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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

Effects of oxamate on the catalytic reactions at pH 8.0. a-c) The saturation curves for oxamate. The reaction velocities for FNLDH (a), PALDH (b), and ECLDH (c) were measured in 50 mM Bicine-NaOH buffer (pH 8.0) containing 0.1 mM NADH, 2.5 mM (for FNLDH), 1.2 mM (for PALDH), or 7.5 mM (for ECLDH) pyruvate, and the indicated concentration of oxamate. The data for FNLDH, PALDH, and ECLDH were interpreted using the equation for competitive-type, mix-type, and noncompetitive-type inhibition of allosteric enzyme. The kinetic parameters were as follows; FNLDH: Ki = 51 ± 10 (mM), kcat’ = 80 ± 20 (s−1), nH’ = 1.1 ± 0.1, and Kact = 9.9 ± 3 (mM). PALDH: KiKi’ / (Ki + Ki’) = 2.0 ± 0.6 (mM), kcat’ = 470 ± 100 (s−1), nH’ = 1.7 ± 0.2, and Kact = 1.7 ± 0.4 (mM). ECLDH: Ki = 80 ± 3 (mM). The lines indicate the calculated saturation curves obtained with kinetic parameters. d-f) the saturation curves for pyruvate with or without oxamate. The reaction velocities for FNLDH (d), PALDH (e), and ECLDH (f) were measured in 50 mM Bicine-NaOH buffer (pH 8.0) in the presence of 0.1 mM NADH and the indicated concentrations of pyruvate with no effector (open circles), or 20 mM (for FNLDH), 2.5 mM (for PALDH), or 30 mM (for ECLDH) oxamate (closed circles). The lines indicate the calculated saturation curves obtained with kinetic parameters.
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Figure 5: Effects of oxamate on the catalytic reactions at pH 8.0. a-c) The saturation curves for oxamate. The reaction velocities for FNLDH (a), PALDH (b), and ECLDH (c) were measured in 50 mM Bicine-NaOH buffer (pH 8.0) containing 0.1 mM NADH, 2.5 mM (for FNLDH), 1.2 mM (for PALDH), or 7.5 mM (for ECLDH) pyruvate, and the indicated concentration of oxamate. The data for FNLDH, PALDH, and ECLDH were interpreted using the equation for competitive-type, mix-type, and noncompetitive-type inhibition of allosteric enzyme. The kinetic parameters were as follows; FNLDH: Ki = 51 ± 10 (mM), kcat’ = 80 ± 20 (s−1), nH’ = 1.1 ± 0.1, and Kact = 9.9 ± 3 (mM). PALDH: KiKi’ / (Ki + Ki’) = 2.0 ± 0.6 (mM), kcat’ = 470 ± 100 (s−1), nH’ = 1.7 ± 0.2, and Kact = 1.7 ± 0.4 (mM). ECLDH: Ki = 80 ± 3 (mM). The lines indicate the calculated saturation curves obtained with kinetic parameters. d-f) the saturation curves for pyruvate with or without oxamate. The reaction velocities for FNLDH (d), PALDH (e), and ECLDH (f) were measured in 50 mM Bicine-NaOH buffer (pH 8.0) in the presence of 0.1 mM NADH and the indicated concentrations of pyruvate with no effector (open circles), or 20 mM (for FNLDH), 2.5 mM (for PALDH), or 30 mM (for ECLDH) oxamate (closed circles). The lines indicate the calculated saturation curves obtained with kinetic parameters.

Mentions: Oxamate, an inert pyruvate analogue, inhibited the reactions of the three enzymes at pH 7.0, where the enzymes showed no marked homotropic cooperativity, in different manners, i.e. an apparently competitive manner with pyruvate for FNLDH, and a mixed manner for PALDH and ECLDH (Figure 4). This suggests that oxamate is bound not only to the catalytic site, but also to unknown allosteric sites in PALDH and ECLDH, whereas it is bound mostly to the catalytic site in FNLDH. Oxamate apparently enhanced the reactions of FNLDH and PALDH, and exhibited the highest activating effects at 20 mM and 2.5 mM, respectively (Figure 5a, b), whereas oxamate inhibited the ECLDH reaction (Figure 5c), at pH 8.0, where the three enzymes exhibited marked homotropic cooperativity. FNLDH and PALDH showed no significant homotropic (positive or negative) cooperativity in the presence of 20 mM and 2.5 mM oxamate, respectively (Figure 5d, e). In ECLDH, the inhibition occurred in a noncompetitive manner toward pyruvate (Figure 5f), suggesting that it is mostly bound to the allosteric site of ECLDH at pH 8.0.


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)

Effects of oxamate on the catalytic reactions at pH 8.0. a-c) The saturation curves for oxamate. The reaction velocities for FNLDH (a), PALDH (b), and ECLDH (c) were measured in 50 mM Bicine-NaOH buffer (pH 8.0) containing 0.1 mM NADH, 2.5 mM (for FNLDH), 1.2 mM (for PALDH), or 7.5 mM (for ECLDH) pyruvate, and the indicated concentration of oxamate. The data for FNLDH, PALDH, and ECLDH were interpreted using the equation for competitive-type, mix-type, and noncompetitive-type inhibition of allosteric enzyme. The kinetic parameters were as follows; FNLDH: Ki = 51 ± 10 (mM), kcat’ = 80 ± 20 (s−1), nH’ = 1.1 ± 0.1, and Kact = 9.9 ± 3 (mM). PALDH: KiKi’ / (Ki + Ki’) = 2.0 ± 0.6 (mM), kcat’ = 470 ± 100 (s−1), nH’ = 1.7 ± 0.2, and Kact = 1.7 ± 0.4 (mM). ECLDH: Ki = 80 ± 3 (mM). The lines indicate the calculated saturation curves obtained with kinetic parameters. d-f) the saturation curves for pyruvate with or without oxamate. The reaction velocities for FNLDH (d), PALDH (e), and ECLDH (f) were measured in 50 mM Bicine-NaOH buffer (pH 8.0) in the presence of 0.1 mM NADH and the indicated concentrations of pyruvate with no effector (open circles), or 20 mM (for FNLDH), 2.5 mM (for PALDH), or 30 mM (for ECLDH) oxamate (closed circles). The lines indicate the calculated saturation curves obtained with kinetic parameters.
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Figure 5: Effects of oxamate on the catalytic reactions at pH 8.0. a-c) The saturation curves for oxamate. The reaction velocities for FNLDH (a), PALDH (b), and ECLDH (c) were measured in 50 mM Bicine-NaOH buffer (pH 8.0) containing 0.1 mM NADH, 2.5 mM (for FNLDH), 1.2 mM (for PALDH), or 7.5 mM (for ECLDH) pyruvate, and the indicated concentration of oxamate. The data for FNLDH, PALDH, and ECLDH were interpreted using the equation for competitive-type, mix-type, and noncompetitive-type inhibition of allosteric enzyme. The kinetic parameters were as follows; FNLDH: Ki = 51 ± 10 (mM), kcat’ = 80 ± 20 (s−1), nH’ = 1.1 ± 0.1, and Kact = 9.9 ± 3 (mM). PALDH: KiKi’ / (Ki + Ki’) = 2.0 ± 0.6 (mM), kcat’ = 470 ± 100 (s−1), nH’ = 1.7 ± 0.2, and Kact = 1.7 ± 0.4 (mM). ECLDH: Ki = 80 ± 3 (mM). The lines indicate the calculated saturation curves obtained with kinetic parameters. d-f) the saturation curves for pyruvate with or without oxamate. The reaction velocities for FNLDH (d), PALDH (e), and ECLDH (f) were measured in 50 mM Bicine-NaOH buffer (pH 8.0) in the presence of 0.1 mM NADH and the indicated concentrations of pyruvate with no effector (open circles), or 20 mM (for FNLDH), 2.5 mM (for PALDH), or 30 mM (for ECLDH) oxamate (closed circles). The lines indicate the calculated saturation curves obtained with kinetic parameters.
Mentions: Oxamate, an inert pyruvate analogue, inhibited the reactions of the three enzymes at pH 7.0, where the enzymes showed no marked homotropic cooperativity, in different manners, i.e. an apparently competitive manner with pyruvate for FNLDH, and a mixed manner for PALDH and ECLDH (Figure 4). This suggests that oxamate is bound not only to the catalytic site, but also to unknown allosteric sites in PALDH and ECLDH, whereas it is bound mostly to the catalytic site in FNLDH. Oxamate apparently enhanced the reactions of FNLDH and PALDH, and exhibited the highest activating effects at 20 mM and 2.5 mM, respectively (Figure 5a, b), whereas oxamate inhibited the ECLDH reaction (Figure 5c), at pH 8.0, where the three enzymes exhibited marked homotropic cooperativity. FNLDH and PALDH showed no significant homotropic (positive or negative) cooperativity in the presence of 20 mM and 2.5 mM oxamate, respectively (Figure 5d, e). In ECLDH, the inhibition occurred in a noncompetitive manner toward pyruvate (Figure 5f), suggesting that it is mostly bound to the allosteric site of ECLDH at pH 8.0.

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