Structural Basis of Bifunctional CTP/dCTP Synthase.

[Display omitted] • Cryo-EM resolves the structure of Drosophila CTP/dCTP synthase (CTPS) with dNTPs at 2.7 Å. • dATP acts similarly to ATP as a substrate in CTPS. • dUTP is a less efficient substrate than UTP for CTPS. • dGTP is a weaker regulator than GTP for CTPS. • CTP and dCTP induce CTPS filamentation and inhibition. • Model explains CTPS dual functionality. The final step in the de novo synthesis of cytidine 5′-triphosphate (CTP) is catalyzed by CTP synthase (CTPS), which can form cytoophidia in all three domains of life. Recently, we have discovered that CTPS binds to ribonucleotides (NTPs) to form filaments, and have successfully resolved the structures of Drosophila melanogaster CTPS bound with NTPs. Previous biochemical studies have shown that CTPS can bind to deoxyribonucleotides (dNTPs) to produce 2′-deoxycytidine-5′-triphosphate (dCTP). However, the structural basis of CTPS binding to dNTPs is still unclear. In this study, we find that Drosophila CTPS can also form filaments with dNTPs. Using cryo-electron microscopy, we are able to resolve the structure of Drosophila melanogaster CTPS bound to dNTPs with a resolution of up to 2.7 Å. By combining these structural findings with biochemical analysis, we compare the binding and reaction characteristics of NTPs and dNTPs with CTPS. Our results indicate that the same enzyme can act bifunctionally as CTP/dCTP synthase in vitro , and provide a structural basis for these activities. [ABSTRACT FROM AUTHOR]