Auxiliary glyceraldehyde-3-phosphate dehydrogenases shape central carbon metabolism in Pseudomonas putida

Glyceraldehyde-3-phosphate dehydrogenases (GapDHs) are key enzymes in central carbon metabolism, yet their functional diversity extends beyond canonical glycolysis and gluconeogenesis. The soil bacterium Pseudomonas putida KT2440 encodes four GapDH isoenzymes (i.e., GapA, GapB, GapC, and GapD). GapA and GapB have been recently described as gatekeepers of metabolic segregation, with GapA driving glycolysis and GapB associated with gluconeogenesis. However, the physiological roles of GapC and GapD remain poorly defined. In this work, we combined targeted mutagenesis, growth phenotyping, in vitro biochemical analysis, proteomics, and metabolomics to dissect the functions of these lesser-studied GapDH isoforms. GapC and GapD have distinct structural features, diverging from the canonical GapA and GapB enzymes. Physiological characterization of gap-deletion mutants of P. putida across multiple glycolytic and gluconeogenic substrates showed that GapC and GapD exert substrate-specific effects and contribute to network-wide redox balancing. Proteomic and metabolomic profiling further indicated that GapC and GapD are integrated into specialized metabolic modules, supporting auxiliary functions that complement the dominant roles of GapA and GapB. This case of metabolic redundancy at the GapDH node highlights the versatility of central carbon metabolism in bacteria adapted to changing environmental conditions.

Bacteria often carry multiple isoenzymes for the same metabolic step, yet the reasons for such redundancy remain poorly understood. By dissecting the roles of GapDH isoforms in P. putida, we show that these enzymes are not functionally equivalent but rather partition their activity across glycolytic, gluconeogenic, and auxiliary functions. GapC and GapD provide secondary capacity under specific conditions that complement the known roles of GapA and GapB, which dominate glycolysis and gluconeogenesis, respectively. Growth phenotyping, together with proteomic and metabolomic analyses, revealed that isoenzyme specialization is reflected in both substrate-dependent abundance patterns and metabolite profiles. This study underscores the evolutionary diversification of central metabolic enzymes in environmental bacteria and their coordinated regulation across nutritional states. Furthermore, our findings provide a framework for engineering carbon flux in Pseudomonas and related bacteria, with implications for metabolic engineering applications targeting diverse feedstocks.