Purification and Characterisation of Malate Dehydrogenase From Synechocystis sp. PCC 6803: Biochemical Barrier of the Oxidative Tricarboxylic Acid Cycle

Abstract

Cyanobacteria possess an atypical tricarboxylic acid (TCA) cycle with various bypasses. Previous studies have suggested that a cyclic flow through the TCA cycle is not essential for cyanobacteria under normal growth conditions. The cyanobacterial TCA cycle is, thus, different from that in other bacteria, and the biochemical properties of enzymes in this TCA cycle are less understood. In this study, we reveal the biochemical characteristics of malate dehydrogenase (MDH) from Synechocystis sp. PCC 6803 MDH (SyMDH). The optimal temperature of SyMDH activity was 45-50°C and SyMDH was more thermostable than MDHs from other mesophilic microorganisms. The optimal pH of SyMDH varied with the direction of the reaction: pH 8.0 for the oxidative reaction and pH 6.5 for the reductive reaction. The reductive reaction catalysed by SyMDH was activated by magnesium ions and fumarate, indicating that SyMDH is regulated by a positive feedback mechanism. The K_m-value of SyMDH for malate was approximately 210-fold higher than that for oxaloacetate and the K_m-value for NAD⁺ was approximately 19-fold higher than that for NADH. The catalytic efficiency of SyMDH for the reductive reaction, deduced from k_cat-values, was also higher than that for the oxidative reaction. These results indicate that SyMDH is more efficient in the reductive reaction in the TCA cycle, and it plays key roles in determining the direction of the TCA cycle in this cyanobacterium.

Keywords: TCA cycle; biochemistry; cyanobacteria; malate dehydrogenase; metabolic enzyme.

FIGURE 1

(A) Purification of GST-tagged SyMDH. Proteins were electrophoresed on a 12% SDS-PAGE gel. The gel was stained with InstantBlue. Arrowheads indicate the molecular weight. (B) The effect of pH on SyMDH activity. Red square represents the specific activity in the oxidative reaction (malate to oxaloacetate). Blue triangle represents the specific activity in the reductive reaction (oxaloacetate to malate). Data represent the relative values of the mean from three independent experiments. (C) The effect of temperature on SyMDH activity. Red square represents the specific activity in the oxidative reaction (malate to oxaloacetate). Blue triangle represents the specific activity in the reductive reaction (oxaloacetate to malate). Data represent the relative values of the mean from three independent experiments.

FIGURE 2

Enzyme assay of SyMDH in the oxidative reaction in vitro. (A) Activity was measured by varying the malate concentration at a fixed NAD⁺ concentration (8.0 mM). The graphs show the mean ± SD obtained from three independent experiments. (B) Activity was measured by varying the NAD⁺ concentration at a fixed malate concentration (4.0 mM). The graphs show the mean ± SD obtained from three independent experiments.

FIGURE 3

Enzyme assay of SyMDH in the reductive reaction in vitro. (A) Activity was measured by varying the oxaloacetate concentration at a fixed NADH concentration (0.1 mM). The graphs show the mean ± SD obtained from three independent experiments. (B) Activity was measured by varying the NADH concentration at a fixed oxaloacetate concentration (0.1 mM). The graphs show the mean ± SD obtained from three independent experiments.

FIGURE 4

Effects of various metal ions and compounds on the SyMDH in the reductive reaction in vitro. 10 μg of SyMDH was pre-incubated with 100 mM potassium phosphate (pH 6.5), 0.1 mM NADH, 0.1 mM oxaloacetate and effectors, at 45°C. The graphs show the mean ± SD obtained from three independent experiments. Activity of SyMDH in the absence of effectors was set at 100%. Ca, CaCl₂; Mn, MnCl₂⋅4H₂O; Co, Co(NO₃)₂⋅6H₂O; Zn, ZnSO₄⋅7H₂O; Cu, CuSO₄⋅5H₂O; K, KCl; Na, NaCl; Mg, MgCl₂; Asp, L-Aspartate; Cit, Citrate; Mal, L-Malate; Suc, Succinate; Lac, L-lactate; Fum, Fumarate; Pyr, Pyruvate; Pep, Phosphoenolpyruvate.

FIGURE 5

The K_m and V_max-values for oxaloacetate in the presence of 10 mM fumarate and 10 mM magnesium ion in vitro. (A) Saturation curves of the activity of SyMDH. Blue line indicates mock, green line indicates presence of fumarate, and red line indicates the presence of magnesium. The graph shows the mean of three independent experiments. (B) K_m (mean ± SD) (units/mg protein) values in the presence of 10 mM fumarate and 10 mM magnesium ion, obtained from three independent experiments. (C) V_max (mean ± SD) values for oxaloacetate, obtained from three independent experiments. Mock indicates the enzymatic activity in the absence of additional compounds.

FIGURE 6

Thermal profiles of SyMDH in the oxidative reaction in vitro. (A) Lineweaver–Burk plot of the SyMDH activity in the oxidative reaction at 20–50°C. Blue, red, green, and purple lines indicate condition at 20, 30, 40, and 50°C, respectively. The graph shows the mean of three independent experiments. (B) K_m (mean ± SD) values for malate were obtained from three independent experiments by varying the temperature (20–50°C). (C) V_max (mean ± SD) (units/mg protein) values for malate were obtained from three independent experiments by varying the temperature (20–50°C).

FIGURE 7

Thermal profiles of SyMDH in the reductive reaction in vitro. (A) Lineweaver–Burk plot of the SyMDH activity in the reductive reaction at 20–50°C. Blue, red, green, and purple lines indicate condition at 20, 30, 40, and 50°C, respectively. The graph shows the mean of three independent experiments. (B) K_m (mean ± SD) values for oxaloacetate were obtained from three independent experiments by varying the temperature (20–50°C). (C) V_max (mean ± SD) (units/mg protein) values for oxaloacetate were obtained from three independent experiments by varying the temperature (20–50°C).