title: New determinants in the catalytic mechanism of nucleoside hydrolases from the structures of two isozymes from Sulfolobus solfataricus.
abstract: The purine- and pyrimidine-specific nucleoside hydrolases (NHs) from the archaeon Sulfolobus solfataricus participate in the fundamental pathway of nucleotide catabolism and function to maintain adequate levels of free nitrogenous bases for cellular function. The two highly homologous isozymes display distinct specificities toward nucleoside substrates, and both lack the amino acids employed for activation of the leaving group in the hydrolytic reaction by the NHs characterized thus far. We determined the high-resolution crystal structures of the purine- and pyrimidine-specific NHs from S. solfataricus to reveal that both enzymes belong to NH structural homology group I, despite the different substrate specificities. A Na(+) ion is bound at the active site of the pyrimidine-specific NH instead of the prototypical Ca(2+), delineating a role of the metals in the catalytic mechanism of NHs in the substrate binding rather than nucleophile activation. A conserved His residue, which regulates product release in other homologous NHs, provides crucial interactions for leaving group activation in the archaeal isozymes. Modeling of the enzyme-substrate interactions suggests that steric exclusion and catalytic selection underlie the orthogonal base specificity of the two isozymes.
authors.affiliation: Biocrystallography Unit, Department of Immunology, Transplantation, and Infectious Diseases, Scientific Institute San Raffaele, via Olgettina 58, 20132 Milan, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Minici
authors.forename: Giovanna
authors.initials: G
authors.lastname: Cacciapuoti
authors.forename: Ester
authors.initials: E
authors.lastname: De Leo
authors.forename: Marina
authors.initials: M
authors.lastname: Porcelli
authors.forename: Massimo
authors.initials: M
authors.lastname: Degano
mesh_terms: D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D009705:Nucleosides / Q000378:metabolism; D011487:Protein Conformation; D011687:Purines / Q000378:metabolism; D011743:Pyrimidines / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000737:chemistry / Q000201:enzymology*
chemical_list: D007527:Isoenzymes; D009705:Nucleosides; D011687:Purines; D011743:Pyrimidines; D012964:Sodium; D009699:N-Glycosyl Hydrolases