Fig. 1: Substrate recognition and catalytic pathway of CD-NTases.

A In the conserved canonical catalytic mechanism of nucleotidyltransferases, the ribose O3’ (or O2’) is deprotonated and stabilized by divalent cation “Metal A” before its attack at the P atom of the α-phosphate. The negative charge developed on the α-phosphate is neutralized by divalent cation “Metal B”, which also binds to the β and γ-phosphate and leaves with the diphosphate group. Metal A and Metal B could be either Mg²⁺ or Mn²⁺. B In CD-NTases, the cage-like architecture helps retaining and reorienting the intermediate within the active site. In this example of VcDncV with ATP as the acceptor and GTP as the donor, the ATP moiety of the intermediate pppApG takes over the original position of GTP, and vice versa, after the first reaction. The guanosine then serves as the acceptor and the ATP part as the donor in the second reaction. C Surface presentation of VcDncV (PDB: 4XJ3). The lid helix (salmon) and the C-terminal loop (green) above the ligand-binding pocket are highlighted and indicated. The donor (yellow) and acceptor (cyan) nucleotide and the two conserved residues in lid helix, lid residue 1 (Lid 1) and lid residue 2 (Lid 2), are shown in sticks. In this study, we identified conserved lid residue 3 (Lid 3) located at the C-terminal loop, which dictates the donor nucleotide specificity.