Structural Basis for Substrate Specificity in Group I Nucleoside Hydrolases^,

Abstract

Enzymes with nucleoside hydrolase activity (NHs) belonging to homology group I either are markedly specific for pyrimidine nucleoside substrates or hydrolyze with comparable efficiencies the N-glycosidic bond in all common nucleosides. The biochemical and structural basis for these differences in substrate specificity is still unknown. Here we characterize the binding interactions between the slowly hydrolyzed substrate inosine and the Escherichia coli pyrimidine-specific NH YeiK using cryotrapping and X-ray crystallography. Guided by the structural features of the Michaelis complex, we show the synergic effect of two specific point mutations in YeiK that increase the catalytic efficiency toward purine nucleosides to values comparable to those of natural nonspecific NHs. We demonstrate that the integrity of an active-site catalytic triad comprised of two hydroxylated amino acids and one histidine residue is a requirement for the highly efficient hydrolysis of inosine by group I NHs. Instead, cleavage of the YeiK-preferred substrate uridine is not affected by mutations at the same locations, suggesting a different fine chemical mechanism for the hydrolysis of the two nucleoside substrates. Our study provides for the first time direct evidence that distinct subsets of amino acid residues are involved in the hydrolysis of purine or pyrimidine nucleosides in group I NHs.

Structure and Function of Nucleoside Hydrolases from Physcomitrella patens and Maize Catalyzing the Hydrolysis of Purine, Pyrimidine, and Cytokinin Ribosides

“…However, the strongly reduced turnover rate is probably a consequence of ineffective stabilization of the negative charge in the leaving group, leading to an increased energy barrier to reach the transition state. Because Tyr-241 is fully conserved among plant NRHs, it is most likely involved in this process and in line with similar findings on the equivalent residue in YeiK from E. coli and CfNRH (Iovane et al, 2008). As expected from the structure, the E247A variant resembles the wild-type enzyme regarding activity.…”

“…However, a slight shift of helix a11, such as that observed on substrate binding in bacterial NRHs ( Fig. 5A; Iovane et al, 2008), could bring its imidazole side chain into direct contact with the nucleobase, which is supported by mutagenesis results. The K m values for the Y241A variant are higher but not as much as for the three mutants above.…”

“…These residues belong to a particular a9 helix (equivalent to the a11 helix in plant NRHs) that is known to move upon substrate binding in CU-NRHs from E. coli (Giabbai and Degano, 2004;Muzzolini et al, 2006;Iovane et al, 2008;Garau et al, 2010). This triad binds the substrate and allows the efficient protonation of the N7 atom of the purine base (the leaving group).…”

“…Crystal structures are available for empty NRH or in complex with inhibitors from Crithidia fasciculata (CfNRH; Degano et al, 1998), Leishmania major (LmNRH; Shi et al, 1999), and Trypanosoma vivax (TvNRH; Versées et al, 2001Versées et al, , 2002. The structures of two CU-NRHs from Escherichia coli, namely YeiK (Iovane et al, 2008) and YbeK (rihA; Muzzolini et al, 2006;Garau et al, 2010), are also available. NRHs are believed to catalyze N-glycosidic bond cleavage by a direct displacement mechanism.…”

Nicotinamide Riboside and Nicotinic Acid Riboside Salvage in Fungi and Mammals

“…Although both enzymes convert NR to Nam with a specificity constant that is 8 -13% as great as the corresponding inosine reaction, bovine but not yeast Pnp is also an NaR phosphorylase. Despite a recent claim that Urh1 is a uridine-specific nucleoside hydrolase (22), our kinetic analysis of recombinant Urh1 demonstrates a 100-fold greater specificity for NR than for uridine. Urh1 also functions in vitro and in vivo as an NaR hydrolase such that Nrk-independent NaR salvage in yeast principally depends on Urh1.…”