases from Clostridium absonum.

  Ferrandi E.E., Bertolesi G.M., Polentini F., Negri A., Riva S., Monti D.

  Nicotinamide adenine dinucleotide phosphate-dependent 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenases (7β-HSDH) from Clostridium absonum catalyze the epimerization of primary bile acids through 7-keto bile acid intermediates and may be suitable as biocatalysts for th ... >> More

  Nicotinamide adenine dinucleotide phosphate-dependent 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenases (7β-HSDH) from Clostridium absonum catalyze the epimerization of primary bile acids through 7-keto bile acid intermediates and may be suitable as biocatalysts for the synthesis of bile acids derivatives of pharmacological interest. C. absonum 7α-HSDH has been purified to homogeneity and the N-terminal sequence has been determined by Edman sequencing. After PCR amplifications of a gene fragment with degenerate primers, cloning of the complete gene (786 nt) has been achieved by sequencing of C. absonum genomic DNA. The sequence coding for the 7β-HSDH (783 nt) has been obtained by sequencing of the genomic DNA region flanking the 5' termini of 7α-HSDH gene, the two genes being contiguous and presumably part of the same operon. After insertion in suitable expression vectors, both HSDHs have been successfully produced in recombinant form in Escherichia coli, purified by affinity chromatography and submitted to kinetic analysis for determination of Michaelis constants (K (m)) and specificity constants (k (cat)/K (m)) in the presence of various bile acids derivatives. Both enzymes showed a very strong substrate inhibition with all the tested substrates. The lowest K (S) values were observed with chenodeoxycholic acid and 12-ketochenodeoxycholic acid as substrates in the case of 7α-HSDH, whereas ursocholic acid was the most effective inhibitor of 7β-HSDH activity. << Less

  Appl. Microbiol. Biotechnol. 95:1221-1223(2012) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

• Characterization and regulation of the NADP-linked 7 alpha-hydroxysteroid dehydrogenase gene from Clostridium sordellii.

  Coleman J.P., Hudson L.L., Adams M.J.

  A bile acid-inducible NADP-linked 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) from Clostridium sordellii ATCC 9714 was purified 310-fold by ion-exchange, gel filtration, and dye-ligand affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purifi ... >> More

  A bile acid-inducible NADP-linked 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) from Clostridium sordellii ATCC 9714 was purified 310-fold by ion-exchange, gel filtration, and dye-ligand affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified enzyme showed one predominant peptide band (30,000 Da). The N-terminal sequence was determined, and the corresponding oligonucleotides were synthesized and used to screen EcoRI and HindIII genomic digests of C. sordellii. Two separate fragments (4,500 bp, EcoRI; 3,200 bp, HindIII) were subsequently cloned by ligation to pUC19 and transformation into Escherichia coli DH5 alpha-MCR. The EcoRI fragment was shown to contain a truncated 7 alpha-HSDH gene, while the HindIII fragment contained the entire coding region. E. coli clones containing the HindIII insert expressed high levels of an NADP-linked 7 alpha-HSDH. Nucleotide sequence analyses suggest that the 7 alpha-HSDH is encoded by a monocistronic transcriptional unit, with DNA sequence elements resembling rho-independent terminators located in both the upstream and downstream flanking regions. The transcriptional start site was located by primer extension analysis. Northern (RNA) blot analysis indicated that induction is mediated at the transcriptional level in response to the presence of bile acid in the growth medium. In addition, growth-phase-dependent expression is observed in uninduced cultures. Analysis of the predicted protein sequence indicates that the enzyme can be classified in the short-chain dehydrogenase group. << Less

  J. Bacteriol. 176:4865-4874(1994) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Hepatic reduction of the secondary bile acid 7-oxolithocholic acid is mediated by 11beta-hydroxysteroid dehydrogenase 1.

  Odermatt A., Da Cunha T., Penno C.A., Chandsawangbhuwana C., Reichert C., Wolf A., Dong M., Baker M.E.

  The oxidized bile acid 7-oxoLCA (7-oxolithocholic acid), formed primarily by gut micro-organisms, is reduced in human liver to CDCA (chenodeoxycholic acid) and, to a lesser extent, UDCA (ursodeoxycholic acid). The enzyme(s) responsible remained unknown. Using human liver microsomes, we observed en ... >> More

  The oxidized bile acid 7-oxoLCA (7-oxolithocholic acid), formed primarily by gut micro-organisms, is reduced in human liver to CDCA (chenodeoxycholic acid) and, to a lesser extent, UDCA (ursodeoxycholic acid). The enzyme(s) responsible remained unknown. Using human liver microsomes, we observed enhanced 7-oxoLCA reduction in the presence of detergent. The reaction was dependent on NADPH and stimulated by glucose 6-phosphate, suggesting localization of the enzyme in the ER (endoplasmic reticulum) and dependence on NADPH-generating H6PDH (hexose-6-phosphate dehydrogenase). Using recombinant human 11β-HSD1 (11β-hydroxysteroid dehydrogenase 1), we demonstrate efficient conversion of 7-oxoLCA into CDCA and, to a lesser extent, UDCA. Unlike the reversible metabolism of glucocorticoids, 11β-HSD1 mediated solely 7-oxo reduction of 7-oxoLCA and its taurine and glycine conjugates. Furthermore, we investigated the interference of bile acids with 11β-HSD1-dependent interconversion of glucocorticoids. 7-OxoLCA and its conjugates preferentially inhibited cortisone reduction, and CDCA and its conjugates inhibited cortisol oxidation. Three-dimensional modelling provided an explanation for the binding mode and selectivity of the bile acids studied. The results reveal that 11β-HSD1 is responsible for 7-oxoLCA reduction in humans, providing a further link between hepatic glucocorticoid activation and bile acid metabolism. These findings also suggest the need for animal and clinical studies to explore whether inhibition of 11β-HSD1 to reduce cortisol levels would also lead to an accumulation of 7-oxoLCA, thereby potentially affecting bile acid-mediated functions. << Less

  Biochem. J. 436:621-629(2011) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Carboxyl-terminal and Arg38 are essential for activity of the 7alpha-hydroxysteroid dehydrogenase from Clostridium absonum.

  Lou D., Wang B., Tan J., Zhu L.

  7α-hydroxysteroid dehydrogenase (7α-HSDH, EC 1.1.1.159), one of the short-chain dehydrogenase/reductase superfamily, catalyzes the dehydrogenation of C7 hydroxyl group of the steroid skeleton of bile acids. Clostridium absonum 7α-HSDH (Ca 7α-HSDH) was cloned and heterologously expressed in Escheri ... >> More

  7α-hydroxysteroid dehydrogenase (7α-HSDH, EC 1.1.1.159), one of the short-chain dehydrogenase/reductase superfamily, catalyzes the dehydrogenation of C7 hydroxyl group of the steroid skeleton of bile acids. Clostridium absonum 7α-HSDH (Ca 7α-HSDH) was cloned and heterologously expressed in Escherichia coli. The function of carboxyterminal (C-terminal) and Arg38 of Ca 7α-HSDH was investigated through truncations and site-directed mutagenesis, respectively. When 2 and 6 amino acids of C-terminal were removed, the catalytic efficiency (k(cat)/K(m)) of Ca 7α-HSDH remained 19.1% and 2.5%, respectively. Furthermore, the activity could not be detected after 8, 14 and 17 amino acids were deleted. No activity could be detected with coenzyme either NADP(+) or NAD(+) after replacement of arginine at position 38 by aspartic acid. The metal ions Mg(2+) (50 mM), Na(+) (200 mM) and K(+) (500 mM) could maximally improve the activity of Ca 7α-HSDH by 61.4%, 64.7% and 105.7%, respectively. The activity had no significant change after incubation at 4 or 25 °C for 108 h, but decreased dramatically at 37 °C. Our study confirmed that C-terminal and Arg38 were essential for the catalytic function of Ca 7α-HSDH and the enzyme activity can be improved by metal ions. << Less

  Protein Pept. Lett. 21:894-900(2014) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.