The Experts below are selected from a list of 189 Experts worldwide ranked by ideXlab platform
J O Mcintyre - One of the best experts on this subject based on the ideXlab platform.
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r 3 Hydroxybutyrate Dehydrogenase selective phosphatidylcholine binding by the c terminal domain
Biochemistry, 2000Co-Authors: C Loebhennard, J O McintyreAbstract:(R)-3-Hydroxybutyrate 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...
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phosphatidylcholine activation of human heart r 3 Hydroxybutyrate Dehydrogenase mutants lacking active center sulfhydryls site directed mutagenesis of a new recombinant fusion protein
Biochemistry, 2000Co-Authors: D Chelius, Sidney Fleischer, C Loebhennard, J O Mcintyre, Andrew R Marks, S De, S Hahn, M M Jehl, J Moeller, Reinhard PhilippAbstract:(R)-3-Hydroxybutyrate 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)-3-Hydroxybutyrate (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...
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wild type and mutant human heart r 3 Hydroxybutyrate Dehydrogenase expressed in insect cells
Biochemistry, 1996Co-Authors: David Green, Sidney Fleischer, Andrew R Marks, J O McintyreAbstract:(R)-3-Hydroxybutyrate 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 ...
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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, 1996Co-Authors: K Klein, Sidney Fleischer, J O Mcintyre, B Rudy, Wolfgang E TrommerAbstract:: The interaction of phospholipid with (R)-3-Hydroxybutyrate 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)-3-Hydroxybutyrate 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)-3-Hydroxybutyrate 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)-3-Hydroxybutyrate 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.
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effect of selective thiol group derivatization on enzyme kinetics of r 3 Hydroxybutyrate Dehydrogenase
Biochemical Journal, 1993Co-Authors: L A Dalton, J O Mcintyre, Sidney FleischerAbstract:(R)-3-Hydroxybutyrate Dehydrogenase (BDH) is a phosphatidylcholine-requiring tetrameric enzyme with two thiol groups (SH-1 and SH-2) per protomer. By first protecting the more rapidly reacting thiol group (SH-1) with diamide [1,1'-azobis-(NN'-dimethylformamide), DM] to form DM(SH-1)BDH, SH-2 can be selectively derivatized by reaction with maleimide reagents such as 4-maleimido-2,2,6,6-tetramethyl-piperidine-N-oxyl (MSL), which gives DM(SH-1)MSL(SH-2)BDH. Reduction with dithiothreitol (DTT) regenerates SH-1, yielding MAL(SH-2)BDH (where MAL is the diamagnetic reduction product of MSL-BDH and DTT). The enzymic activity of DM(SH-1)BDH is decreased to approx. 4% relative to the native purified enzyme, and the apparent Km for substrate, KmBOH, is increased approx. 100-fold. Reduction of DM(SH-1)BDH with DTT regenerates SH-1 and restores normal enzymic function. Modification of SH-2 with piperidinylmaleimide [MAL(SH-2)BDH] diminishes enzymic activity to approx. 35% of its original value, but has no significant effect on apparent KmBOH. The doubly derivatized enzyme, DM(SH-1)MSL(SH-2)BDH, has lower enzymic activity [about half that for DM(SH-2)BDH] and a yet higher apparent KmBOH than DM(SH-1)BDH. Derivatization of SH-2 with different maleimide reagents results in diminished activity approximately proportional to the size of the maleimide substituent, suggesting that this inhibition is steric. Whereas modification of SH-1 results in marked changes in kinetic parameters (increased apparent Km and reduced apparent Vmax), derivatization of SH-2 has a lesser effect on enzymic function. Thus SH-1 is postulated to be closer to the active centre than is SH-2, although neither is involved in catalysis, since: (1) the activity of the derivatized enzyme is not abolished; and (2) activity can be enhanced by increasing substrate (and cofactor) concentrations.
Trevor C Charles - One of the best experts on this subject based on the ideXlab platform.
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characterization of bdha encoding the enzyme d 3 Hydroxybutyrate Dehydrogenase from sinorhizobium sp strain ngr234
Fems Microbiology Letters, 2005Co-Authors: Punita Aneja, Trevor C CharlesAbstract: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 -3-Hydroxybutyrate 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 -3-Hydroxybutyrate Dehydrogenase activity, and was unable to grow on D -3-Hydroxybutyrate as sole carbon source. NGRPA2 exhibited symbiotic defects on Leucaena but not on Vigna, Macroptilium or Tephrosia host plants. Furthermore, the D -3-Hydroxybutyrate 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.
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poly 3 Hydroxybutyrate degradation in rhizobium sinorhizobium meliloti isolation and characterization of a gene encoding 3 Hydroxybutyrate Dehydrogenase
Journal of Bacteriology, 1999Co-Authors: Punita Aneja, Trevor C CharlesAbstract:We have cloned and sequenced the 3-Hydroxybutyrate 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 3-Hydroxybutyrate as a sole carbon source. The R. meliloti bdhA gene is able to confer upon Escherichia coli the ability to utilize 3-Hydroxybutyrate as a sole carbon source. An R. meliloti bdhA mutant accumulates poly-3-Hydroxybutyrate 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 - One of the best experts on this subject based on the ideXlab platform.
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phosphatidylcholine activation of human heart r 3 Hydroxybutyrate Dehydrogenase mutants lacking active center sulfhydryls site directed mutagenesis of a new recombinant fusion protein
Biochemistry, 2000Co-Authors: D Chelius, Sidney Fleischer, C Loebhennard, J O Mcintyre, Andrew R Marks, S De, S Hahn, M M Jehl, J Moeller, Reinhard PhilippAbstract:(R)-3-Hydroxybutyrate 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)-3-Hydroxybutyrate (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...
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wild type and mutant human heart r 3 Hydroxybutyrate Dehydrogenase expressed in insect cells
Biochemistry, 1996Co-Authors: David Green, Sidney Fleischer, Andrew R Marks, J O McintyreAbstract:(R)-3-Hydroxybutyrate 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 ...
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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, 1996Co-Authors: K Klein, Sidney Fleischer, J O Mcintyre, B Rudy, Wolfgang E TrommerAbstract:: The interaction of phospholipid with (R)-3-Hydroxybutyrate 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)-3-Hydroxybutyrate 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)-3-Hydroxybutyrate 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)-3-Hydroxybutyrate 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.
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effect of selective thiol group derivatization on enzyme kinetics of r 3 Hydroxybutyrate Dehydrogenase
Biochemical Journal, 1993Co-Authors: L A Dalton, J O Mcintyre, Sidney FleischerAbstract:(R)-3-Hydroxybutyrate Dehydrogenase (BDH) is a phosphatidylcholine-requiring tetrameric enzyme with two thiol groups (SH-1 and SH-2) per protomer. By first protecting the more rapidly reacting thiol group (SH-1) with diamide [1,1'-azobis-(NN'-dimethylformamide), DM] to form DM(SH-1)BDH, SH-2 can be selectively derivatized by reaction with maleimide reagents such as 4-maleimido-2,2,6,6-tetramethyl-piperidine-N-oxyl (MSL), which gives DM(SH-1)MSL(SH-2)BDH. Reduction with dithiothreitol (DTT) regenerates SH-1, yielding MAL(SH-2)BDH (where MAL is the diamagnetic reduction product of MSL-BDH and DTT). The enzymic activity of DM(SH-1)BDH is decreased to approx. 4% relative to the native purified enzyme, and the apparent Km for substrate, KmBOH, is increased approx. 100-fold. Reduction of DM(SH-1)BDH with DTT regenerates SH-1 and restores normal enzymic function. Modification of SH-2 with piperidinylmaleimide [MAL(SH-2)BDH] diminishes enzymic activity to approx. 35% of its original value, but has no significant effect on apparent KmBOH. The doubly derivatized enzyme, DM(SH-1)MSL(SH-2)BDH, has lower enzymic activity [about half that for DM(SH-2)BDH] and a yet higher apparent KmBOH than DM(SH-1)BDH. Derivatization of SH-2 with different maleimide reagents results in diminished activity approximately proportional to the size of the maleimide substituent, suggesting that this inhibition is steric. Whereas modification of SH-1 results in marked changes in kinetic parameters (increased apparent Km and reduced apparent Vmax), derivatization of SH-2 has a lesser effect on enzymic function. Thus SH-1 is postulated to be closer to the active centre than is SH-2, although neither is involved in catalysis, since: (1) the activity of the derivatized enzyme is not abolished; and (2) activity can be enhanced by increasing substrate (and cofactor) concentrations.
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monoclonal antibodies for structure function studies of r 3 Hydroxybutyrate Dehydrogenase a lipid dependent membrane bound enzyme
Biochemical Journal, 1993Co-Authors: N Latruffe, J O Mcintyre, T M Duncan, P Adami, C E Carter, Chi Fu, M Melin, Sidney FleischerAbstract:Monoclonal antibodies (mAbs) have been used to study structure-function relationships of (R)-3-Hydroxybutyrate Dehydrogenase (BDH) (EC 1.1.1.30), a lipid-requiring mitochondrial membrane enzyme with an absolute and specific requirement for phosphatidylcholine (PC) for enzymic activity. The purified enzyme (apoBDH, devoid of phospholipid and thereby inactive) can be re-activated with preformed phospholipid vesicles containing PC or by short-chain soluble PC. Five of six mAbs cross-react with BDH from bovine heart and rat liver, including two mAbs to conformational epitopes. One mAb was found to be specific for the C-terminal sequence of BDH and served to: (1) map endopeptidase cleavage and epitope sites on BDH; and (2) demonstrate that the C-terminus is essential for the activity of BDH. Carboxypeptidase cleavage of only a few (< or = 14) C-terminal amino acids from apoBDH (as detected by the loss of C-terminal epitope for mAb 3-10A) prevents activation by either bilayer or soluble PC. Further, for BDH in bilayers containing PC, the C-terminus is protected from carboxy-peptidase cleavage, whereas in bilayers devoid of PC the C-terminus is cleaved, and subsequent activation by PC is precluded. We conclude that: (1) the C-terminus of BDH is essential for enzymic activity, consistent with the prediction, from primary sequence analysis, that the PC-binding site is in the C-terminal domain of BDH; and (2) the allosteric activation of BDH by PC in bilayers protects the C-terminus from carboxypeptidase cleavage, indicative of a PC-induced conformational change in the enzyme.
Nobutoshi Kiba - One of the best experts on this subject based on the ideXlab platform.
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determination of 3 Hydroxybutyrate in serum by flow injection analysis using a co immobilized 3 Hydroxybutyrate Dehydrogenase nadh oxidase reactor and a chemiluminometer
Analytica Chimica Acta, 1994Co-Authors: Nobutoshi Kiba, Hidekazu Koemado, Junko Inagaki, Motohisa FurusawaAbstract:Abstract A flow-injection system with a co-immobilized enzyme reactor is described for the determination of 3-Hydroxybutyrate. 3-Hydroxybutyrate Dehydrogenase and NADH oxidase were immobilized on aminated poly(vinyl alcohol) beads and packed into a stainless-steel column (5 cm × 4 mm i.d.). Serum was diluted 10-fold with borate buffer (pH 9.4) and ultrafiltered. Filtrate (20 μl) was injected into the carrier stream. The hydrogen peroxide produced was detected chemiluminometrically via a luminol-hexacyanoferrate(III) reaction. The calibration graph was linear for 2 × 10−7 M-5 × 10−4 M; the detection limit was 8 × 10−8 M and the sample throughput was 30 h−1 without carryover.
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Determination of 3-Hydroxybutyrate in serum by flow-injection analysis using a co-immobilized 3-Hydroxybutyrate Dehydrogenase/NADH oxidase reactor and a chemiluminometer
Analytica Chimica Acta, 1994Co-Authors: Nobutoshi Kiba, Hidekazu Koemado, Junko Inagaki, Motohisa FurusawaAbstract:Abstract A flow-injection system with a co-immobilized enzyme reactor is described for the determination of 3-Hydroxybutyrate. 3-Hydroxybutyrate Dehydrogenase and NADH oxidase were immobilized on aminated poly(vinyl alcohol) beads and packed into a stainless-steel column (5 cm × 4 mm i.d.). Serum was diluted 10-fold with borate buffer (pH 9.4) and ultrafiltered. Filtrate (20 μl) was injected into the carrier stream. The hydrogen peroxide produced was detected chemiluminometrically via a luminol-hexacyanoferrate(III) reaction. The calibration graph was linear for 2 × 10−7 M-5 × 10−4 M; the detection limit was 8 × 10−8 M and the sample throughput was 30 h−1 without carryover.
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flow injection determination of 3 Hydroxybutyrate in serum with an immobilized 3 Hydroxybutyrate Dehydrogenase reactor and fluorescence detection
Talanta, 1994Co-Authors: Nobutoshi Kiba, Hidekazu Koemado, Motohisa FurusawaAbstract:Abstract A flow-injection system with an immobilized enzyme reactor is proposed for the determination of 3-Hydroxybutyrate. 3-Hydroxybutyrate Dehydrogenase is immobilized on aminated poly(vinyl alcohol) beads and packed into a stainless-steel column (4 cm × 4 mm I.D.). Serum is diluted and filtered. Sample solution (20 μl) is injected into the carrier stream [4m M NAD + in glycine buffer (pH 9.3)]. The NADH formed is detected at 465 nm (excitation at 340 nm). The calibration graph is linear for 0.7–500μ M 3-Hydroxybutyrate; the detection limit is 0.5μ M .
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flow injection determination of 3 hdroxybutyrate in serum with an immobilized 3 Hydroxybutyrate Dehydrogenase reactor and chemiluminescence detection
Analytica Chimica Acta, 1994Co-Authors: Nobutoshi Kiba, Hidekazu Koemado, Motoshisa FurusawaAbstract:Abstract A flow-injection system for the determination of 3-Hydroxybutyrate in serum is described. 3-Hydroxybutyrate Dehydrogenase is immobilized on poly(vinyl alcohol) beads and incorporated in a flow-injection system. 1-Methoxy-5-methylphenazinium methylsulphate reacts with enzymatically generated NADH to give H 2 O 2 , which is detected chemiluminometrically with the reaction of luminol and hexacynoferrate(III). Serum is diluted and filtered through an ultrafiltration membrane. The system responds linearly to injected samples (80 μl) in the concentration range 0.5–300 μM; the detection limit is 0.1 μM. The within-day relative standard deviation ( n = 90) for 58 μM 3-Hydroxybutyrate in serum is 0.8%. The maximum throughout is 20 samples per hour. The immobilized enzyme is stable for at least 1 month.
Young Joo Yeon - One of the best experts on this subject based on the ideXlab platform.
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Structure-based Mutational Studies of D-3-Hydroxybutyrate Dehydrogenase for Substrate Recognition of Aliphatic Hydroxy Acids with a Variable Length of Carbon Chain
Biotechnology and Bioprocess Engineering, 2019Co-Authors: Jeong Geol Na, Young Joo YeonAbstract:Native 3-Hydroxybutyrate Dehydrogenase from Alcaligenes faecalis can catalyze the reversible reduction of acetoacetate, a four carbon chain oxo acid. This enzyme has been engineered to enable the reduction of levulinic acid, with one carbon longer than acetoacetate. In this study, the native and engineered enzymes were subjected to the catalysis of oxo acids with a carbon chain length of 3 to 8, in order to examine the capability of the enzyme to work on various platform chemicals. The engineered enzyme could reduce the C_7 and C_8 oxo acids whereas the wild-type had no activity on these substrates. Docking simulation has indicated Tyr155 and Ser142 are key residues for the catalysis. In addition, stable hydrogen bond formation between Gln196 and the substrates affects the turnover rate. Mutation sites in the engineered enzyme were focused on creating larger active site volume for substrates with extended chain lengths. Both qualitative and quantitative structural basis for the enzyme substrate specificity on alpha, beta, gamma and omega hydroxy acids could be elucidated.
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Structural basis for the substrate specificity of 3-Hydroxybutyrate Dehydrogenase
Biotechnology and Bioprocess Engineering, 2016Co-Authors: Young Joo Yeon, Hyung-yeon Park, Kyungmoon Park, Hyun June ParkAbstract:The substrate specificity of 3-Hydroxybutyrate Dehydrogenase from Alcaligenes faecalis with a non-native substrate, levulinic acid, was studied by analysis of the enzyme-substrate molecular interactions. The relation between structural and kinetic parameters was investigated considering the catalytic mechanism of the enzyme. The effects of key positive mutations (H144L, H144L/W187F) on the catalytic activity of the enzyme were studied by employing a surface analysis of its interatomic contacts between the enzyme and substrate atoms. The results revealed that the alteration of hydrogen bond network and rearrangement of the hydrophobic interactions between the active site and substrate molecule are the key structural basis for the change of the substrate specificity of 3-Hydroxybutyrate Dehydrogenase toward levulinic acid. With this approach, the structural basis for the substrate specificity of the enzyme could be elucidated in a quantitative manner.
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Effect of His-tag location on the catalytic activity of 3-Hydroxybutyrate Dehydrogenase
Biotechnology and Bioprocess Engineering, 2014Co-Authors: Young Joo Yeon, Hyun June Park, Hyung-yeon ParkAbstract:The effect of hexahistidine-tag (His-tag) location at either the C or N-terminus on the catalytic activity of 3-Hydroxybutyrate Dehydrogenase (3HBDH) from Alcaligenes faecalis was studied. The kinetic parameters of 3HBDHs with C and N-terminal His-tags were investigated, and the enzyme with an N-terminal His-tag was found to have approximately 1,200-fold higher catalytic efficiency than its C-terminal counterpart. Furthermore, the effect of His-tag location on the catalytic activity of 3 engineered variants of 3HBDH that were previously developed for the conversion of levulinic acid to 4-hydroxyvaleric acid was also investigated. All of the N-terminal variants exhibited higher catalytic efficiency for levulinic acid than did the C-terminal counterparts. The structural basis of the His-tag effect was studied by investigating the structure of 3HBDH obtained from in silico His-tag modification, and the results revealed that the modification of the C-terminal structure could deform the hinge region of the active site entry loop, disrupting the catalytic motion of the enzyme. In contrast, due to the location of the N-terminus far from the active site of the enzyme, the catalytic activity of the enzyme was not severely affected by the N-terminal His-tag.
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enzymatic reduction of levulinic acid by engineering the substrate specificity of 3 Hydroxybutyrate Dehydrogenase
Bioresource Technology, 2013Co-Authors: Young Joo Yeon, Hyung-yeon ParkAbstract:Abstract Enzymatic reduction of levulinic acid (LA) was performed for the synthesis of 4-hydroxyvaleric acid (4HV) – a monomer of bio-polyester and a precursor of bio-fuels – using 3-Hydroxybutyrate Dehydrogenase (3HBDH) from Alcaligenes faecalis . Due to the catalytic inactivity of the wild-type enzyme toward LA, engineering of the substrate specificity of the enzyme was performed. A rational design approach with molecular docking simulation was applied, and a double mutant, His144Leu/Trp187Phe, which has catalytic activity ( k cat / K m = 578.0 min −1 M −1 ) toward LA was generated. Approximately 57% conversion of LA to 4HV was achieved with this double mutant in 24 h, while no conversion was achieved with the wild-type enzyme.
