Dynamic formation of the malate dehydrogenase/citrate synthase multienzyme complex

Abstract

The enzymes of Krebs tricarboxylic acid (TCA) cycle form multi‐enzyme complexes mediating metabolite (substrate) channeling. As metabolite channeling can enhance the pathway reactions, the multi‐enzyme complexes are postulated to regulate metabolic flux by dynamic association and dissociation. Especially, the interaction between malate dehydrogenase (MDH) and citrate synthase (CS) is expected to have significant impact on the TCA cycle flux since the forward reaction of MDH to produce oxaloacetate from malate is thermodynamically infeasible with physiological metabolite concentrations. However, the dynamics of this multi‐enzyme complex and its relationship with metabolic pathway fluxes have not been demonstrated in vivo. To this end, plate reader‐based split‐luciferase assay was developed to monitor the dynamics of the protein‐protein interaction in living yeast cells and was applied to observe the dynamics of MDH/CS multi‐enzyme complex during the transition of metabolic status. The sequences coding N‐ and C‐ terminus of highly sensitive NanoLUC luciferase were introduced into the yeast genome by homologous recombination to fuse these peptides to the C‐terminus of CS (CIT1) and MDH (MDH1) enzymes, respectively. The resulted yeast strain showed decent fluorescent signals while wild‐type cells showed no background signal. The substrate concentration and cell concentration in the culture were optimized to obtain constant signals for 120 min of measurement. Using this system, the interaction of MDH1/CIT1 complex was monitored following the changes of growth condition which are expected to induce the alteration in the TCA cycle metabolic flux. In the preliminary results, we observed the alteration of luminescence signal in some conditions which are known to modulate the TCA cycle flux. We are currently testing various conditions to analyze the relationship between the dynamics of the multi‐enzyme complex and metabolic fluxes in the TCA cycle and adjacent pathways. We are also trying to identify the factors affecting the interaction of the enzymes. Support or Funding Information This study is supported by National Science Foundation, Faculty Early Career Development Program 1845451 to T.O.