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.
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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
Dawes EA, Senior PJ (1973) The role and regulation of energy reserve polymers in microorganisms. Adv Microb Physiol 10:135–266
Anderson AJ, Dawes EA (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54:450–472
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
Gazzotti P, Bock HGO, Fleischer S (1974) Role of lecithin in D-3-hydroxybutyrate dehydrogenase function. Biochem Biophys Res Commun 58:309–315
Williamson DH, Mellamby JH, Krebs HA (1962) Enzymic determination of D(−) beta-hydroxybutyric acid and acetoacetic acid in blood. Biochem J 82:90–96
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
Matyskova I, Kovar J, Racek P (1985) Purification and properties of D-3-hydroxybutyrate dehydrogenase from Paracoccus denitrificanss. Biochim Biophys Acta 839:300–307
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
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
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
Latruffe N, Gaudemer Y (1974) Beta-hydoxybutyrate dehydrogenase, properties and compared roles (in french). Ann Scient Univ Besançon. 3th serie. fasc 11:3–18
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
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
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:4668–4673
Towbin H, Stahelin T, Gordon J (1992) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocel procedure and applications. Biotechnology 24:145–149
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
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
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
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
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
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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.
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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
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DOI: https://doi.org/10.1007/s00284-009-9568-7