title: A novel hyperthermostable 5'-deoxy-5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus.
abstract: 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 Km (0.7 microm) for 5'-deoxy-5'-methylthioadenosine. The enzyme is highly thermophilic with an optimum temperature of 120 degrees C and extremely thermostable with an apparent Tm of 112 degrees 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.
authors.affiliation: Dipartimento di Biochimica e Biofisica F. Cedrangolo, Seconda Università di Napoli, Italy. giovanna.cacciapuoti@unina2.it
authors.forename: Giovanna
authors.initials: G
authors.lastname: Cacciapuoti
authors.forename: Sabrina
authors.initials: S
authors.lastname: Forte
authors.forename: Maria Angela
authors.initials: MA
authors.lastname: Moretti
authors.forename: Assunta
authors.initials: A
authors.lastname: Brio
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Zappia
authors.forename: Marina
authors.initials: M
authors.lastname: Porcelli
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics; D013816:Thermodynamics
chemical_list: D011683:Purine-Nucleoside Phosphorylase; C021784:5'-methylthioadenosine phosphorylase