Functional redundancy driven by isocitrate dehydrogenase enhances metabolic robustness in Pseudomonas putida KT2440

BACKGROUND: Prokaryotic isocitrate dehydrogenase (PIDH) is a conserved and highly versatile enzyme regulating carbon flux between the TCA cycle and the glyoxylate shunt. While most mesophilic organisms encode a single PIDH, the industrial chassis Pseudomonas putida KT2440 harbours two isoforms: a monomeric (IDH) and a dimeric (ICD). The physiological significance of this dual-system in mesophiles remains poorly understood, yet it may provide a framework for understanding metabolic flexibility and flux regulation in biotechnological applications. RESULTS: Here, we biochemically characterized both PIDHs and, through modelling and site-directed mutagenesis, identified key residues (Ser133, Asn136, and Arg140) critical for monomeric catalytic activity and substrate binding. Genetic analysis revealed that the monomeric IDH is essential for growth, whereas the dimeric ICD appears to facilitate high-flux oxidative metabolism. System-level analysis integrating transcriptomics and flux modelling further demonstrated that these isoforms might fuel distinct TCA functional modes. Our results suggest that IDH sustains a basal, carbon-independent mode critical for biosynthetic precursor supply. In contrast, ICD drives a high-flux oxidative mode under glycolytic conditions and is post-translationally inactivated under gluconeogenic conditions, favouring the glyoxylate cycle. CONCLUSIONS: Taken together, these findings support a model in which specialized isoenzymes with conditional redundancy, rather than simple redundancy, in P. putida. This dual-enzyme system enables the cell to balance energy production with biosynthetic demands, which might facilitate metabolic adaptation under fluctuating environmental conditions. Our results provide a potential modular framework for engineering central metabolism to optimize precursor provisioning in microbial cell factories.