Function, kinetic properties, crystallization, and regulation of microbial malate dehydrogenase

Abstract

Abstract:Malate dehydrogenase (MDH) is an enzyme widely distributed among living organisms and is a key protein in the central oxidative pathway. It catalyzes the interconversion between malate and oxaloacetate using NAD + or NADP + as a cofactor. Surprisingly, this enzyme has been extensively studied in eukaryotes but there are few reports about this enzyme in prokaryotes. It is necessary to review the relevant information to gain a better understanding of the function of this enzyme. Our review of the data generated from studies in bacteria shows much diversity in their molecular properties, including weight, oligomeric states, cofactor and substrate binding affinities, as well as differences in the direction of the enzymatic reaction. Furthermore, due to the importance of its function, the transcription and activity of this enzyme are rigorously regulated. Crystal structures of MDH from different bacterial sources led to the identification of the regions involved in substrate and cofactor binding and the residues important for the dimer-dimer interface. This structural information allows one to make direct modifications to improve the enzyme catalysis by increasing its activity, cofactor binding capacity, substrate specificity, and thermostability. A comparative analysis of the phylogenetic reconstruction of MDH reveals interesting facts about its evolutionary history, dividing this superfamily of proteins into two principle clades and establishing relationships between MDHs from different cellular compartments from archaea, bacteria, and eukaryotes.

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

“…The affinity of oxaloacetate and NADH for Sy MDH was higher than the affinity of malate and NAD + , respectively ( Table 1 ). Generally, bacterial MDHs show higher affinity for oxaloacetate than malate ( Takahashi-Íñiguez et al, 2016 ), and Sy MDH was consistent with this. When comparing the substrate affinity among bacterial MDHs, the K m (malate)/ K m (oxaloacetate) ratio in descending order is as follows: N. europaea (250) , Synechocystis 6803 (210), Syntrophic propionate-oxidising bacterium strain MPOB (80.0), and Methanobacterium thermoautotrophicum (13.3) ( Table 2 ).…”

“…The reductive reaction catalysed by bacterial MDHs is inhibited by TCA cycle metabolites, such as excess oxaloacetate and divalent metal ions ( Takahashi-Íñiguez et al, 2016 ). Therefore, we measured the activity of Sy MDH in the reductive reaction in the presence of various effectors.…”

Acid-resistant genes of oral plaque microbiome from the functional metagenomics

“…Whether the detailed mechanism is consistent with E. coli requires further study. In addition, this protein can catalyze the interconversion between malate and oxaloacetate with the help of cofactor NAD + or NADP + and plays a critical role in the central oxidative pathway of living organisms [33]. In gram-negative bacteria, this protein generally forms a dimeric molecule and participates in the tricarboxylic acid (TCA) cycle.…”

Conformational changes on substrate binding revealed by structures of Methylobacterium extorquens malate dehydrogenase

“…The kinetic parameters for MexMDH-His 8 were calculated using the decrease in absorbance caused by the enzymatic oxidation of NADH to NAD + (" = 6.22 mM À1 cm À1 ) in the presence of oxaloacetate. We obtained a Michaelis constant (K m ) of 36.8 AE 0.6 mM for oxaloacetate, which compares favorably to a serine-type methanotroph (Rozova et al, 2015) and is twofold to tenfold lower then those observed for MDHs found in many aerobic bacteria (Rozova et al, 2015;Takahashi-Íñ iguez et al, 2016). MexMDH catalyzes the reduction of oxaloacetic acid with a k cat of (4.6 AE 0.1) Â 10 2 s À1 , yielding a catalytic efficiency k cat / K m of (1.3 AE 0.3) Â 10 7 M À1 s À1 .…”