Structural evolution of the selectivity clamp confers ADPR-PP specificity in Namat, a phage nicotinamide ADP-ribose transferase.

Phages and bacteria engage in an evolutionary arms race, in which NAD⁺ depletion serves as a potent bacterial defense. The phage NAD⁺ reconstitution pathway 1 (NARP1) counteracts this strategy via ADP-ribose phosphorylase (Adps) and nicotinamide ADP-ribose transferase (Namat), which restores NAD⁺ by repurposing the products of NAD⁺-depletion systems. Here, we dissect how Namat, the key ligase of NARP1, combines a conserved Nampt-like catalytic core with a specialized adenine ring-binding selectivity clamp to overcome host immunity. We determine its crystal structures bound to nicotinamide (NAM) and NAD⁺, combined with mutational, biochemical, and phylogenetic analyses. The structures reveal a "selectivity clamp," consisting of a variable loop and a conserved helix, that enforces strict specificity for ADP-ribose pyrophosphate (ADPR-PP) over phosphoribosylpyrophosphate (PRPP), the substrate of nicotinamide phosphoribosyltransferase (Nampt). Functional assays show that both the catalytic center and the selectivity clamp are essential for NAD⁺ biosynthesis and for counteracting NAD⁺-depleting defense. Guided by these insights, we identify bacterial homologs of NARP1 with similar enzymatic activity. These findings define the structural basis of Namat substrate selectivity and refine our understanding of NAD⁺ metabolism in host-phage interactions.