Kinetic characterization and thermostability of C. elegans cytoplasmic and mitochondrial malate dehydrogenases

Malate dehydrogenase (MDH) catalyzes the conversion of NAD+ and malate to NADH and oxaloacetate in the last step of the citric acid cycle. Eukaryotes have at least two MDH isozymes, one that is imported into the mitochondria and one that remains in the cytoplasm. We overexpressed and purified Caenorhabditis elegans cytoplasmic MDH-1 (F46E10.10) and mitochondrial MDH-2 (F20H11.3) in E. coli. Our goal was to compare the kinetic and structural properties of these enzymes because C. elegans can survive adverse environmental conditions, such as lack of food and elevated temperatures. In steady-state enzyme kinetics assays, we determined that the KM values for oxaloacetate were 54 and 52 M, and the KM values for NADH were 61 and 107 M, for MDH-1 and MDH-2, respectively. We partially purified endogenous MDH from a mixed population of worms and separated MDH-1 from MDH-2 using anion exchange chromatography. Both endogenous enzymes had a KM for oxaloacetate similar to that of the corresponding recombinant enzyme. The reaction velocities of the recombinant enzymes had slightly different temperature-dependencies: MDH-1 and MDH-2 had maximum activity at 40 {degrees}C and 35 {degrees}C, respectively. In a thermotolerance assay, MDH-1 was much more thermostable than MDH-2. Molecular homology modeling predicted that MDH-1 had more salt-bridges between the subunits than mammalian MDH1 enzymes, and these ionic interactions may contribute to its thermostability. In contrast, the MDH-2 homology model predicted fewer ionic interaction between the subunits compared to mammalian MDH2 enzymes. These results suggest that the increased structural stability of MDH-1 may facilitate its ability to remain active in adverse environmental conditions. In contrast, MDH-2 may use other strategies, such as protein binding partners, to function under similar conditions.