Skip to main content
SpringerLink
Log in

Structural and Catalytic Properties of the D-3-Hydroxybutyrate Dehydrogenase from Pseudomonas aeruginosa

  • Published:
Current Microbiology Aims and scope Submit manuscript

Abstract

To put forward BDH from Pseudomonas aeruginosa’s enzymatic properties, we report a two-step purification of BDH and its gene sequencing allowing the investigation of its structural properties. Purification of BDH was achieved, using ammonium sulfate fractionation and Blue Sepharose CL-6B affinity chromatography. SDS–PAGE analysis reveals a MM of 29 kDa, whereas the native enzyme showed a MM of 120 kDa suggesting a homotetrameric structure. BDH encoding gene sequence shows a nucleotide open reading frame sequence of 771 bp encoding a 265 amino acid residues polypeptide chain. The modeling analysis of the three dimensional structure fits with the importance of amino acids in the catalysis reaction especially a strictly conserved tetrad. Amino-acid residues in interaction with the coenzyme NAD+ were also identified.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Trautmann M, Lepper PM, Haller M (2005) Ecology of Pseudomonas aeruginosa in the intensive care unit and the evolving role of water outlets as a reservoir of the organism. Am J Infect Control 33:41–49

    Article  Google Scholar 

  2. Dawes EA, Senior PJ (1973) The role and regulation of energy reserve polymers in microorganisms. Adv Microb Physiol 10:135–266

    Article  CAS  PubMed  Google Scholar 

  3. Anderson AJ, Dawes EA (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54:450–472

    CAS  PubMed  Google Scholar 

  4. Wise JB, Lehninger AL (1962) The stability of mitochondrial D-3-hydroxybutyric dehydrogenase and its relationships to the respiratory chain. J Biol Chem 237:1363–1670

    CAS  PubMed  Google Scholar 

  5. Gazzotti P, Bock HGO, Fleischer S (1974) Role of lecithin in D-3-hydroxybutyrate dehydrogenase function. Biochem Biophys Res Commun 58:309–315

    Article  CAS  PubMed  Google Scholar 

  6. Williamson DH, Mellamby JH, Krebs HA (1962) Enzymic determination of D(−) beta-hydroxybutyric acid and acetoacetic acid in blood. Biochem J 82:90–96

    CAS  PubMed  Google Scholar 

  7. Bergmeyer HU, Gawehn K, Klotzsch H, Krebs HA, Williamson DH (1967) Purification and properties of crystalline 3-hydroxybutyrate dehydrogenase from Rhodopseudomonas spheroides. Biochem J 102:423–431

    CAS  PubMed  Google Scholar 

  8. Matyskova I, Kovar J, Racek P (1985) Purification and properties of D-3-hydroxybutyrate dehydrogenase from Paracoccus denitrificanss. Biochim Biophys Acta 839:300–307

    CAS  Google Scholar 

  9. Takanashi M, Shibahara T, Shiraki M, Saito T (2004) Biochemical and genetic characterization of a D-3-hydroxybutyrate dehydrogenase from Acidovorax sp. Strain SA1. J Biosci Bioeng 1:78–81

    Google Scholar 

  10. Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  11. Mountassif D, Andreoletti P, El Kebbaj Z, Moutaouakki A, Cherkaoui-Malki M, Latruffe N, El Kebbaj MS (2008) Immunoaffinity purification and characterization of mitochondrial membrane-bound D-3-hydroxybutyrate dehydrogenase from Jaculus orientalis. BMC Biochem 9(1):e26

    Article  Google Scholar 

  12. Latruffe N, Gaudemer Y (1974) Beta-hydoxybutyrate dehydrogenase, properties and compared roles (in french). Ann Scient Univ Besançon. 3th serie. fasc 11:3–18

    Google Scholar 

  13. Cleland WW (1963) The kinetics of enzymes catalysed reaction with two or more substrates or products, nomenclature and rate equations. Biochim Biophys Acta 67:104–137

    Article  CAS  PubMed  Google Scholar 

  14. Hedrick JL, Smith AJ (1968) Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch Biochem Biophys 126:155–164

    Article  CAS  PubMed  Google Scholar 

  15. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:4668–4673

    Article  Google Scholar 

  16. Towbin H, Stahelin T, Gordon J (1992) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocel procedure and applications. Biotechnology 24:145–149

    CAS  PubMed  Google Scholar 

  17. Coquard C, Adami P, Cherkaoui Malki M, Fellmann D, Latruffe N (1987) Immunological study of the tissue expression of D-3-hydroxybutyrate dehydrogenase, a ketone body converting enzyme. Biol Cell 181:381–388

    Google Scholar 

  18. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98

    CAS  Google Scholar 

  19. Ito K, Nakajima Y, Ichihara E, Ogawa K, Katayama N, Nakashima K, Yoshimoto T (2005) D-3-hydroxybutyrate dehydrogenase from Pseudomonas fragi: molecular cloning of the enzyme gene and crystal structure of the enzyme. J Mol Biol 355:722–733

    Article  PubMed  Google Scholar 

  20. Burnett BK, Khorana HG (1985) A rapid and efficient procedure for the purification of mitochondrial D-3-hydroxybutyrate dehydrogenase. Biochim Biophys Acta 815:51–56

    Article  CAS  PubMed  Google Scholar 

  21. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

Supported by the Regional Council of Burgundy and IFR no. 100, the Programme Thématique d’Appui à la Recherche Scientifique-Morocco, Biologie No.134, and the AI franco-marocaine MA/05/134.

Author information

Authors and Affiliations

  1. INSERM U866; Université de Bourgogne; LBMC (Biochimie Métabolique et Nutritionnelle), Faculté des Sciences, 6 Bd Gabriel, 21000, Dijon cedex, France

    Driss Mountassif, Pierre Andreoletti, Mustapha Cherkaoui-Malki & Norbert Latruffe

  2. Laboratoire de Biochimie et Biologie Moléculaire, Université Hassan II - Aïn Chock, Faculté des Sciences Aïn Chock, km 8 route d’El Jadida, BP. 5366, Maarif, Casablanca, Morocco

    Driss Mountassif & M’hammed Saïd El Kebbaj

Authors
  1. Driss Mountassif
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pierre Andreoletti
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mustapha Cherkaoui-Malki
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Norbert Latruffe
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. M’hammed Saïd El Kebbaj
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Norbert Latruffe.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mountassif, D., Andreoletti, P., Cherkaoui-Malki, M. et al. Structural and Catalytic Properties of the D-3-Hydroxybutyrate Dehydrogenase from Pseudomonas aeruginosa . Curr Microbiol 61, 7–12 (2010). https://doi.org/10.1007/s00284-009-9568-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00284-009-9568-7

Keywords

Search

Navigation