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, N⁵-methylglutamine. Untargeted metabolomics revealed that liver-specific GS deletion and its pharmacological inhibition in mice suppress hepatic and circulating levels of N⁵-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 N⁵-methylglutamine over glutamine. N⁵-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 N⁵-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.

谷氨酰胺合成酶 (GS) 活性从原核生物到人类都是保守的，其中从谷氨酸和氨中依赖 ATP 产生谷氨酰胺对于神经传递和氨解毒至关重要。在这里，我们证明哺乳动物 GS 使用谷氨酸和甲胺来产生甲基化谷氨酰胺类似物N ⁵ -甲基谷氨酰胺。非靶向代谢组学揭示小鼠肝脏特异性 GS 缺失及其药理学抑制可抑制N ^{5 -}甲基谷氨酰胺的肝脏和循环水平。 GS 的这种替代活性在人重组酶和细胞中得到了证实，其中活性位点 (R324C) 的致病性突变促进了N ⁵ -甲基谷氨酰胺而非谷氨酰胺的合成。 N ⁵ -甲基谷氨酰胺在循环中被检测到，其水平由微生物组维持，正如使用无菌小鼠所证明的那样。最后，我们发现N ^{5 -}甲基谷氨酰胺的尿液水平与 β-连环蛋白驱动的肝癌模型中的肿瘤负荷和 GS 表达相关，突出了这种未表征的代谢物的转化潜力。