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3 Hydroxybutyrate Dehydrogenase

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J O Mcintyre – 1st expert on this subject based on the ideXlab platform

  • r 3 Hydroxybutyrate Dehydrogenase selective phosphatidylcholine binding by the c terminal domain
    Biochemistry, 2000
    Co-Authors: C Loebhennard, J O Mcintyre

    Abstract:

    (R)-3Hydroxybutyrate Dehydrogenase (BDH) is a lipid-requiring mitochondrial enzyme that has a specific requirement of phosphatidylcholine (PC) for function. The C-terminal domain (CTBDH) of human heart BDH (residues 195−297) has now been expressed in Escherichia coli as a chimera with a soluble protein, glutathione S-transferase (GST), yielding GST-CTBDH, a novel fusion protein that has been purified and shown to selectively bind to PC vesicles. Both recombinant human heart BDH (HH-Histag-BDH) and GST-CTBDH (but not GST) form well-defined protein−lipid complexes with either PC or phosphatidylethanolamine (PE)/diphosphatidylglycerol (DPG) vesicles (but not with digalactosyl diglyceride vesicles) as demonstrated by flotation in sucrose gradients. The protein−PC complexes are stable to 0.5 M NaCl, but complexes of either HH-Histag-BDH or GST-CTBDH with PE/DPG vesicles are dissociated by salt treatment. Thrombin cleavage of GST-CTBDH, either before or after reconstitution with PC vesicles, yields CTBDH (12 1…

  • phosphatidylcholine activation of human heart r 3 Hydroxybutyrate Dehydrogenase mutants lacking active center sulfhydryls site directed mutagenesis of a new recombinant fusion protein
    Biochemistry, 2000
    Co-Authors: D Chelius, C Loebhennard, J O Mcintyre, Sidney Fleischer, Andrew R Marks, S De, S Hahn, M M Jehl, J Moeller, Reinhard Philipp

    Abstract:

    (R)-3Hydroxybutyrate Dehydrogenase (BDH) is a lipid-requiring mitochondrial enzyme with a specific requirement of phosphatidylcholine (PC) for function. A plasmid has been constructed to express human heart (HH) BDH in Escherichia coli as a hexahistidine-tagged fusion protein (HH-Histag-BDH). A rapid two-step affinity purification yields active HH-Histag-BDH (and six mutants) with high specific activity (∼130 μmol of NAD+ reduced·min-1·mg-1). HH-Histag-BDH has no activity in the absence of phospholipid and exhibits a specific requirement of PC for function. The HH-Histag-BDH−PC complex (and HH-BDH derived therefrom by enterokinase cleavage) has apparent Michaelis constants (Km values) for NAD+, NADH, (R)-3Hydroxybutyrate (HOB), and acetoacetate (AcAc) similar to those for bovine heart or rat liver BDH. A computed structural model of HH-BDH predicts the two active center sulfhydryls to be C69 (near the adenosine moiety of NAD) and C242. With both sulfhydryls derivatized, BDH has minimal activity, but sit…

  • wild type and mutant human heart r 3 Hydroxybutyrate Dehydrogenase expressed in insect cells
    Biochemistry, 1996
    Co-Authors: David Green, Sidney Fleischer, Andrew R Marks, J O Mcintyre

    Abstract:

    (R)-3Hydroxybutyrate Dehydrogenase (BDH) is a lipid-requiring mitochondrial enzyme with a specific requirement of phosphatidylcholine (PC) for function. PC is an allosteric activator that enhances NAD(H) binding to BDH. The enzyme serves as a paradigm to study specific lipid−protein interactions in membranes. Analysis of the primary sequence of BDH, as determined by molecular cloning, predicts that lipid binding and substrate specificity are contributed by the C-terminal third of the protein [Marks, A. R., McIntyre, J. O., Duncan, T. M., Erdjument-Bromage, H., Tempst, P., & Fleischer, S. (1992) J. Biol. Chem. 267, 15459−15463]. The mature form of human heart BDH has now been expressed in catalytically active form in insect cells (Sf 9, Spodoptera frugiperda) transfected with BDH-cDNA in baculovirus. Endogenous PC in the insect cells fulfills the lipid requirement for the expressed BDH since enzymatic activity is lost upon digestion with phospholipase A2 and restored selectively by reconstitution with PC …

Trevor C Charles – 2nd expert on this subject based on the ideXlab platform

  • characterization of bdha encoding the enzyme d 3 Hydroxybutyrate Dehydrogenase from sinorhizobium sp strain ngr234
    Fems Microbiology Letters, 2005
    Co-Authors: Punita Aneja, Trevor C Charles

    Abstract:

    Abstract A genomic library of Sinorhizobium sp. strain NGR234 was introduced into Escherichia coli LS5218, a strain with a constitutively active pathway for acetoacetate degradation, and clones that confer the ability to utilize D –3Hydroxybutyrate as a sole carbon source were isolated. Subcloning experiments identified a 2.3 kb EcoRI fragment that retained complementing ability, and an ORF that appeared orthologous with known bdhA genes was located within this fragment. The deduced NGR234 BdhA amino acid sequence revealed 91% identity to the Sinorhizobium meliloti BdhA. Site-directed insertion mutagenesis was performed by introduction of a ΩSmSp cassette at a unique EcoRV site within the bdhA coding region. A NGR234 bdhA mutant, NGRPA2, was generated by homogenotization, utilizing the sacB gene-based lethal selection procedure. This mutant was devoid of d –3Hydroxybutyrate Dehydrogenase activity, and was unable to grow on D –3Hydroxybutyrate as sole carbon source. NGRPA2 exhibited symbiotic defects on Leucaena but not on Vigna, Macroptilium or Tephrosia host plants. Furthermore, the D –3Hydroxybutyrate utilization phenotype of NGRPA2 was suppressed by presence of plasmid-encoded multiple copies of the S. meliloti acsA2 gene. The glpK–bdhA–xdhA gene organization and the bdhA–xdhA operon arrangement observed in S. meliloti are also conserved in NGR234.

  • poly 3 Hydroxybutyrate degradation in rhizobium sinorhizobium meliloti isolation and characterization of a gene encoding 3 Hydroxybutyrate Dehydrogenase
    Journal of Bacteriology, 1999
    Co-Authors: Punita Aneja, Trevor C Charles

    Abstract:

    We have cloned and sequenced the 3Hydroxybutyrate Dehydrogenase-encoding gene (bdhA) from Rhizobium (Sinorhizobium) meliloti. The gene has an open reading frame of 777 bp that encodes a polypeptide of 258 amino acid residues (molecular weight 27,177, pI 6.07). The R. meliloti Bdh protein exhibits features common to members of the short-chain alcohol Dehydrogenase superfamily. bdhA is the first gene transcribed in an operon that also includes xdhA, encoding xanthine oxidase/Dehydrogenase. Transcriptional start site analysis by primer extension identified two transcription starts. S1, a minor start site, was located 46 to 47 nucleotides upstream of the predicted ATG start codon, while S2, the major start site, was mapped 148 nucleotides from the start codon. Analysis of the sequence immediately upstream of either S1 or S2 failed to reveal the presence of any known consensus promoter sequences. Although a ς54 consensus sequence was identified in the region between S1 and S2, a corresponding transcript was not detected, and a rpoN mutant of R. meliloti was able to utilize 3Hydroxybutyrate as a sole carbon source. The R. meliloti bdhA gene is able to confer upon Escherichia coli the ability to utilize 3Hydroxybutyrate as a sole carbon source. An R. meliloti bdhA mutant accumulates poly-3Hydroxybutyrate to the same extent as the wild type and shows no symbiotic defects. Studies with a strain carrying a lacZ transcriptional fusion to bdhA demonstrated that gene expression is growth phase associated.

Sidney Fleischer – 3rd expert on this subject based on the ideXlab platform

  • phosphatidylcholine activation of human heart r 3 Hydroxybutyrate Dehydrogenase mutants lacking active center sulfhydryls site directed mutagenesis of a new recombinant fusion protein
    Biochemistry, 2000
    Co-Authors: D Chelius, C Loebhennard, J O Mcintyre, Sidney Fleischer, Andrew R Marks, S De, S Hahn, M M Jehl, J Moeller, Reinhard Philipp

    Abstract:

    (R)-3Hydroxybutyrate Dehydrogenase (BDH) is a lipid-requiring mitochondrial enzyme with a specific requirement of phosphatidylcholine (PC) for function. A plasmid has been constructed to express human heart (HH) BDH in Escherichia coli as a hexahistidine-tagged fusion protein (HH-Histag-BDH). A rapid two-step affinity purification yields active HH-Histag-BDH (and six mutants) with high specific activity (∼130 μmol of NAD+ reduced·min-1·mg-1). HH-Histag-BDH has no activity in the absence of phospholipid and exhibits a specific requirement of PC for function. The HH-Histag-BDH−PC complex (and HH-BDH derived therefrom by enterokinase cleavage) has apparent Michaelis constants (Km values) for NAD+, NADH, (R)-3Hydroxybutyrate (HOB), and acetoacetate (AcAc) similar to those for bovine heart or rat liver BDH. A computed structural model of HH-BDH predicts the two active center sulfhydryls to be C69 (near the adenosine moiety of NAD) and C242. With both sulfhydryls derivatized, BDH has minimal activity, but sit…

  • wild type and mutant human heart r 3 Hydroxybutyrate Dehydrogenase expressed in insect cells
    Biochemistry, 1996
    Co-Authors: David Green, Sidney Fleischer, Andrew R Marks, J O Mcintyre

    Abstract:

    (R)-3Hydroxybutyrate Dehydrogenase (BDH) is a lipid-requiring mitochondrial enzyme with a specific requirement of phosphatidylcholine (PC) for function. PC is an allosteric activator that enhances NAD(H) binding to BDH. The enzyme serves as a paradigm to study specific lipid−protein interactions in membranes. Analysis of the primary sequence of BDH, as determined by molecular cloning, predicts that lipid binding and substrate specificity are contributed by the C-terminal third of the protein [Marks, A. R., McIntyre, J. O., Duncan, T. M., Erdjument-Bromage, H., Tempst, P., & Fleischer, S. (1992) J. Biol. Chem. 267, 15459−15463]. The mature form of human heart BDH has now been expressed in catalytically active form in insect cells (Sf 9, Spodoptera frugiperda) transfected with BDH-cDNA in baculovirus. Endogenous PC in the insect cells fulfills the lipid requirement for the expressed BDH since enzymatic activity is lost upon digestion with phospholipase A2 and restored selectively by reconstitution with PC …

  • specific interaction of r 3 Hydroxybutyrate Dehydrogenase with membrane phosphatidylcholine as studied by esr spectroscopy in oriented phospholipid multibilayers coenzyme binding enhances the interaction with phosphatidylcholine
    Biochemistry, 1996
    Co-Authors: K Klein, J O Mcintyre, B Rudy, Sidney Fleischer, Wolfgang E Trommer

    Abstract:

    : The interaction of phospholipid with (R)-3Hydroxybutyrate Dehydrogenase, a phosphatidylcholine-requiring membrane enzyme, has been studied using ESR spectroscopy of spin-labeled lipids, both as ordered multibilayers and in lipid vesicle suspensions (liposomes). Partially oriented phospholipid multibilayers were prepared from lipid vesicles composed of a 1:1 mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Vesicles containing (R)-3Hydroxybutyrate Dehydrogenase yielded active preparations of the enzyme in such multibilayers. With increasing protein/lipid ratio, the order of the multibilayers was disrupted as monitored by ESR spectroscopy with a spin-labeled analogue of PC, 5-doxyl-PC (5 mol %, 10% of total PC) as a probe. The outer peak separation of 5-doxyl-PC varied with the lipid/protein ratio. The lower the ratio, the larger was the separation, with higher activity enzyme being more effective in exerting this effect. When 5-doxylstearic acid was substituted for 5-doxyl-PC or when the enzyme was inactive, the 2A(zz) value stayed practically constant at its lower limit (about 54 G). Multilayers composed of 81% PE, 11% diphosphatidylglycerol (DPG), and 8% 5-doxyl-PC (no unlabeled PC present) gave similar results. With this lipid mixture, the maximal 2A(zz) value (about 61 G) was reached at lower protein/lipid ratios, although the enzymic activity of (R)-3Hydroxybutyrate Dehydrogenase is reduced to 40% in this system. The outer peak separation also depended on the presence of the coenzyme, NAD+, and 2-methylmalonate. The latter enhances binding of NAD+ about 100-fold by forming a ternary complex. With this ternary complex, the 2A(zz) values were increased unless the maximal values had been reached already in the absence of coenzyme. In all these experiments only a single ESR spectral component was observed. Similar results were obtained for the enzyme in liposomes, although the effect was less pronounced apparently due to the higher mobility of the probe. It is concluded that PC is motionally restricted by (R)-3Hydroxybutyrate Dehydrogenase and yet is in rapid exchange with the bulk lipid on the ESR time scale. PC is required for formation of tight and functional complexes with NAD [Rudy et al. (1989) Biochemistry 28, 5354-5366], and such complexes strengthen the interaction of the enzyme with PC.