title: Thermal unfolding of nucleoside hydrolases from the hyperthermophilic archaeon Sulfolobus solfataricus: role of disulfide bonds.
abstract: Nucleoside hydrolases are metalloproteins that hydrolyze the N-glycosidic bond of β-ribonucleosides, forming the free purine/pyrimidine base and ribose. We report the stability of the two hyperthermophilic enzymes Sulfolobus solfataricus pyrimidine-specific nucleoside hydrolase (SsCU-NH) and Sulfolobus solfataricus purine-specific inosineadenosine- guanosine nucleoside hydrolase (SsIAG-NH) against the denaturing action of temperature and guanidine hydrochloride by means of circular dichroism and fluorescence spectroscopy. The guanidine hydrochloride-induced unfolding is reversible for both enzymes as demonstrated by the analysis of the refolding process by activity assays and fluorescence measurements. The evidence that the denaturation of SsIAG-NH carried out in the presence of reducing agents proved to be reversible indicates that the presence of disulfide bonds interferes with the refolding process of this enzyme. Both enzymes are highly thermostable and no thermal unfolding transition can be obtained up to 108°C. SsIAG-NH is thermally denatured under reducing conditions (T(m)=93°C) demonstrating the contribution of disulfide bridges to enzyme thermostability.
authors.affiliation: Dipartimento di Biochimica e Biofisica F. Cedrangolo, Seconda Universita di Napoli, Napoli, Italy. marina.porcelli@unina2.it
authors.forename: Marina
authors.initials: M
authors.lastname: Porcelli
authors.forename: Ester
authors.initials: E
authors.lastname: De Leo
authors.forename: Pompea
authors.initials: P
authors.lastname: Del Vecchio
authors.forename: Francesca
authors.initials: F
authors.lastname: Fuccio
authors.forename: Giovanna
authors.initials: G
authors.lastname: Cacciapuoti
mesh_terms: D004220:Disulfides / Q000737:chemistry*; D004795:Enzyme Stability; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000378:metabolism; D058767:Protein Unfolding; D011743:Pyrimidines / Q000378:metabolism; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology*; D013696:Temperature
chemical_list: D004220:Disulfides; D011743:Pyrimidines; D009699:N-Glycosyl Hydrolases; C030986:pyrimidine