Nitrogen metabolism profiling reveals cell state-specific pyrimidine synthesis pathway choice

Stable isotope tracing assays track few metabolites, yet cells use many nutrients to sustain nitrogen metabolism. Here, we create a platform for tracing 30 nitrogen isotope-labeled metabolites in parallel to enable a system-level understanding of cellular nitrogen metabolism. This platform reveals that while primitive cells engage both de novo and salvage pyrimidine synthesis pathways, differentiated cells nearly exclusively salvage uridine. This link between cell state and pyrimidine synthesis pathway preference persists in murine and human tissues. Mechanistically, we find that S1900 phosphorylation of CAD, the first enzyme of the de novo pathway, is induced by uridine deprivation in differentiated cells and constitutively enriched in primitive cells. Mimicking CAD S1900 phosphorylation in differentiated cells constitutively activates de novo pyrimidine synthesis, while blocking this modification impairs the cellular response to uridine starvation. Collectively, we establish a method for nitrogen metabolism profiling and define a mechanism of cell state-specific pyrimidine synthesis pathway choice.