What is malate dehydrogenase

In this study, Sy MDH was purified, and its biochemical functions were demonstrated for the first time, revealing unique regulatory mechanisms of Sy MDH.

Sequence integrity was confirmed by sequencing. Expression vectors were transformed into E. Two litres of E. Affinity chromatography was performed for protein purification as described in a previous study Osanai et al. The purified protein was mixed with 1 mL of assay solution mM potassium phosphate buffer [pH 8.

For the cell extract assay, cells from 1 L culture were collected by centrifugation and resuspend in mM potassium phosphate buffer pH7. V max and K m - values were determined using a Lineweaver—Burk double reciprocal plot. Results were plotted as a graph of the rate of reaction against the concentration of substrate and coenzyme using Kaleida Graph ver. When the data did not show substrate inhibition, we performed curve fitting used the Michaelis—Menten equation Eq.

When the data exhibited substrate inhibition, we performed curve fitting using the modified Michaelis—Menten equation Eq. Sy MDH activity in the oxidative reaction malate to oxaloacetate was the highest at pH 8. Sy MDH activity in the reductive reaction oxaloacetate to malate was the highest at pH 6. Kinetic parameters of Sy MDH were determined by a Lineweaver—Burk double reciprocal plot using the specific activity values in Figures 2 , 3.

These results are summarised in Tables 1 , 2. Sy MDH displayed approximately 1. The catalytic efficiency of the reductive reaction was higher than that of the oxidation reaction for both the substrate and the coenzyme. These results are summarised in Supplementary Tables S1, S2. The gel was stained with InstantBlue.

Arrowheads indicate the molecular weight. 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. Enzyme assay of Sy MDH in the oxidative reaction in vitro. Enzyme assay of Sy MDH in the reductive reaction in vitro. Table 2. Comparison of K m -values of MDHs in various microorganisms. Therefore, we measured the activity of Sy MDH in the reductive reaction in the presence of various effectors. With the exception of cobalt, magnesium, and copper ions, all other metal ions showed little effect on Sy MDH Figure 4.

Among the metal ions tested, only copper ions reduced the activity of Sy MDH. Sy MDH activity could not be measured in the presence of 10 mM calcium, manganese, cobalt, zinc, or copper ions due to the formation of a precipitate Figure 4.

Sy MDH activity with oxaloacetate at a concentration of 0. To strengthen the validity of our results, we also performed biochemical assays using cell extracts Supplementary Figure S3a. Effects of various metal ions and compounds on the Sy MDH in the reductive reaction in vitro.

The K m and V max -values for oxaloacetate in the presence of 10 mM fumarate and 10 mM magnesium ion in vitro. 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.

Mock indicates the enzymatic activity in the absence of additional compounds. The K m and the V max -values for malate tend to decrease as the temperature rise, although the V max -values less dependent on the temperature Figure 6 and Supplementary Figure S4.

On the contrary, the K m and the V max -values for oxaloacetate increased as the temperature rise Figure 7 and Supplementary Figure S5. Thermal profiles of Sy MDH in the oxidative reaction in vitro. Thermal profiles of Sy MDH in the reductive reaction in vitro. Cyanobacteria utilise a reductive branch of TCA cycle and excrete succinate under dark anaerobic conditions Hasunuma et al.

The intracellular pH of cyanobacteria decreases during the transition from light to dark conditions Coleman and Coleman, ; Mangan et al. Therefore, to adapt to the drastic changes in primary metabolism during the light and dark cycle, Sy MDH is thought to shift its substrate affinity according to the intracellular pH.

Sy MDH was stable at a wide range of temperature, being particularly tolerant to high temperatures Figure 1C. Generally, an enzymatic reaction is promoted by increasing temperature, because the kinetic energy of the reactants increases.

However, an enzyme denature at high temperatures. Besides Synechocystis , microorganisms having the MDHs with the optimal temperature much higher than the optimal growth temperature are S. These results are indicative of the diversity of regulation among MDHs. You could not be signed in. Sign In Forgot password?

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New issue alert. Receive exclusive offers and updates from Oxford Academic. Related articles in Google Scholar. Related articles in PubMed Coadministration of gallic acid abates zearalenone-mediated defects in male rat's reproductive function. A number of non-mammalian malate dehydrogenases have been included in the references. See also Schwartz Molecular weight : 70, Thorne and Kaplan According to Devenyi et al. Composition : The molecule consists of two polypeptide chains.

There are two coenzyme binding sites per 70, daltons Eberhardt and Wolfe Active center studies include those of Chen and Engel , Foster and Harrison and , et al.

See also Codd who reports a possible binding site for FMN. For halophilic MDH details, see Halophilic malate dehydrogenase.

See also:. The secondary structure of a single subunit contains a wrapped by. Near the sodium bound end, 4 small anti-parallel beta sheets and 1 small alpha helix enable a turn in the residue chain.

Opposite the sodium bound ligand, 6 alpha helices point towards a common point, three on each side of the beta sheet backbone. As for the 3D structure, the enzyme forms a sort of for the substrate to bind. The mechanism of catalysis is dependent on.