Abstract 2011 The structural and functional effects of phosphorylation on human mitochondrial malate dehydrogenase

Malate dehydrogenase (human mitochondrial MDH: hMDH2), the final enzymatic component of the tricarboxylic acid (TCA) cycle, plays an essential role in cellular metabolism and energy production. Previous studies have indicated allosteric regulation of hMDH2 by TCA cycle intermediates, and mass spectrometry analysis has identified phosphorylation at 32 unique residues. While the impact of phosphorylation on the structure and function of related dehydrogenases is known, such as lactate dehydrogenase or pyruvate dehydrogenase, the effects on hMDH2 remain unexplored. In this study, solvent-exposed residues aligned in key domains of MDH were selected, including those associated with NADH binding, dimer binding, predicted interfaces with citrate synthase, and proximity to the mobile loop of the catalytic site-specifically, sites S8, S45, Y56, and T85. Through site-directed mutagenesis, aspartic acid replaced the wild-type residue to act as a phosphomimic. Wild-type and mutant hMDH2 proteins were expressed and purified as his-tagged recombinant proteins. The specific activity, Km, and Vmax were measured for each phosphomimic: hMDH2 S8D, hMDH2 S45D, hMDH2 Y56D, and hMDH2 T85D. While all phosphomimics exhibited increased activity at basic pH and decreased activity at acidic pH, differences among them were noted. In the presence of TCA cycle intermediates at cellular concentrations, the hMDH2 phosphomimics displayed varied and pronounced activities, the details of which will be discussed further. Structural analysis through fast thermal melts revealed that the four phosphomutants underwent a native conformation change at lower temperatures compared to wild-type hMDH2. Small-angle X-ray scattering (SAXS) demonstrated that hMDH2 S8D and hMDH2 S45D maintained an unchanged secondary structure, similar to wild-type hMDH2, while hMDH2 Y56D and hMDH2 T85D exhibited a greater conformational change associated with unfolding, oligomerization, or aggregation. Size-exclusion chromatography revealed changes in quaternary structure due to phosphorylation, except for hMDH2 S8D and hMDH2 T85D, which maintained dimers like wild-type hMDH2. Conversely, hMDH2 S45D and hMDH2 Y56D exhibited dimer and oligomeric characteristics, respectively, indicating a drastic alteration in quaternary structure through phosphorylation. Our results confirm that phosphorylation significantly impacts both the structure and function of hMDH2, particularly under physiological pH conditions and in the presence of allosteric regulators. This suggests that hMDH2 undergoes regulation through an unknown structural change controlled by phosphorylation, with the exact mechanism yet to be fully understood.