Active site plasticity revealed from the structure of the enterobacterial N-ribohydrolase RihA bound to a competitive inhibitor

Here, we report the first crystal structure of a CU-NH bound to a competitive inhibitor, the complex between the Escherichia coli enzyme RihA bound to 3, 4-diaminophenyl-iminoribitol (DAPIR) to a resolution of 2.1 ？. The ligand can bind at the active site in two distinct orientations, and the stabilization of two flexible active site regions is pivotal to establish the interactions required for substrate discrimination and catalysis.A comparison with the product-bound RihA structure allows a rationalization of the structural rearrangements required for an enzymatic catalytic cycle, highlighting a substrate-assisted cooperative motion, and suggesting a yet overlooked role of the conserved His82 residue in modulating product release. Differences in the structural features of the active sites in the two homologous CU-NHs RihA and RihB from E. coli provide a rationale for their fine differences in substrate specificity. These new findings hint at a possible role of CU-NHs in the breakdown of modified nucleosides derived from RNA molecules.Pyrimidine-preferring nucleoside hydrolases (CU-NHs) are members of the broad family of Ca2+-dependent hydrolases that catalyze the cleavage of the N-glycosidic bond in nucleosides [1,2]. Enzymes with NH activity have been isolated from several different organisms, ranging from bacteria to fungi, nematodes, insects, and plants [3-7]. The biological function of these enzymes in both prokaryotes and eukaryotes is still controversial, and the widespread presence of NH-encoding genes can either be ascribed to a conserved role in general nucleotide catabolism. For instance, several purine or pyrimidine-auxotrophic parasites such as protozoa rely on nucleoside hydrolases to recycle nitrogenous bases uptaken from the host, lacking nucleoside phosphorylase activity [7]. In addition, NHs are also apparently involved in species-specific processes. Purine-specific NHs appear to modulate sporulation in spore-forming bacteria, such as Bacillus cere