Pyruvate kinase directly generates GTP in glycolysis, supporting growth and contributing to guanosine toxicity.

Guanosine triphosphate (GTP) is essential for macromolecular biosynthesis, and its intracellular levels are tightly regulated in bacteria. Loss of the alarmone (p)ppGpp disrupts GTP regulation in Bacillus subtilis, causing cell death in the presence of exogenous guanosine and underscoring the critical importance of GTP homeostasis. To investigate the basis of guanosine toxicity, we performed a genetic selection for spontaneous mutations that suppress this effect, uncovering an unexpected link between GTP synthesis and glycolysis. In particular, we identified suppressor mutations in pyk, which encodes pyruvate kinase, a glycolytic enzyme. Metabolomic analysis revealed that inactivating pyruvate kinase prevents guanosine toxicity by reducing GTP levels. Although traditionally associated with ATP generation via substrate-level phosphorylation, B. subtilis pyruvate kinase in vitro was found to produce GTP and UTP approximately 10 and three times more efficiently than ATP, respectively. This efficient GTP/UTP synthesis extends to Enterococcus faecalis and Listeria monocytogenes, challenging the conventional understanding of pyruvate kinase's primary role in ATP production. These findings support a model in which glycolysis directly contributes to GTP synthesis, fueling energy-demanding processes, such as protein translation. Finally, we observed a synergistic essentiality of the Δndk Δpyk double mutant specifically on glucose, indicating that pyruvate kinase and nucleoside diphosphate kinase are the major contributors to nucleoside triphosphate production and complement each other during glycolysis. Our work highlights the critical role of nucleotide selectivity in pyruvate kinase and its broader implications in cellular physiology.

Importance: In this study, we reveal that pyruvate kinase, a key glycolytic enzyme, primarily generates GTP from GDP in Bacillus subtilis, relative to other nucleotide triphosphates, such as ATP. This finding, uncovered through genetic selection for mutants that suppress toxic GTP overaccumulation, challenges the conventional understanding that pyruvate kinase predominantly produces ATP via substrate-level phosphorylation. The substantial role of GTP production by pyruvate kinase suggests a model where glycolysis rapidly and directly supplies GTP as the energy currency to power high GTP-demanding processes such as protein synthesis. Our results underscore the importance of nucleotide selectivity (ATP vs GTP vs UTP) in shaping the physiological state and fate of the cell, prompting further exploration into the mechanisms and broader implications of this selective nucleotide synthesis.