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bioRxiv · 10.1101/2023.10.19.563106

Filamentation and inhibition of prokaryotic CTP synthase

Guo, C.-J.·Wang, Z.-X.·Liu, J.-L.
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Abstract

CTP synthase (CTPS) plays a pivotal role in the de novo synthesis of CTP, a fundamental building block for RNA and DNA, which is essential for life. CTPS is capable of directly binding to all four nucleotide triphosphates: ATP, UTP, CTP, and GTP. 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 virus or prokaryotic pathogens. Utilizing cryo-electron microscopy, we have determined the structure of Escherichia coli CTPS (ecCTPS) filament in complex with CTP, NADH, and the covalent inhibitor DON, achieving a resolution of 2.8[A]. We construct a phylogenetic tree based on differences in filament-forming interfaces and design a variant to validate our hypothesis, providing an evolutionary perspective on the CTPS filament formation. Our computational analysis reveals a solvent-accessible ammonia tunnel upon DON binding. By comparative structural analysis, we discern a distinct mode of CTP binding of ecCTPS, differing from eukaryotic counterparts. Combining biochemical assays and structural analysis, we determine and validate the synergistic inhibitory effects of CTP with NADH or adenine on CTPS. Our results expand our comprehension of diverse regulatory aspects of CTPS and lay a foundation for the design of specific inhibitors targeting prokaryotic CTPS.

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Guo, C.-J., Wang, Z.-X., Liu, J.-L.. 2023-10-19. Filamentation and inhibition of prokaryotic CTP synthase. https://doi.org/10.1101/2023.10.19.563106

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