TAILIEUCHUNG - Báo cáo khoa học: Biochemical and structural characterization of mammalian-like purine nucleoside phosphorylase from the Archaeon Pyrococcus furiosus

We report here the characterization of the first mammalian-like purine nucleoside phosphorylase from the hyperthermophilic archaeonPyrococcus furiosus (PfPNP). The gene PF0853 encoding PfPNP was cloned and expressed in Escherichia coliand the recombinant protein was purified to homogeneity. | ễFEBS Journal Biochemical and structural characterization of mammalian-like purine nucleoside phosphorylase from the Archaeon Pyrococcus furiosus Giovanna Cacciapuoti1 Sabrina Gorassini1 Maria Fiorella Mazzeo2 Rosa Anna Siciliano2 Virginia Carbone2 Vincenzo Zappia1 and Marina Porcelli1 1 Dipartimento di Biochimica e Biofisica F. Cedrangolo Seconda Universita di Napoli Italy 2 Centro di Spettrometria di Massa Proteomica e Biomolecolare Istituto di Scienze dell Alimentazione delCNR Avellino Italy Keywords CXC motif 5 -deoxy-5 -methylthioadenosine phosphorylase disulfide bonds hyperthermostability purine nucleoside phosphorylase Correspondence G. Cacciapuoti Dipartimento di Biochimica e Biofisica F. Cedrangolo Seconda University di Napoli Via Costantinopoli 16 80138 Napoli Italy Fax Tel 39 081 5667519 E-mail Received 2 February 2007 revised 6 March 2007 accepted 12 March 2007 doi We report here the characterization of the first mammalian-like purine nucleoside phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus PfPNP . The gene PF0853 encoding PfPNP was cloned and expressed in Escherichia coli and the recombinant protein was purified to homogeneity. PfPNP is a homohexamer of 180 kDa which shows a much higher similarity with 5 -deoxy-5 -methylthioadenosine phosphorylase MTAP than with purine nucleoside phosphorylase PNP family members. Like human PNP PfPNP shows an absolute specificity for inosine and guanosine. PfPNP shares 50 identity with MTAP from PfMTAP . The alignment of the protein sequences of PfPNP and PfM-TAP indicates that only four residue changes are able to switch the specificity of PfPNP from a 6-oxo to a 6-amino purine nucleoside phosphorylase still maintaining the same overall active site organization. PfPNP is highly thermophilic with an optimum temperature of 120 C and is characterized by extreme thermodynamic stability Tm 110 C that increases to 120 C in the

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