Hepatic glutamine synthetase controls N(5)-methylglutamine in homeostasis and cancer

Glutamine synthetase (GS) activity is conserved from prokaryotes to humans, where the ATP-dependent production of glutamine from glutamate and ammonia is essential for neurotransmission and ammonia detoxification. Here, we show that mammalian GS uses glutamate and methylamine to produce a methylated glutamine analog, N(5)-methylglutamine. Untargeted metabolomics revealed that liver-specific GS deletion and its pharmacological inhibition in mice suppress hepatic and circulating levels of N(5)-methylglutamine. This alternative activity of GS was confirmed in human recombinant enzyme and cells, where a pathogenic mutation in the active site (R324C) promoted the synthesis of N(5)-methylglutamine over glutamine. N(5)-methylglutamine is detected in the circulation, and its levels are sustained by the microbiome, as demonstrated by using germ-free mice. Finally, we show that urine levels of N(5)-methylglutamine correlate with tumor burden and GS expression in a β-catenin-driven model of liver cancer, highlighting the translational potential of this uncharacterized metabolite. [Image: see text]