title: Purification and characterization of extremely thermophilic and thermostable 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus. Purine nucleoside phosphorylase activity and evidence for intersubunit disulfide bonds.
abstract: 5'-Methylthioadenosine phosphorylase from Sulfolobus solfataricus, a thermoacidophilic archaeon optimally growing at 87 degrees C, has been purified to homogeneity. Reducing agents are not required for catalytic activity. The enzyme has a molecular mass of 160 kDa and is composed of six apparently identical subunits of 27 kDa. The NH2-terminal sequence shows high homology (50%) with the NH2-terminal sequence of Escherichia coli purine nucleoside phosphorylase. Physicochemical and kinetic features are reported. 5'-Methylthioadenosine phosphorylase is highly thermophilic, with an optimum temperature of 120 degrees C. The enzyme is characterized by extreme thermal stability, remaining completely active after 2 h at 100 degrees C and showing half-inactivation times of 15 and 5 min when incubated at 130 and 140 degrees C, respectively. An apparent melting temperature of 132 degrees C has been calculated. After 24 h of incubation at room temperature no loss of activity is detected in the presence of 9 M urea, 4 M guanidine hydrochloride, 0.075% SDS, 50% methanol, 50% ethanol, 50% dimethylformamide, 1 M NaCl, and 1% Triton X-100. Data are also reported on the enzyme's resistance to proteolysis and on the effect of salts, detergents, solvents, and reducing agents on enzyme thermostability. Labeling experiments with iodo[2-14C]acetic acid resulted in the incorporation of approximately 12 mol of labeled iodoacetate/mol of protein, indicating the presence of six disulfide bonds that, on the basis of SDS-polyacrylamide gel electrophoresis, are probably positioned intersubunits, resulting in the organization of the enzyme into two trimers. 5'-Methylthioadenosine (MTA) phosphorylase is endowed with a broad substrate specificity, being able to phosphorolytically cleave inosine, guanosine, and adenosine with a better efficiency than MTA, allowing us to hypothesize that in S. solfataricus the same enzyme is responsible for the catabolism of MTA and of these purine nucleosides.
authors.affiliation: Institute of Biochemistry of Macromolecules, Faculty of Medicine, Second University of Naples, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Cacciapuoti
authors.forename: M
authors.initials: M
authors.lastname: Porcelli
authors.forename: C
authors.initials: C
authors.lastname: Bertoldo
authors.forename: M
authors.initials: M
authors.lastname: De Rosa
authors.forename: V
authors.initials: V
authors.lastname: Zappia
mesh_terms: D000595:Amino Acid Sequence; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry / Q000302:isolation & purification*; D016415:Sequence Alignment; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology*
chemical_list: D011683:Purine-Nucleoside Phosphorylase; C021784:5'-methylthioadenosine phosphorylase