Impact of Phosphorylation on MDH2 Structure, Function and Protein

Abstract

Mitochondrial malate dehydrogenase (MDH2) is an enzyme utilizing the NAD/NADH cofactor system in the citric acid cycle to reversibly oxidize malate to oxaloacetate, where citrate synthase (CS) uses oxaloacetate to produce citrate. Coupling of the MDH2‐CS of the two reactions is critical as the thermodynamics of MDH2 alone would not drive the cycle forward. However, shuttling of carbon through the cycle during gluconeogenesis necessitates an uncoupling of the two proteins, allowing for the reversal of the reaction. Thus, a mechanism that favors the known interaction and disrupts substrate channeling through these two enzymes must exist. We performed an analysis of putative MDH2 phosphorylation sites, identified the potential kinases, and conducted a meta‐analysis of mass spectrometry data bases of phosphorylation. We have identified 13 predicted, and 30 identified phosphorylated sites of mammalian MDH2. Further analysis of possible interaction sites and mapping functional domains with these phosphorylation sites highlight the important yet unknown role of phosphorylation to regulate MDH structure and function. In order to better understand MDH2‐CS interactions and MDH function, we synthesized four phosphomimics (S/T‐D) using site directed mutagenesis on a codon optimized human MDH2 gene in a His‐tagged, pET28a construct for bacterial expression. Using purified protein, we show impact of each mutation structure with circular dichroism and the thermal melting profile of the phosphomimics and the wildtype hMDH2. The impact of phosphorylation on MHD function was also studied by comparing kinetic values. Lastly the impact of the mutations on MDH‐CS binding was analyzed using a micro‐protein thermal shift assay.Support or Funding InformationNSF EHR‐IUSE Project support NSF‐1726932