胞苷三磷酸合酶 (CTPS) 包含氨连接酶结构域和谷氨酰胺氨基转移酶结构域，催化从头 CTP 生物合成的最后一步。CTPS 的活性受四个核苷酸和谷氨酰胺的结合调节。虽然谷氨酰胺作为氨供体用于 UTP 到 CTP 的 ATP 依赖性转化，但第四个核苷酸 GTP 充当变构激活剂。已经提出模型来解释氨连接酶结构域活性位点的作用机制以及由 GTP 结合引起的构象变化。然而，实际的 GTP/ATP/UTP 结合模式和相关的构象变化尚未完全揭示。在这里，我们报告了在果蝇中发现四种核苷酸和谷氨酰胺类似物 6-diazo-5-oxo-L-norleucine 的结合模式近原子分辨率的低温电子显微镜 CTPS。GTP 与周围残基之间的相互作用表明 GTP 通过直接阻断氨泄漏和稳定氨通道来协调两个域的反应。此外，我们观察到 ATP 依赖性 UTP 磷酸化中间体并确定氨连接酶上的相互作用残基。在 ATP 结合位点的非规范 CTP 结合表明另一层反馈抑制。我们的研究结果不仅描绘了所有底物存在下 CTPS 的结构，而且完成了我们对变构调节和 CTP 合成的潜在机制的理解。

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Cytidine triphosphate synthase (CTPS), which comprises an ammonia ligase domain and a glutamine amidotransferase domain, catalyzes the final step of de novo CTP biosynthesis. The activity of CTPS is regulated by the binding of four nucleotides and glutamine. While glutamine serves as an ammonia donor for the ATP-dependent conversion of UTP to CTP, the fourth nucleotide GTP acts as an allosteric activator. Models have been proposed to explain the mechanisms of action at the active site of the ammonia ligase domain and the conformational changes derived by GTP binding. However, actual GTP/ATP/UTP binding modes and relevant conformational changes have not been revealed fully. Here, we report the discovery of binding modes of four nucleotides and a glutamine analog 6-diazo-5-oxo-L-norleucine in Drosophila CTPS by cryo–electron microscopy with near-atomic resolution. Interactions between GTP and surrounding residues indicate that GTP acts to coordinate reactions at both domains by directly blocking ammonia leakage and stabilizing the ammonia tunnel. Additionally, we observe the ATP-dependent UTP phosphorylation intermediate and determine interacting residues at the ammonia ligase. A noncanonical CTP binding at the ATP binding site suggests another layer of feedback inhibition. Our findings not only delineate the structure of CTPS in the presence of all substrates but also complete our understanding of the underlying mechanisms of the allosteric regulation and CTP synthesis.