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

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.

Keywords: Allosteric regulation; Escherichia coli; Fusobacterium nucleatum; Gram-negative bacteria; NAD-dependent d-lactate dehydrogenase; Pseudomonas aeruginosa.

Figure 1

SDS-PAGE and size-exclusion chromatography of the purified enzymes. a) Lane 1, molecular mass standards (kDa); lane 2, FNLDH; lane 3, PALDH; lane 4, ECLDH. b) Elution of the enzymes from Superdex 200 with the buffers described in ‘Materials and methods’. Solid, dashed, and dotted lines indicate FNLDH, PALDH, and ECLDH, respectively.

Figure 2

pH-stability of FNLDH (a), PALDH (b), and ECLDH (c), and heat-stability of the enzymes (d). a-c) Each enzyme was treated at 30°C for 1 h in sodium citrate (open circles), sodium acetate (closed circles), MES-NaOH (open triangles), MOPS-NaOH (closed triangles), HEPES-NaOH (open squares), Bicine-NaOH (N,N-Bis(2-hydroxyethyl)glycine) (closed squares), CHES-NaOH (N-cyclohexyl-2-aminoethanesulfonic acid) (open diamonds), and CAPS-NaOH (N-cyclohexyl-3-aminopropanesulfonic acid) (closed diamonds) buffers. d) FNLDH (white circles and solid lines), PALDH (grey circles and dashed lines), and ECLDH (black circles and dotted lines) were treated at various temperatures for 30 min in the buffers described in ‘Materials and methods’.

Figure 3

pH dependence of kinetic parameters on pyruvate reduction. White, grey, and black circles indicate FNLDH, PALDH, and ECLDH, respectively. The k_cat(a), S_0.5(b), and k_cat/S_0.5 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.

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: k_cat = 79 ± 1 (s⁻¹), K_m = 0.39 ± 0.01 (mM), and K_i = 9.4 ± 0.2 (mM). PALDH: k_cat = 410 ± 10 (s⁻¹), K_m = 0.074 ± 0.006 (mM), K_i = 0.29 ± 0.04 (mM), and K_i’ = 0.33 ± 0.04 (mM). ECLDH: k_cat = 670 ± 10 (s⁻¹), K_m = 5.6 ± 0.3 (mM), K_i = 20 ± 2 (mM), and K_i’ = 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.

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: K_i = 51 ± 10 (mM), k_cat’ = 80 ± 20 (s⁻¹), n_H’ = 1.1 ± 0.1, and K_act = 9.9 ± 3 (mM). PALDH: K_iK_i’ / (K_i + K_i’) = 2.0 ± 0.6 (mM), k_cat’ = 470 ± 100 (s⁻¹), n_H’ = 1.7 ± 0.2, and K_act = 1.7 ± 0.4 (mM). ECLDH: K_i = 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.

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).