Citrate synthase condensation requires catalytic events in Corynebacterium glutamicum

Protein condensation regulates cellular metabolism. Although many enzymes are highly structured proteins, the molecular mechanisms underlying their condensation remain poorly understood. Here, we report the discovery and characterization of Corynebacterium glutamicum citrate synthase (CgCS) condensates in relation to extracellular glutamate overproduction. CgCS, which does not possess typical intrinsically disordered sequences, formed droplet-like condensates in C. glutamicum. CgCS condensates were observed in growing cells and dispersed in stationary phase cells. Condensates were not formed for the CgCS variants that lacked a catalytic residue, and the degree of condensation was correlated with catalytic activity. Oxaloacetate, a substrate of CS, was a crucial factor affecting condensation. Both substrates (oxaloacetate and acetyl-CoA) affected the saturation concentration of CgCS in a catalytic residue-dependent manner. We suggest that conformational heterogeneity generated through catalytic events is important for condensation, i.e., catalysis-dependent condensation. This study provides insights into the mechanisms underlying condensation of enzymes lacking intrinsically disordered sequences. The molecular mechanisms underlying protein condensation remain poorly understood. Here the authors discover and characterise the dynamic condensation of citrate synthase in C. glutamicum, showing condensation is dependent on catalytic activity and substrate binding.