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

Abstract

Abstract 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.