Applied Microbiology and Biotechnology

, Volume 95, Issue 5, pp 1221–1233 | Cite as

In search of sustainable chemical processes: cloning, recombinant expression, and functional characterization of the 7α- and 7β-hydroxysteroid dehydrogenases from Clostridium absonum

  • Erica Elisa Ferrandi
  • Giulia Maria Bertolesi
  • Fausto Polentini
  • Armando Negri
  • Sergio Riva
  • Daniela MontiEmail author
Biotechnologically relevant enzymes and proteins


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.


Hydroxysteroid dehydrogenases Clostridium absonum Bile acids Cloning Biochemical characterization Substrate inhibition 



We thank Dr. Elena Fossati (Concordia University, Montreal), Dr. Matteo Piazza (University of Milano-Bicocca), and Dr. Chiara Dugoni (University of Modena & Reggio Emilia) for valuable contribution to protein purification and characterization. Prodotti Chimici Alimentari S.p.A. is gratefully acknowledged for financial support to EEF.

Supplementary material

253_2011_3798_MOESM1_ESM.doc (2.1 mb)
ESM 1 Doc 2,135 kb


  1. Akao T, Akao T, Kobashi K (1987) Purification and characterization of 7β-hydroxysteroid dehydrogenase from Ruminococcus sp. of human intestine. J Biochem 102(3):613–619Google Scholar
  2. Baron SF, Franklund CV, Hylemon PB (1991) Cloning, sequencing, and expression of the gene coding for bile acid 7a-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708. J Bacteriol 173:4558–4569Google Scholar
  3. Bennett MJ, McKnight SL, Coleman JP (2003) Cloning and characterization of the NAD-dependent 7α-hydroxysteroid dehydrogenase from Bacteroides fragilis. Curr Microbiol 47:475–484CrossRefGoogle Scholar
  4. Beuers U (2005) Hepatic overlap syndromes. J Hepatol 42:S93–S99CrossRefGoogle Scholar
  5. Bovara R, Canzi E, Carrea G, Pilotti A, Riva S (1993) Enzymatic α/β inversion of the C-7-hydroxyl of steroids. J Org Chem 58:499–501CrossRefGoogle Scholar
  6. Bovara R, Carrea G, Riva S, Secundo F (1996) A new enzymatic route to the synthesis of 12-ketoursodeoxycholic acid. Biotechnol Lett 18:305–308CrossRefGoogle Scholar
  7. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  8. Coleman JP, Hudson LL, Adams MJ (1994) Characterization and regulation of the NADP-linked 7α-hydroxysteroid dehydrogenase gene from Clostridium sordellii. J Bacteriol 176:4865–4874Google Scholar
  9. Cornish-Bowden A (1995) Fundamentals of enzyme kinetics. Portland Press, LondonGoogle Scholar
  10. Edenharder R, Pfützner A, Hammann R (1989) Characterization of NAD-dependent 3 alpha- and 3 beta-hydroxysteroid dehydrogenase and of NADP-dependent 7 beta-hydroxysteroid dehydrogenase from Peptostreptococcus productus. Biochim Biophys Acta 1004(2):230–238CrossRefGoogle Scholar
  11. Fossati E, Riva S (2006) Stereoselective modifications of polyhydroxylated steroids. In: Patel RN (ed) Biocatalysis in the pharmaceutical and biotechnology industries. CRC Press, Boca Raton, Florida, pp 591–604Google Scholar
  12. Fossati E, Polentini F, Carrea G, Riva S (2006) Exploitation of the alcohol dehydrogenase-acetone NADP-regeneration system for the enzymatic preparative-scale production of 12-ketochenodeoxycholic acid. Biotechnol Bioeng 93:1216–1220CrossRefGoogle Scholar
  13. Hylemon PB, Zhou H, Pandak WM, Ren S, Gil G, Dent P (2009) Bile acids as regulatory molecules. J Lipid Res 50:1509–1520CrossRefGoogle Scholar
  14. Iida T, Chang FC (1982) Potential bile acid metabolites. 7. 3,7,12-Trihydroxy-5β-cholanic acids and related compounds. J Org Chem 47:2972–2978CrossRefGoogle Scholar
  15. Kavanagh KL, Jörnvall H, Persson B, Oppermann U (2008) The SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes. Cell Mol Life Sci 65:3895–3906CrossRefGoogle Scholar
  16. Kristan K, Stojan J, Adamski J, Rizner TL (2007) Rational design of novel mutants of fungal 17 beta-hydroxy steroid dehydrogenase. J Biotechnol 129:123–130CrossRefGoogle Scholar
  17. Laemmli UK (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  18. Lepercq P, Gérard P, Béguet F, Grill J-P, Relano P, Cayuela C, Juste C (2004) Isolates from normal human intestinal flora but not lactic acid bacteria exhibit 7α- and 7β-hydroxysteroid dehydrogenase activities. Microb Ecol Health Dis 16(4):195–201CrossRefGoogle Scholar
  19. Liu L, Aigner A, Schmid RD (2010) Identification, cloning, heterologous expression, and characterization of a NADPH-dependent 7β-hydroxysteroid dehydrogenase from Collinsella aerofaciens. Appl Microbiol Biotechnol 90:127–135CrossRefGoogle Scholar
  20. Macdonald IA, Roach PD (1981) Bile salt induction of 7α and 7β-hydroxysteroid dehydrogenases in Clostridium absonum. Biochim Biophys Acta 665:262–269CrossRefGoogle Scholar
  21. Macdonald IA, Sutherland JD (1983) Further studies on the bile salt induction of 7α- and 7β-hydroxysteroid dehydrogenases in Clostridium absonum. Biochim Biophys Acta 750:397–403CrossRefGoogle Scholar
  22. Macdonald IA, Hutchison DM, Forrest TP (1981) Formation of urso- and ursodeoxy-cholic acids from primary bile acids by Clostridium absonum. J Lipid Res 22:458–466Google Scholar
  23. Macdonald IA, White BA, Hylemon PB (1983) Separation of 7α- and 7β-hydroxysteroid dehydrogenase activities from Clostridium absonum ATCC# 27555 and cellular response of this organism to bile acid inducers. J Lipid Res 24:1119–1126Google Scholar
  24. Monti D, Ferrandi EE, Zanellato I, Hua L, Polentini F, Carrea G, Riva S (2009) One-pot multienzymatic synthesis of 12-ketoursodeoxycholic acid: subtle cofactor specificities rule the reaction equilibria of five biocatalysts working in a row. Adv Synth Catal 351:1303–1311CrossRefGoogle Scholar
  25. Mukhopadhyay S, Maitra U (2004) Chemistry and biology of bile acids. Curr Sci 87:1666–1683Google Scholar
  26. Pedrini P, Andreotti E, Guerrini A, Dean M, Fantin G, Giovannini PP (2006) Xanthomonas maltophilia CBS 897.97 as a source of new 7β- and 7α-hydroxysteroid dehydrogenases and cholylglycine hydrolase: improved biotransformations of bile acids. Steroids 71:189–198CrossRefGoogle Scholar
  27. Pellicciari R, Gioiello A, Macchiarulo A, Thomas C, Rosatelli E, Natalini B, Sardella R, Pruzanski M, Roda A, Pastorini E, Schoonjans K, Auwerx J (2009) Discovery of 6α-ethyl-23(S)-methylcholic acid (S-EMCA, INT-777) as a potent and selective agonist for the TGR5 receptor, a novel target for diabesity. J Med Chem 52:7958–7961CrossRefGoogle Scholar
  28. Prahba V, Ohri M (2006) Bacterial transformations of bile acids. World J Microbiol Biotechnol 22:191–196CrossRefGoogle Scholar
  29. Ridlon JM, Kang D-J, Hylemon PB (2006) Bile salt biotransformations by human intestinal bacteria. J Lipid Res 47:241–259CrossRefGoogle Scholar
  30. Ridlon JM, Kang D-J, Hylemon PB (2010) Isolation and characterization of a bile acid inducible 7α-dehydroxylating operon in Clostridium hylemonae TN271. Anaerobe 16(2):137–46CrossRefGoogle Scholar
  31. Riva S, Bovara R, Pasta P, Carrea G (1986) Preparative-scale regio- and stereospecific oxidoreduction of cholic acid and dehydrocholic acid catalyzed by hydroxysteroid dehydrogenases. J Org Chem 51:2902–2906CrossRefGoogle Scholar
  32. Rosenberg M, Court D (1979) Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet 13:319–353CrossRefGoogle Scholar
  33. Sambrook J, MacCallum P, Russell D (2001) Molecular cloning: a laboratory manual (third edition). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  34. Tanabe T, Tanaka N, Uchikawa K, Kabashima T, Ito K, Nonaka T, Mitsui Y, Tsuru M, Yoshimoto T (1998) Roles of the Serl46, Tyrl59, and Lysl63 residues in the catalytic action of 7α-hydroxysteroid dehydrogenase from Escherichia coli. J Biochem 124:634–641CrossRefGoogle Scholar
  35. Tanaka N, Nonaka T, Tanabe T, Yoshimoto T, Tsuru D, Mitsui Y (1996) Crystal structures of the binary and ternary complexes of 7α-hydroxysteroid dehydrogenase from Escherichia coli. Biochemistry 35:7715–7730CrossRefGoogle Scholar
  36. Thomas C, Pellicciari R, Pruzanski M, Auwerx J, Schoonjans K (2008) Targeting bile-acid signalling for metabolic diseases. Nat Rev Drug Discov 7:678–693CrossRefGoogle Scholar
  37. Triglia T, Peterson MG, Kemp D (1988) A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucleic Acid Res 16:8186–8186CrossRefGoogle Scholar
  38. Yoshimoto T, Higashi H, Kanatani A, Lin XS, Nagai H, Oyama H, Kurazono K, Tsuru D (1991) Cloning and sequencing of the 7α-hydroxysteroid dehydrogenase gene from Escherichia coli HB101 and characterization of the expressed enzyme. J Bacteriol 173:2173–2179Google Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Erica Elisa Ferrandi
    • 1
  • Giulia Maria Bertolesi
    • 1
  • Fausto Polentini
    • 2
  • Armando Negri
    • 3
  • Sergio Riva
    • 1
  • Daniela Monti
    • 1
    Email author
  1. 1.Istituto di Chimica del Riconoscimento Molecolare, C.N.R.MilanItaly
  2. 2.Prodotti Chimici e Alimentari S. p. A.BasaluzzoItaly
  3. 3.Dipartimento di Patologia Animale Igiene e Sanità Pubblica VeterinariaUniversità degli Studi di MilanoMilanItaly

Personalised recommendations