Crystal structure and functional implications of cyclic di-pyrimidine-synthesizing cGAS/DncV-like nucleotidyltransferases

• Published in: Nature communications Vol. 14; no. 1; p. 5078
• Main Authors: Yang, Chia-Shin, Ko, Tzu-Ping, Chen, Chao-Jung, Hou, Mei-Hui, Wang, Yu-Chuan, Chen, Yeh
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 21.08.2023; Nature Publishing Group; Nature Portfolio
• Subjects: 140/58; 631/326/41/2536; 631/45/607; 631/535/1266; Bacteria; Crystal structure; Cytosine; Humanities and Social Sciences; multidisciplinary; Nucleotides; Phages; Proteins; Pyrimidines; Science; Science (multidisciplinary); Second messengers; Structural analysis; Structure-function relationships; Substrates; Synthesis; Uracil
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-023-40787-9

Purine-containing nucleotide second messengers regulate diverse cellular activities. Cyclic di-pyrimidines mediate anti-phage functions in bacteria; however, the synthesis mechanism remains elusive. Here, we determine the high-resolution structures of cyclic di-pyrimidine-synthesizing c GAS/ D ncV-like n ucleotidyl t ransferases (CD-NTases) in clade E (CdnE) in its apo, substrate-, and intermediate-bound states. A conserved (R/Q)xW motif controlling the pyrimidine specificity of donor nucleotide is identified. Mutation of Trp or Arg from the (R/Q)xW motif to Ala rewires its specificity to purine nucleotides, producing mixed purine-pyrimidine cyclic dinucleotides (CDNs). Preferential binding of uracil over cytosine bases explains the product specificity of cyclic di-pyrimidine-synthesizing CdnE to cyclic di-UMP (cUU). Based on the intermediate-bound structures, a synthetic pathway for cUU containing a unique 2’3’-phosphodiester linkage through intermediate pppU[3’−5’]pU is deduced. Our results provide a framework for pyrimidine selection and establish the importance of conserved residues at the C-terminal loop for the specificity determination of CD-NTases. Here, the authors present structural and functional characterization of bacterial CD-NTases that synthesize cyclic dipyrimidines for phage resistance, revealing a (R/Q)xW motif dictating pyrimidine selection which suggests a sequential pathway for synthesizing 2’3’-cyclic di-UMP.