The molecular basis of ADP-ribose pyrophosphorylation by a phage PRPS-like enzyme in NAD+ reconstitution.

Depletion of cellular NAD+ is an increasingly recognized bacterial antiphage strategy, and many phages counter this pressure by rebuilding NAD+ from its cleavage products. In the NARP1 pathway, Adps converts ADP-ribose (ADPR) and ATP into ADPR-PP, the immediate substrate for Namat-dependent NAD+ synthesis, but how a phosphoribosyl pyrophosphate synthetase (PRPS)-like fold catalyzes this noncanonical reaction has been unknown. Here we report structures of phage Adps in apo, ADPR/ATP-bound pre-catalytic, and ADPR-PP/AMP-bound product states, supported by LC-MS activity assays, kinetics, and mutagenesis. The structures show that Adps uses a conserved interdomain groove to bind the ADPR acceptor, while a remodeled PP loop and catalytic β-hairpin create a donor site that positions the ATP β-γ pyrophosphate next to the distal ribose of ADPR. Lys185 moves toward the reaction center during the transition from substrate to product states, and its substitution abolishes ADPR-PP formation and impairs Adps-Namat-mediated rescue. Comparison with canonical PRPS enzymes indicates that Adps retained an ancestral acceptor-recognition surface while rewiring donor binding and regulatory elements. These findings define the molecular basis of phage ADPR pyrophosphorylation and illustrate how viral enzymes can repurpose conserved nucleotide-metabolic scaffolds to restore NAD+ during host immune attack.