Characterization of a novel 3-hydroxybutyrate dehydrogenase from Ralstonia pickettii T1.
We previously reported that the activities of two 3-hydroxybutyrate dehydrogenases (BDH1 and BDH2) were greatly influenced by culture conditions when Ralstonia pickettii T1, a strain growing on extracellular poly-3-hydroxybutyrate (PHB), was grown on different carbon sources such as 3HB and succinate. In this study, knockout mutants of bdh1 or bdh2 were constructed and characterized under different culture conditions. In addition, a novel BDH (BDH3) was found in bdh2 mutants, and bdh3 was cloned. Apparent kinetic parameters for the substrates of BDH3 indicated that the enzyme is suitable for the oxidation reaction of 3-hydroxybutyrate (3HB) to acetoacetate. In Western blotting, it was clear that BDH3 is produced only in cells grown on 3HB or PHB as a carbon source, while BDH1 and BDH2 are produced in cells grown on various carbon sources such as sugars, amino acids, organic acids, 3HB, and PHB. Both the bdh1 and bdh2 mutants lagged behind the wild type in growth rates when the cells were cultured with 3HB, citrate, succinate, or nutrient broth. A test of sensitivity to diamide as an oxidative stress revealed that the lack of BDH1 or BDH2 caused a decline in the capacity to neutralize the stress. These results suggested that BDH1 and BDH2 are needed to regulate the cytoplasmic redox state as well as to utilize 3HB, while BDH3 is specialized to utilize 3HB. The expression of bdh3 may be coordinately regulated with a gene encoding putative 3HB permease.
Takanashi
Masahiko
M
Laboratory of Molecular Microbiology, Department of Biological Science, Faculty of Science, Kanagawa University, 2946 Tsuchiya, Hiratsuka, Kanagawa, Japan.
Shiraki
Mari
M
Saito
Terumi
T
D020155:3-Hydroxybutyric Acid / Q000378:metabolism; D000090:Acetoacetates / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D019343:Citric Acid; D003001:Cloning, Molecular; D003470:Culture Media; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D020869:Gene Expression Profiling; D055786:Gene Knockout Techniques; D023061:Gene Order; D006884:Hydroxybutyrate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D006885:Hydroxybutyrates / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D010802:Phylogeny; D011091:Polyesters / Q000378:metabolism; D049920:Ralstonia pickettii / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D019802:Succinic Acid / Q000378:metabolism; D000094122:Polyhydroxybutyrates
D020155:3-Hydroxybutyric Acid; D000090:Acetoacetates; D001426:Bacterial Proteins; D019343:Citric Acid; D003470:Culture Media; D004269:DNA, Bacterial; D006884:Hydroxybutyrate Dehydrogenase; D006885:Hydroxybutyrates; D011091:Polyesters; D019802:Succinic Acid; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C016635:acetoacetic acid