title: 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.
authors: Biocrystallography Unit, DIBIT San Raffaele Scientific Institute, via Olgettina 58, 20132 Milan, Italy.
authors: Elena
authors: E
authors: Iovane
authors: Barbara
authors: B
authors: Giabbai
authors: Laura
authors: L
authors: Muzzolini
authors: Vittoria
authors: V
authors: Matafora
authors: Arianna
authors: A
authors: Fornili
authors: Claudia
authors: C
authors: Minici
authors: Francesca
authors: F
authors: Giannese
authors: Massimo
authors: M
authors: Degano
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007288:Inosine / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D014529:Uridine / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D007288:Inosine; D009699:N-Glycosyl Hydrolases; C422988:RihB protein, E coli; C096466:inosine-uridine hydrolase; D014529:Uridine