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Diverse allosteric and catalytic functions of tetrameric d-lactate dehydrogenases from three Gram-negative bacteria.

Furukawa N, Miyanaga A, Togawa M, Nakajima M, Taguchi H - AMB Express (2014)

Bottom Line: NAD-dependent d-lactate dehydrogenases (d-LDHs) reduce pyruvate into d-lactate with oxidation of NADH into NAD(+).Fructose 1,6-bisphosphate and certain divalent metal ions such as Mg(2+) also markedly enhanced the reactions of FNLDH and PALDH, but none of them enhanced the reaction of ECLDH.Thus, our study demonstrates that bacterial d-LDHs have highly divergent allosteric and catalytic properties.

View Article: PubMed Central - HTML - PubMed

Affiliation: Tokyo University of Science, 2641 Yamazaki, Noda 278-8510, Chiba, Japan.

ABSTRACT
NAD-dependent d-lactate dehydrogenases (d-LDHs) reduce pyruvate into d-lactate with oxidation of NADH into NAD(+). Although non-allosteric d-LDHs from Lactobacilli have been extensively studied, the catalytic properties of allosteric d-LDHs from Gram-negative bacteria except for Escherichia coli remain unknown. We characterized the catalytic properties of d-LDHs from three Gram-negative bacteria, Fusobacterium nucleatum (FNLDH), Pseudomonas aeruginosa (PALDH), and E. coli (ECLDH) to gain an insight into allosteric mechanism of d-LDHs. While PALDH and ECLDH exhibited narrow substrate specificities toward pyruvate like usual d-LDHs, FNLDH exhibited a broad substrate specificity toward hydrophobic 2-ketoacids such as 2-ketobutyrate and 2-ketovalerate, the former of which gave a 2-fold higher k cat/S0.5 value than pyruvate. Whereas the three enzymes consistently showed hyperbolic shaped pyruvate saturation curves below pH 6.5, FNLDH and ECLDH, and PALDH showed marked positive and negative cooperativity, respectively, in the pyruvate saturation curves above pH 7.5. Oxamate inhibited the catalytic reactions of FNLDH competitively with pyruvate, and the PALDH reaction in a mixed manner at pH 7.0, but markedly enhanced the reactions of the two enzymes at low concentration through canceling of the apparent homotropic cooperativity at pH 8.0, although it constantly inhibited the ECLDH reaction. Fructose 1,6-bisphosphate and certain divalent metal ions such as Mg(2+) also markedly enhanced the reactions of FNLDH and PALDH, but none of them enhanced the reaction of ECLDH. Thus, our study demonstrates that bacterial d-LDHs have highly divergent allosteric and catalytic properties.

No MeSH data available.


Related in: MedlinePlus

pH dependence of kinetic parameters on pyruvate reduction. White, grey, and black circles indicate FNLDH, PALDH, and ECLDH, respectively. The kcat(a), S0.5(b), and kcat/S0.5 data (c) are plotted logarithmically, and the Hill coefficient (d) is plotted linearly. The buffers used for the assay were sodium acetate buffer (pH 4.5, 5.0, and 5.5; circles), MES-NaOH buffer (pH 5.5, 6.0, and 6.5; triangles), MOPS-NaOH buffer (pH 6.5, 7.0, and 7.5; squares), HEPES-NaOH buffer (pH 7.0, 7.5 and 8.0; diamonds), and Bicine-NaOH buffer (pH 8.0, 8.5, and 9.0; hexagons). We adjusted the pH of each buffer solution prior to the addition of substrate and cofactor, and confirmed that the pH value is not affected even in the presence of high concentrations of substrate.
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Figure 3: pH dependence of kinetic parameters on pyruvate reduction. White, grey, and black circles indicate FNLDH, PALDH, and ECLDH, respectively. The kcat(a), S0.5(b), and kcat/S0.5 data (c) are plotted logarithmically, and the Hill coefficient (d) is plotted linearly. The buffers used for the assay were sodium acetate buffer (pH 4.5, 5.0, and 5.5; circles), MES-NaOH buffer (pH 5.5, 6.0, and 6.5; triangles), MOPS-NaOH buffer (pH 6.5, 7.0, and 7.5; squares), HEPES-NaOH buffer (pH 7.0, 7.5 and 8.0; diamonds), and Bicine-NaOH buffer (pH 8.0, 8.5, and 9.0; hexagons). We adjusted the pH of each buffer solution prior to the addition of substrate and cofactor, and confirmed that the pH value is not affected even in the presence of high concentrations of substrate.

Mentions: The kinetic parameters, kcat, S0.5, kcat/S0.5, and nH values, were determined by pyruvate reduction assaying in the pH range of 4.5-9.0 (Figure 3). The three enzymes consistently showed virtually constant kcat values independently of pH, and PALDH and ECLDH exhibited about 5-fold larger kcat values than FNLDH. The three enzymes also consistently showed constant pyruvate S0.5 values below pH 7.0, and PALDH and FNLDH exhibited approximately one order of magnitude smaller S0.5 values than ECLDH. Above pH 8.0, the three enzymes exhibited increased S0.5 values, depending on the pH, and their kcat/S0.5 values changed mostly according to the changes in the S0.5 values. ECLDH apparently showed lower pH-dependence of the S0.5 value than the other two enzymes, of which PALDH showed slightly higher pH-dependence than FNLDH. Under acidic conditions below pH 6.0, the three enzymes consistently showed hyperbolic shaped pyruvate saturation curves, and no marked cooperativity in pyruvate binding. Under higher pH conditions, however, FNLDH and ECLDH showed markedly sigmoidal saturation curves for pyruvate, i.e., positive homotropic cooperativity, giving maximal nH values of about 2.0. In contrast, PALDH showed negative homotropic cooperativitiy above pH 8.0, giving a minimal nH value of 0.5.


Diverse allosteric and catalytic functions of tetrameric d-lactate dehydrogenases from three Gram-negative bacteria.

Furukawa N, Miyanaga A, Togawa M, Nakajima M, Taguchi H - AMB Express (2014)

pH dependence of kinetic parameters on pyruvate reduction. White, grey, and black circles indicate FNLDH, PALDH, and ECLDH, respectively. The kcat(a), S0.5(b), and kcat/S0.5 data (c) are plotted logarithmically, and the Hill coefficient (d) is plotted linearly. The buffers used for the assay were sodium acetate buffer (pH 4.5, 5.0, and 5.5; circles), MES-NaOH buffer (pH 5.5, 6.0, and 6.5; triangles), MOPS-NaOH buffer (pH 6.5, 7.0, and 7.5; squares), HEPES-NaOH buffer (pH 7.0, 7.5 and 8.0; diamonds), and Bicine-NaOH buffer (pH 8.0, 8.5, and 9.0; hexagons). We adjusted the pH of each buffer solution prior to the addition of substrate and cofactor, and confirmed that the pH value is not affected even in the presence of high concentrations of substrate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230899&req=5

Figure 3: pH dependence of kinetic parameters on pyruvate reduction. White, grey, and black circles indicate FNLDH, PALDH, and ECLDH, respectively. The kcat(a), S0.5(b), and kcat/S0.5 data (c) are plotted logarithmically, and the Hill coefficient (d) is plotted linearly. The buffers used for the assay were sodium acetate buffer (pH 4.5, 5.0, and 5.5; circles), MES-NaOH buffer (pH 5.5, 6.0, and 6.5; triangles), MOPS-NaOH buffer (pH 6.5, 7.0, and 7.5; squares), HEPES-NaOH buffer (pH 7.0, 7.5 and 8.0; diamonds), and Bicine-NaOH buffer (pH 8.0, 8.5, and 9.0; hexagons). We adjusted the pH of each buffer solution prior to the addition of substrate and cofactor, and confirmed that the pH value is not affected even in the presence of high concentrations of substrate.
Mentions: The kinetic parameters, kcat, S0.5, kcat/S0.5, and nH values, were determined by pyruvate reduction assaying in the pH range of 4.5-9.0 (Figure 3). The three enzymes consistently showed virtually constant kcat values independently of pH, and PALDH and ECLDH exhibited about 5-fold larger kcat values than FNLDH. The three enzymes also consistently showed constant pyruvate S0.5 values below pH 7.0, and PALDH and FNLDH exhibited approximately one order of magnitude smaller S0.5 values than ECLDH. Above pH 8.0, the three enzymes exhibited increased S0.5 values, depending on the pH, and their kcat/S0.5 values changed mostly according to the changes in the S0.5 values. ECLDH apparently showed lower pH-dependence of the S0.5 value than the other two enzymes, of which PALDH showed slightly higher pH-dependence than FNLDH. Under acidic conditions below pH 6.0, the three enzymes consistently showed hyperbolic shaped pyruvate saturation curves, and no marked cooperativity in pyruvate binding. Under higher pH conditions, however, FNLDH and ECLDH showed markedly sigmoidal saturation curves for pyruvate, i.e., positive homotropic cooperativity, giving maximal nH values of about 2.0. In contrast, PALDH showed negative homotropic cooperativitiy above pH 8.0, giving a minimal nH value of 0.5.

Bottom Line: NAD-dependent d-lactate dehydrogenases (d-LDHs) reduce pyruvate into d-lactate with oxidation of NADH into NAD(+).Fructose 1,6-bisphosphate and certain divalent metal ions such as Mg(2+) also markedly enhanced the reactions of FNLDH and PALDH, but none of them enhanced the reaction of ECLDH.Thus, our study demonstrates that bacterial d-LDHs have highly divergent allosteric and catalytic properties.

View Article: PubMed Central - HTML - PubMed

Affiliation: Tokyo University of Science, 2641 Yamazaki, Noda 278-8510, Chiba, Japan.

ABSTRACT
NAD-dependent d-lactate dehydrogenases (d-LDHs) reduce pyruvate into d-lactate with oxidation of NADH into NAD(+). Although non-allosteric d-LDHs from Lactobacilli have been extensively studied, the catalytic properties of allosteric d-LDHs from Gram-negative bacteria except for Escherichia coli remain unknown. We characterized the catalytic properties of d-LDHs from three Gram-negative bacteria, Fusobacterium nucleatum (FNLDH), Pseudomonas aeruginosa (PALDH), and E. coli (ECLDH) to gain an insight into allosteric mechanism of d-LDHs. While PALDH and ECLDH exhibited narrow substrate specificities toward pyruvate like usual d-LDHs, FNLDH exhibited a broad substrate specificity toward hydrophobic 2-ketoacids such as 2-ketobutyrate and 2-ketovalerate, the former of which gave a 2-fold higher k cat/S0.5 value than pyruvate. Whereas the three enzymes consistently showed hyperbolic shaped pyruvate saturation curves below pH 6.5, FNLDH and ECLDH, and PALDH showed marked positive and negative cooperativity, respectively, in the pyruvate saturation curves above pH 7.5. Oxamate inhibited the catalytic reactions of FNLDH competitively with pyruvate, and the PALDH reaction in a mixed manner at pH 7.0, but markedly enhanced the reactions of the two enzymes at low concentration through canceling of the apparent homotropic cooperativity at pH 8.0, although it constantly inhibited the ECLDH reaction. Fructose 1,6-bisphosphate and certain divalent metal ions such as Mg(2+) also markedly enhanced the reactions of FNLDH and PALDH, but none of them enhanced the reaction of ECLDH. Thus, our study demonstrates that bacterial d-LDHs have highly divergent allosteric and catalytic properties.

No MeSH data available.


Related in: MedlinePlus