Investigation of isoform‐specific interactions between citrate synthase and malate dehydrogenase

Abstract

Malate dehydrogenase (MDH) and citrate synthase (CS) catalyze subsequent metabolic reactions in the Krebs cycle. MDH and CS interact to form enzyme complexes called metabolons where substrates are directly shuttled from one active site to the other, improving efficiency of the overall metabolic cycle. Prior studies have shown that the mitochondrial isoform (MDH2) has stronger interactions with CS than the cytosolic isoform (MDH1), suggesting that isoformspecific sites of interaction are involved in the formation of the enzyme complex. This study attempts to determine key residues of MDH2 that are responsible for isoform‐specific affinities to CS, allowing for the formation of metabolon complexes. Model metabolon docking complexes between MDH2 and CS were generated computationally in Cluspro, using predicted and published interactions between the two enzymes. Potential interacting residues were identified and then mapped using clustal alignments to identify possible domains of isoformspecific interactions. Previous studies by our lab have shown that CS displays an isoformspecific affinity to MDH. Specifically, CS binds with high affinity to MDH2, moderate affinity to MDH1, and displays undetectable interaction with watermelon glyoxosomal MDH (wgMDH). We identified several regions of residues unique to MDH2 containing shown & predicted interactions with CS. Using docking analysis, we identified five regions of potential isoformspecific interactions. This combined with residue alignment of MDH2, MDH1 and wgMDH isoforms helped to further define potential isoform specific sites of interaction. Mutations exchanging mitochondrial residues for unique cytosolic counterparts or deletion mutations defined by our bioinformatic analysis were designed, generated and expressed. Enzyme kinetic assays were performed to investigate enzymatic activity of mutant constructs for comparison to native MDH1 and MDH2. Secondary structure and thermal stability were investigated using circular dichroism. Interactions between mutant MDH2 constructs and CS were quantified in fluorescence‐based thermal shift assays.Support or Funding InformationNSF EHR‐IUSE Project support NSF‐1726932