Nucleoside hydrolases (NHs) are a class of metalloproteins that catalyze the irreversible hydrolysis of the N-glycosidic bond of β-ribonucleosides forming ribose and purine or pyrimidine base. In the hyperthermophilic archaeon Sulfolobus solfataricus, two NHs have been purified and extensively characterized. Although these enzymes show different substrate specificity, one for purines, the other for pyrimidines, their architectures are similar to that of non-specific NHs previously identified in protozoa. The detailed inspection into the active site of the two homologous enzymes obtained by homology modeling allowed us to infer the role of specific residues in substrate specificity. We report here the site-directed mutagenesis of the Sulfolobus solfataricus purine-specific inosine-adenosine-guanosine nucleoside hydrolase (SsIAG-NH). The double (L221Y/N228V) mutant of SsIAG-NH was expressed in E. coli and purified, and its activity with different substrates was compared to that of the wild-type enzyme. The double substitution modifies the catalytic pattern of the wild-type enzyme affecting both its substrate specificity and catalytic efficiency. Kinetic data obtained for the double mutant are in good agreement with modeling predictions.
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