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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 7.0. The reaction velocities for FNLDH (a), PALDH (b), and ECLDH (c) were measured in 50 mM MOPS-NaOH buffer (pH 7.0) containing 0.1 mM NADH, the indicated concentrations of pyruvate and several concentrations of oxamate. The concentrations of oxamate for FNLDH were 0 mM (open circles), 10 mM (closed circles), 20 mM (open triangles), 30 mM (closed triangles), and 40 mM (open squares). The concentrations of oxamate for PALDH were 0 mM (open circles), 0.1 mM (closed circles), 0.2 mM (open triangles), 0.3 mM (closed triangles), and 0.4 mM (open squares). The concentrations of oxamate for ECLDH were 0 mM (open circles), 5 mM (closed circles), 10 mM (open triangles), 15 mM (closed triangles), and 20 mM (open squares). The reaction velocity and the concentration of pyruvate were plotted reciprocally. The data for PALDH and ECLDH were interpreted using the equation for mixed type inhibition, whereas the data for FNLDH were interpreted using the equation for competitive type inhibition. The kinetic parameters were as follows; FNLDH: kcat = 79 ± 1 (s−1), Km = 0.39 ± 0.01 (mM), and Ki = 9.4 ± 0.2 (mM). PALDH: kcat = 410 ± 10 (s−1), Km = 0.074 ± 0.006 (mM), Ki = 0.29 ± 0.04 (mM), and Ki’ = 0.33 ± 0.04 (mM). ECLDH: kcat = 670 ± 10 (s−1), Km = 5.6 ± 0.3 (mM), Ki = 20 ± 2 (mM), and Ki’ = 47 ± 6 (mM). GraFit ver 7.0.3 was used for non-linear regression and calculation of values. The lines were calculated with kinetic parameters.
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Figure 4: Effects of oxamate on the catalytic reactions at pH 7.0. The reaction velocities for FNLDH (a), PALDH (b), and ECLDH (c) were measured in 50 mM MOPS-NaOH buffer (pH 7.0) containing 0.1 mM NADH, the indicated concentrations of pyruvate and several concentrations of oxamate. The concentrations of oxamate for FNLDH were 0 mM (open circles), 10 mM (closed circles), 20 mM (open triangles), 30 mM (closed triangles), and 40 mM (open squares). The concentrations of oxamate for PALDH were 0 mM (open circles), 0.1 mM (closed circles), 0.2 mM (open triangles), 0.3 mM (closed triangles), and 0.4 mM (open squares). The concentrations of oxamate for ECLDH were 0 mM (open circles), 5 mM (closed circles), 10 mM (open triangles), 15 mM (closed triangles), and 20 mM (open squares). The reaction velocity and the concentration of pyruvate were plotted reciprocally. The data for PALDH and ECLDH were interpreted using the equation for mixed type inhibition, whereas the data for FNLDH were interpreted using the equation for competitive type inhibition. The kinetic parameters were as follows; FNLDH: kcat = 79 ± 1 (s−1), Km = 0.39 ± 0.01 (mM), and Ki = 9.4 ± 0.2 (mM). PALDH: kcat = 410 ± 10 (s−1), Km = 0.074 ± 0.006 (mM), Ki = 0.29 ± 0.04 (mM), and Ki’ = 0.33 ± 0.04 (mM). ECLDH: kcat = 670 ± 10 (s−1), Km = 5.6 ± 0.3 (mM), Ki = 20 ± 2 (mM), and Ki’ = 47 ± 6 (mM). GraFit ver 7.0.3 was used for non-linear regression and calculation of values. The lines were calculated 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 7.0. The reaction velocities for FNLDH (a), PALDH (b), and ECLDH (c) were measured in 50 mM MOPS-NaOH buffer (pH 7.0) containing 0.1 mM NADH, the indicated concentrations of pyruvate and several concentrations of oxamate. The concentrations of oxamate for FNLDH were 0 mM (open circles), 10 mM (closed circles), 20 mM (open triangles), 30 mM (closed triangles), and 40 mM (open squares). The concentrations of oxamate for PALDH were 0 mM (open circles), 0.1 mM (closed circles), 0.2 mM (open triangles), 0.3 mM (closed triangles), and 0.4 mM (open squares). The concentrations of oxamate for ECLDH were 0 mM (open circles), 5 mM (closed circles), 10 mM (open triangles), 15 mM (closed triangles), and 20 mM (open squares). The reaction velocity and the concentration of pyruvate were plotted reciprocally. The data for PALDH and ECLDH were interpreted using the equation for mixed type inhibition, whereas the data for FNLDH were interpreted using the equation for competitive type inhibition. The kinetic parameters were as follows; FNLDH: kcat = 79 ± 1 (s−1), Km = 0.39 ± 0.01 (mM), and Ki = 9.4 ± 0.2 (mM). PALDH: kcat = 410 ± 10 (s−1), Km = 0.074 ± 0.006 (mM), Ki = 0.29 ± 0.04 (mM), and Ki’ = 0.33 ± 0.04 (mM). ECLDH: kcat = 670 ± 10 (s−1), Km = 5.6 ± 0.3 (mM), Ki = 20 ± 2 (mM), and Ki’ = 47 ± 6 (mM). GraFit ver 7.0.3 was used for non-linear regression and calculation of values. The lines were calculated with kinetic parameters.
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Figure 4: Effects of oxamate on the catalytic reactions at pH 7.0. The reaction velocities for FNLDH (a), PALDH (b), and ECLDH (c) were measured in 50 mM MOPS-NaOH buffer (pH 7.0) containing 0.1 mM NADH, the indicated concentrations of pyruvate and several concentrations of oxamate. The concentrations of oxamate for FNLDH were 0 mM (open circles), 10 mM (closed circles), 20 mM (open triangles), 30 mM (closed triangles), and 40 mM (open squares). The concentrations of oxamate for PALDH were 0 mM (open circles), 0.1 mM (closed circles), 0.2 mM (open triangles), 0.3 mM (closed triangles), and 0.4 mM (open squares). The concentrations of oxamate for ECLDH were 0 mM (open circles), 5 mM (closed circles), 10 mM (open triangles), 15 mM (closed triangles), and 20 mM (open squares). The reaction velocity and the concentration of pyruvate were plotted reciprocally. The data for PALDH and ECLDH were interpreted using the equation for mixed type inhibition, whereas the data for FNLDH were interpreted using the equation for competitive type inhibition. The kinetic parameters were as follows; FNLDH: kcat = 79 ± 1 (s−1), Km = 0.39 ± 0.01 (mM), and Ki = 9.4 ± 0.2 (mM). PALDH: kcat = 410 ± 10 (s−1), Km = 0.074 ± 0.006 (mM), Ki = 0.29 ± 0.04 (mM), and Ki’ = 0.33 ± 0.04 (mM). ECLDH: kcat = 670 ± 10 (s−1), Km = 5.6 ± 0.3 (mM), Ki = 20 ± 2 (mM), and Ki’ = 47 ± 6 (mM). GraFit ver 7.0.3 was used for non-linear regression and calculation of values. The lines were calculated 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