tailieunhanh - Báo cáo khoa học: A novel hyperthermostable 5¢-deoxy-5¢-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus

We report herein the first molecular characterization of 5¢-deoxy-5¢-methyl-thio-adenosine phosphorylase II from Sulfolobus solfataricus(SsMTAPII). The isolated gene of SsMTAPII was overexpressed inEscherichia coli BL21. Purified recombinant SsMTAPII is a homohexamer of 180 kDa with an extremely low Km() for 5¢-deoxy-5¢-methylthioadenosine. The enzyme is highly thermophilic with an optimum temperature of 120 C and extremely thermostable with an apparent Tmof 112 C that increases in the presence of substrates | iFEBS Journal A novel hyperthermostable 5 -deoxy-5 -methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus Giovanna Cacciapuoti1 Sabrina Forte1 Maria Angela Moretti2 Assunta Brio1 Vincenzo Zappia1 and Marina Porcelli1 1 Dipartimento di Biochimica e Biofisica F. Cedrangolo Seconda University di Napoli Italy 2 Centro Regionale di Competenza in Biotecnologie Industriali BioTekNet Seconda Universita di Napoli Italy Keywords 5 -deoxy-5 -methylthioadenosine phosphorylase disulfide bonds hyperthermostability purine nucleoside phosphorylase Sulfolobus solfataricus Correspondence G. Cacciapuoti Dipartimento di Biochimica e Biofisica F. Cedrangolo Seconda University di Napoli Via Costantinopoli 16 80138 Napoli Italy Fax 39 081 5667519 Tel 39 081 5667519 E-mail Received 14 January 2005 revised 16 February 2005 accepted 17 February 2005 doi We report herein the first molecular characterization of 5 -deoxy-5 -methyl-thio-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 pM 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 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 .