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

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 µ m ) 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.