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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.

5′‐Deoxy‐5′‐methylthioadenosine phosphorylase II from Sulfolobus solfataricus (SsMTAPII) and purine nucleoside phosphorylase from Pyrococcus furiosus (PfPNP) are hyperthermophilic purine nucleoside phosphorylases stabilized by intrasubunit disulfide bonds. In their C‐terminus, both enzymes harbour a CXC motif analogous to the CXXC motif present at the active site of eukaryotic protein disulfide isomerase. By monitoring the refolding of SsMTAPII, PfPNP and their mutants lacking the C‐terminal cysteine pair after guanidine‐induced unfolding, we demonstrated that the CXC motif is required for the folding of these enzymes. Moreover, two synthesized CXC‐containing peptides with the same amino acid sequences present in the C‐terminal regions of SsMTAPII and PfPNP were found to act as in vitro catalysts of oxidative folding. These small peptides are involved in the folding of partially refolded SsMTAPII, PfPNP and their CXC‐lacking mutants, with a concomitant recovery of their catalytic activity, thus indicating that the CXC motif is necessary to obtain complete reversibility from the unfolded state of the two proteins. The two CXC‐containing peptides are also able to reactivate scrambled RNaseA. The data obtained in the present study represent the first example of how the CXC motif improves both stability and folding in hyperthermophilic proteins with disulfide bonds.

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