A novel hyperthermostable 5′-deoxy-5′-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus.

Cacciapuoti, Giovanna; Forte, Sabrina; Moretti, Maria Angela; Brio, Assunta; Zappia, Vincenzo; Porcelli, Marina

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 µm) for 5′-deoxy-5′-methylthioadenosine. The enzyme is highly thermophilic with an optimum temperature of 120 °C and extremely thermostable with an apparent Tm 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.

PHOSPHORYLASES; ESCHERICHIA coli; TEMPERATURE; ENZYMES; MUTAGENESIS; PROTEOLYTIC enzymes

FEBS Journal, 2005, Vol 272, Issue 8, p1886

1742-464X

Academic Journal

10.1111/j.1742-4658.2005.04619.x