Lysine pyruvylation couples glycolytic flux to epigenetic regulation

Post-translational modifications (PTMs) dynamically regulate protein function, with metabolite-driven PTMs linking metabolism to protein regulation^1,2. We have previously discovered lysine lactylation, showing that lactate can directly modify proteins and influence cancer progression^3,4. Recently, pyruvate, another glycolytic metabolite, was shown to directly modify STAT1 at lysine 201, thereby suppressing type I interferon signalling⁵. Yet, the enzyme governing this modification, its substrate landscape and potential roles beyond innate immunity remain entirely unexplored. Here we report the systematic characterization of lysine pyruvylation (Kpy). Through biochemical and proteomic approaches, we establish the widespread existence of this modification, identifying 88 Kpy sites in mammalian cells. We investigate the dynamic regulation of Kpy upon metabolic perturbations and find that Kpy fluctuates with changes in glycolytic flux and pyruvate levels. Furthermore, we identify sirtuin 3 (SIRT3) as responsible for removing Kpy, while histone acetyltransferase 1 (HAT1) and p300 (EP300) catalyse its addition. Finally, we explore the function of Kpy in transcriptional regulation. Overall, Kpy expands the repertoire of metabolite-driven PTMs and provides insights into how pyruvate directly modulates protein function.