No abstract

Nucleoside hydrolases (NHs; EC 3.2.2.-) catalyze the irreversible hydrolysis of the N-glycosidic bond of b-ribonucleosides, forming ribose and the free purine or pyrimidine base [1][2][3]. All characterized members are metalloproteins with a unique central b-sheet topology and a cluster of aspartate residues (DXDXXXDD motif) at the N-terminus of the enzyme [2][3][4][5].In nature, a widespread distribution of NHs in different protozoa [6][7][8][9][10][11] We report the characterization of the pyrimidine-specific ribonucleoside hydrolase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsCU-NH). The gene SSO0505 encoding SsCU-NH was cloned and expressed in Escherichia coli and the recombinant protein was purified to homogeneity. SsCU-NH is a homotetramer of 140 kDa that recognizes uridine and cytidine as substrates. SsCU-NH shares 34% sequence identity with pyrimidine-specific nucleoside hydrolase from E. coli YeiK. The alignment of the amino acid sequences of SsCU-NH with nucleoside hydrolases whose 3D structures have been solved indicates that the amino acid residues involved in the calcium-and ribose-binding sites are preserved. SsCU-NH is highly thermophilic with an optimum temperature of 100°C and is characterized by extreme thermodynamic stability (T m = 106°C) and kinetic stability (100% residual activity after 1 h incubation at 90°C). Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is necessary for the integrity of the active site. The structure of the enzyme determined by homology modeling provides insight into the proteolytic analyses as well as into mechanisms of thermal stability. This is the first nucleoside hydrolase from Archaea.Abbreviations Cf, Crithidia

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.