Structural basis of dinucleotide substrate recognition and catalysis by the Nudix effector RipN from Ralstonia...

The type III secreted effector RipN from Ralstonia solanacearum is a Nudix hydrolase that suppresses plant immunity by targeting host dinucleotide metabolism. Although RipN preferentially hydrolyzes NADH, the structural basis of its substrate specificity and catalytic mechanism has remained unclear. Here, we report the crystal structures of the full-length RipN and a truncated mutant, RipN^E220AΔN81. RipN adopts a Nudix fold featuring a composite substrate-binding pocket formed by the conserved catalytic core and surrounding structural elements. CASTp analysis indicates that this cavity is well suited to accommodate bulky dinucleotide substrates. Molecular docking analyses reveal that NADH, ADP-ribose and FAD share a conserved binding mode centered on the adenosyl diphosphate scaffold, which is coordinated indirectly through Mg²⁺ ions. We further identify Tyr318 and Glu384 as key substrate-discriminating residues that mediate base-specific interactions and steric exclusion, enabling RipN to efficiently hydrolyze NADH while excluding closely related metabolites such as NADPH and UDP-glucose. Based on structural and docking data, we propose a Glu220-dependent, Mg²⁺-assisted catalytic mechanism involving activation of a conserved water molecule for phosphoanhydride bond cleavage. Together, these findings provide mechanistic insight into how RipN selectively targets host dinucleotide metabolites and illustrate how a conserved Nudix scaffold is adapted for effector-specific functions.