三磷酸胞苷合成酶(CTPS)在三磷酸胞苷(CTP)的从头合成中起关键作用,RNA和DNA的基本构建块,对生命至关重要。CTPS能够直接结合所有四种核苷酸三磷酸:腺嘌呤三磷酸,尿苷三磷酸,CTP,和三磷酸胍.此外,CTPS可以在体内形成胞嘧啶,在体外形成代谢丝,正在经历多层次的监管。CTPS被认为是对抗病毒或原核病原体入侵或感染的潜在治疗靶标。利用低温电子显微镜,我们确定了与CTP复合的大肠杆菌CTPS(ecCTPS)细丝的结构,烟酰胺腺嘌呤二核苷酸(NADH),和共价抑制剂6-重氮-5-氧代-1-正亮氨酸(DON),达到2.9的分辨率。我们根据细丝形成界面的差异构建了一个系统发育树,并设计了一个变体来验证我们的假设,提供了CTPS细丝形成的进化视角。我们的计算分析揭示了DON结合时溶剂可进入的氨隧道。通过比较结构分析,我们发现ecCTPS的CTP结合模式不同于真核生物对应物。结合生化分析和结构分析,我们确定并验证了CTP与NADH或腺嘌呤对CTPS的协同抑制作用。我们的结果扩展了我们对CTPS的各种调控方面的理解,并为设计靶向原核CTPS的特异性抑制剂奠定了基础。
Cytidine triphosphate synthase (CTPS) plays a pivotal role in the de novo synthesis of cytidine triphosphate (CTP), a fundamental building block for RNA and DNA that is essential for life. CTPS is capable of directly binding to all four nucleotide triphosphates: adenine triphosphate, uridine triphosphate, CTP, and guanidine triphosphate. Furthermore, CTPS can form cytoophidia in vivo and metabolic filaments in vitro, undergoing regulation at multiple levels. CTPS is considered a potential therapeutic target for combating invasions or infections by viral or prokaryotic pathogens. Utilizing cryo-electron microscopy, we determined the structure of Escherichia coli CTPS (ecCTPS) filament in complex with CTP, nicotinamide adenine dinucleotide (NADH), and the covalent inhibitor 6-diazo-5-oxo- l-norleucine (DON), achieving a resolution of 2.9 Å. We constructed a phylogenetic tree based on differences in filament-forming interfaces and designed a variant to validate our hypothesis, providing an evolutionary perspective on CTPS filament formation. Our computational analysis revealed a solvent-accessible ammonia tunnel upon DON binding. Through comparative structural analysis, we discern a distinct mode of CTP binding of ecCTPS that differs from eukaryotic counterparts. Combining biochemical assays and structural analysis, we determined and validated the synergistic inhibitory effects of CTP with NADH or adenine on CTPS. Our results expand our comprehension of the diverse regulatory aspects of CTPS and lay a foundation for the design of specific inhibitors targeting prokaryotic CTPS.