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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 intermediary metabolites and ions at pH 8.0. a) Effects of intermediary metabolites and ions on FNLDH (white boxes), PALDH (grey boxes), and ECLDH (black boxes). The activities 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 1 mM indicated effectors. b-d) the saturation curves for FBP or MgCl2. The reaction velocities for FNLDH (b), PALDH (c), and ECLDH (d) 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 concentrations of FBP (open circles) or MgCl2 (closed circles). The lines indicate the calculated saturation curves obtained with apparent kinetic parameters (Table 2). The data of MgCl2 saturation curve of ECLDH was not fitted with any equations used in this study. e-g) the saturation curves for pyruvate with or without FBP or MgCl2. The reaction velocities for FNLDH (e), PALDH (f), and ECLDH (g) were measured in 50 mM Bicine-NaOH buffer (pH 8.0) containing 0.1 mM NADH, the indicated concentrations of pyruvate with no effector (white circles), 10 mM (for FNLDH and PALDH) or 5 mM (for ECLDH) FBP (grey circles), and 5 mM (for FNLDH and PALDH) or 2.4 mM (for ECLDH) MgCl2 (black circles). The lines indicate the calculated saturation curves obtained with kinetic parameters (Table 3).
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Figure 6: Effects of intermediary metabolites and ions at pH 8.0. a) Effects of intermediary metabolites and ions on FNLDH (white boxes), PALDH (grey boxes), and ECLDH (black boxes). The activities 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 1 mM indicated effectors. b-d) the saturation curves for FBP or MgCl2. The reaction velocities for FNLDH (b), PALDH (c), and ECLDH (d) 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 concentrations of FBP (open circles) or MgCl2 (closed circles). The lines indicate the calculated saturation curves obtained with apparent kinetic parameters (Table 2). The data of MgCl2 saturation curve of ECLDH was not fitted with any equations used in this study. e-g) the saturation curves for pyruvate with or without FBP or MgCl2. The reaction velocities for FNLDH (e), PALDH (f), and ECLDH (g) were measured in 50 mM Bicine-NaOH buffer (pH 8.0) containing 0.1 mM NADH, the indicated concentrations of pyruvate with no effector (white circles), 10 mM (for FNLDH and PALDH) or 5 mM (for ECLDH) FBP (grey circles), and 5 mM (for FNLDH and PALDH) or 2.4 mM (for ECLDH) MgCl2 (black circles). The lines indicate the calculated saturation curves obtained with kinetic parameters (Table 3).

Mentions: Bacterial allosteric l-LDHs are commonly activated by FBP, which usually induces drastic improvement of the substrate S0.5 values of the enzymes (Garvie [1980]). In addition, Lactobacillus caseil-LDH requires some divalent metal ions (e.g., Mn2+) (Arai et al. [2011]), and the Thermus caldophilus enzyme is activated also by citrate under slightly acidic conditions (Taguchi et al. [1984]). We therefore evaluated the effects of FBP, citrate and divalent metal ions at pH 8.0 in the presence of the S0.5 pyruvate. FBP and citrate (1.0 mM) slightly enhanced the catalytic reactions of the three d-LDHs, and divalent metal ions such as Mg2+ and Mn2+ more markedly enhanced the reactions of FNLDH and PALDH (Figure 6a). In the case of FNLDH, Mg2+ and FBP showed significant activation effects, and Mg2+ gave a 2.8-fold smaller S0.5 value (1.5 mM) than FBP (4.2 mM) for the enzyme activation (Figure 6 and Table 2). In the presence of 10 mM FBP or 5 mM Mg2+, FNLDH exhibited hyperbolic pyruvate saturation curves, the pyruvate S0.5 (Km) value being reduced by approximately 1.4-fold or twice, respectively (Figure 6, and Table 3). For PALDH, Mg2+ and FBP also showed activation effects, and Mg2+ gave a 75-fold smaller S0.5 value (0.16 mM) than FBP (12 mM) for the enzyme activation (Figure 6 and Table 2). In this case, FBP and Mg2+ also consistently reduced the pyruvate S0.5 value significantly, and the negative cooperativity in pyruvate binding (Figure 6, and Table 3). In the case of ECLDH, FBP and Mg2+ exhibited only slight activation effects on the enzyme reaction, and the latter even markedly inhibited the reaction at high concentrations (Figure 6, and Tables 2 and 3).


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 intermediary metabolites and ions at pH 8.0. a) Effects of intermediary metabolites and ions on FNLDH (white boxes), PALDH (grey boxes), and ECLDH (black boxes). The activities 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 1 mM indicated effectors. b-d) the saturation curves for FBP or MgCl2. The reaction velocities for FNLDH (b), PALDH (c), and ECLDH (d) 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 concentrations of FBP (open circles) or MgCl2 (closed circles). The lines indicate the calculated saturation curves obtained with apparent kinetic parameters (Table 2). The data of MgCl2 saturation curve of ECLDH was not fitted with any equations used in this study. e-g) the saturation curves for pyruvate with or without FBP or MgCl2. The reaction velocities for FNLDH (e), PALDH (f), and ECLDH (g) were measured in 50 mM Bicine-NaOH buffer (pH 8.0) containing 0.1 mM NADH, the indicated concentrations of pyruvate with no effector (white circles), 10 mM (for FNLDH and PALDH) or 5 mM (for ECLDH) FBP (grey circles), and 5 mM (for FNLDH and PALDH) or 2.4 mM (for ECLDH) MgCl2 (black circles). The lines indicate the calculated saturation curves obtained with kinetic parameters (Table 3).
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Figure 6: Effects of intermediary metabolites and ions at pH 8.0. a) Effects of intermediary metabolites and ions on FNLDH (white boxes), PALDH (grey boxes), and ECLDH (black boxes). The activities 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 1 mM indicated effectors. b-d) the saturation curves for FBP or MgCl2. The reaction velocities for FNLDH (b), PALDH (c), and ECLDH (d) 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 concentrations of FBP (open circles) or MgCl2 (closed circles). The lines indicate the calculated saturation curves obtained with apparent kinetic parameters (Table 2). The data of MgCl2 saturation curve of ECLDH was not fitted with any equations used in this study. e-g) the saturation curves for pyruvate with or without FBP or MgCl2. The reaction velocities for FNLDH (e), PALDH (f), and ECLDH (g) were measured in 50 mM Bicine-NaOH buffer (pH 8.0) containing 0.1 mM NADH, the indicated concentrations of pyruvate with no effector (white circles), 10 mM (for FNLDH and PALDH) or 5 mM (for ECLDH) FBP (grey circles), and 5 mM (for FNLDH and PALDH) or 2.4 mM (for ECLDH) MgCl2 (black circles). The lines indicate the calculated saturation curves obtained with kinetic parameters (Table 3).
Mentions: Bacterial allosteric l-LDHs are commonly activated by FBP, which usually induces drastic improvement of the substrate S0.5 values of the enzymes (Garvie [1980]). In addition, Lactobacillus caseil-LDH requires some divalent metal ions (e.g., Mn2+) (Arai et al. [2011]), and the Thermus caldophilus enzyme is activated also by citrate under slightly acidic conditions (Taguchi et al. [1984]). We therefore evaluated the effects of FBP, citrate and divalent metal ions at pH 8.0 in the presence of the S0.5 pyruvate. FBP and citrate (1.0 mM) slightly enhanced the catalytic reactions of the three d-LDHs, and divalent metal ions such as Mg2+ and Mn2+ more markedly enhanced the reactions of FNLDH and PALDH (Figure 6a). In the case of FNLDH, Mg2+ and FBP showed significant activation effects, and Mg2+ gave a 2.8-fold smaller S0.5 value (1.5 mM) than FBP (4.2 mM) for the enzyme activation (Figure 6 and Table 2). In the presence of 10 mM FBP or 5 mM Mg2+, FNLDH exhibited hyperbolic pyruvate saturation curves, the pyruvate S0.5 (Km) value being reduced by approximately 1.4-fold or twice, respectively (Figure 6, and Table 3). For PALDH, Mg2+ and FBP also showed activation effects, and Mg2+ gave a 75-fold smaller S0.5 value (0.16 mM) than FBP (12 mM) for the enzyme activation (Figure 6 and Table 2). In this case, FBP and Mg2+ also consistently reduced the pyruvate S0.5 value significantly, and the negative cooperativity in pyruvate binding (Figure 6, and Table 3). In the case of ECLDH, FBP and Mg2+ exhibited only slight activation effects on the enzyme reaction, and the latter even markedly inhibited the reaction at high concentrations (Figure 6, and Tables 2 and 3).

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