5¢-Deoxy-5¢-methylthioadenosine phosphorylase (MTAP) (EC 2.4.2.28) catalyzes the reversible phosphorolysis to free adenine and 5-methylthioribose-1-phosphate [1] of 5¢-deoxy-5¢-methylthioadenosine (MTA), a sulfur-containing nucleoside generated from S-adenosylmethionine. In eukaryotes, polyamine biosynthesis represents the major pathway of MTA formation: two moles of MTA are released per mole of spermine and one mole of MTA per mole of spermidine [2,3]. A phosphorolytic breakdown of the We report herein the first molecular characterization of 5¢-deoxy-5¢-methylthio-adenosine phosphorylase II from Sulfolobus solfataricus (SsMTAPII). The isolated gene of SsMTAPII was overexpressed in Escherichia coli BL21. Purified recombinant SsMTAPII is a homohexamer of 180 kDa with an extremely low K m (0.7 lm) for 5¢-deoxy-5¢-methylthioadenosine. The enzyme is highly thermophilic with an optimum temperature of 120°C and extremely thermostable with an apparent T m of 112°C that increases in the presence of substrates. The enzyme is characterized by high kinetic stability and remarkable SDS resistance and is also resistant to guanidinium chloride-induced unfolding with a transition midpoint of 3.3 m after 22-h incubation. Limited proteolysis experiments indicated that the only one 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. Moreover, the binding of 5¢-deoxy-5¢-methylthioadenosine induces a conformational transition that protected the enzyme against protease inactivation. By site-directed mutagenesis we demonstrated that Cys259, Cys261 and Cys262 play an important role in the enzyme stability since the mutants C259S ⁄ C261S and C262S show thermophilicity and thermostability features significantly lower than those of the wild-type enzyme. In order to get insight into the physiological role of SsMTAPII a comparative kinetic analysis with the homologous 5¢-deoxy-5¢-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) was carried out. Finally, the alignment of the protein sequence of SsMTAPII with those of SsMTAP and human 5¢-deoxy-5¢-methylthioadenosine phosphorylase (hMTAP) shows several key residue changes that may account why SsMTAPII, unlike hMTAP, is able to recognize adenosine as substrate.Abbreviations hMTAP, human 5¢-deoxy-5¢-methylthioadenosine phosphorylase; IPTG, isopropyl-b-D-thiogalactoside; MTA, 5¢-deoxy-5¢-methylthioadenosine; MTAP, 5¢-deoxy-5¢-methylthioadenosine phosphorylase; PfMTAP, 5¢-deoxy-5¢-methylthioadenosine phosphorylase from Pyrococcus furiosus; PNP, purine nucleoside phosphorylase; SsMTAP, 5¢-deoxy-5¢-methylthioadenosine phosphorylase from Sulfolobus solfataricus; SsMTAPII, 5¢-deoxy-5¢-methylthioadenosine phosphorylase II from Sulfolobus solfataricus.

5'-Methylthioadenosine phosphorylase (MTAP) was purified to homogeneity from the hyperthermophilic archaeon Pyrococcus furiosus. The protein is a homoexamer of 180 kDa. The enzyme is highly thermoactive, with an optimum temperature of 125 degrees C, and extremely thermostable, retaining 98% residual activity after 5 h at 100 degrees C and showing a half-life of 43 min at 130 degrees C. In the presence of 100 mM phosphate, the apparent T(m) (137 degrees C) increases to 139 degrees C. The enzyme is extremely stable to proteolytic cleavage and after incubation with protein denaturants, detergents, organic solvents, and salts even at high temperature. Thiol groups are not involved in the catalytic process, whereas disulfide bond(s) are present, since incubation with 0.8 M dithiothreitol significantly reduces the thermostability of the enzyme. N-Terminal sequence analysis of the purified enzyme is 100% identical to the predicted amino acid sequence of the gene PF0016 from the partially sequenced P. furiosus genome. The deduced amino acid sequence of the gene revealed a high degree of identity (52%) with human MTAP. Nevertheless, unlike human MTAP, MTAP from P. furiosus is not specific for 5'-methylthioadenosine, since it phosphorolytically cleaves adenosine, inosine, and guanosine. The calculated k(cat)/ K(m) values for 5'-methylthioadenosine and adenosine, about 20-fold higher than for inosine and guanosine, indicate that 6-amino purine nucleosides are preferred substrates of MTAP from P. furiosus. The structural features and the substrate specificity of MTAP from P. furiosus document that it represents a 5'-methylthioadenosine-metabolizing enzyme different from those previously characterized among Archaea, Bacteria, and Eukarya. The functional and structural relationships among MTAP from P. furiosus, human MTAP, and two putative MTAPs from P. furiosus and Sulfolobus solfataricus are discussed here for the first time.

The extremely heat-stable 5¢-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, expressed to high levels in Escherichia coli, and purified to homogeneity by heat precipitation and affinity chromatography. The recombinant enzyme was subjected to a kinetic analysis including initial velocity and product inhibition studies. The reaction follows an ordered Bi-Bi mechanism and phosphate binding precedes nucleoside binding in the phosphorolytic direction. 5¢-Methylthioadenosine phosphorylase from Pyrococcus furiosus is a hexameric protein with five cysteine residues per subunit. Analysis of the fragments obtained after digestion of the protein alkylated without previous reduction identified two intrasubunit disulfide bridges. The enzyme is very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 3.0 M after 22 h incubation. This value decreases to 2.0 M in the presence of 30 mM dithiothreitol, furnishing evidence that disulfide bonds are needed for protein stability. The guanidinium chloride-induced unfolding is completely reversible as demonstrated by the analysis of the refolding process by activity assays, fluorescence measurements and SDS/PAGE. The finding of multiple disulfide bridges in 5¢-methylthioadenosine phosphorylase from Pyrococcus furiosus argues strongly that disulfide bond formation may be a significant molecular strategy for stabilizing intracellular hyperthermophilic proteins.Keywords: disulfide bonds; hyperthermostability; 5¢-methyl thioadenosine phosphorylase; purine nucleoside phosphorylase; Pyrococcus furiosus.Hyperthermophilic enzymes which retain their structure and function near the boiling point of water have been, over the past decade, the object of extensive studies on protein stabilization, folding and evolutionary aspects [1][2][3][4]. Moreover, their unique structure-function properties of high thermostability are potentially significant for developing biotechnological applications [5,6]. Thus, there is a great deal of interest in studies on the biochemical adaptation of hyperthermophiles whose enzymes provide unique models for the study and understanding of the evolution of enzymes in terms of structure, specificity and catalytic properties.Much work has been done to identify the structural determinants of the enhanced stability of hyperthermophilic proteins. Several mechanisms of thermal stabilization have been proposed, among which additional networks of salt bridges and hydrogen bonds, improved packing density and enhanced secondary structure are the most cited [2][3][4][7][8][9]. In spite of this, no general rules have been established to date, and it has been concluded that each protein evolves individually through a limited number of factors that occur at different levels, also involving the amino acid sequence and the quaternary structure of the proteins.In recent years, growing attention has been paid to the presence of disulfide bonds in intracellular hy...