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Istituto di scienza dell'alimentazione

Torna all'elenco Contributi in rivista anno 2011

Contributo in rivista

Tipo: Articolo in rivista

Titolo: Unraveling the structural and functional differences between purine nucleoside phosphorylase and 5'-deoxy-5'-methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus.

Anno di pubblicazione: 2011

Autori: Cacciapuoti G, Marabotti A, Fuccio F, Porcelli M.

Affiliazioni autori: Giovanna Cacciapuoti (Dip. Biochim. Biofis. Seconda UniversitÓ di Napoli), Anna Marabotti (ITB-CNR & ISA-CNR), Francesca Fuccio (Dip. Biochim. Biofis. Seconda UniversitÓ di Napoli), Marina Porcelli (Dip. Biochim. Biofis. Seconda UniversitÓ di Napoli)

Autori CNR:

  • ANNA MARABOTTI
  • ANNA MARABOTTI

Abstract: Purine nucleoside metabolism in the archaeon Pyrococcus furiosus is catalyzed by purine nucleoside phosphorylase (PfPNP) and 5?-deoxy-5?-methylthioadenosine phosphorylase (PfMTAP). These enzymes, characterized by 50% amino acid sequence identity, show non-common features of thermophilicity and thermostability and are stabilized by intramolecular disulfide bonds. PfPNP is highly specific for 6-oxopurine nucleosides while PfMTAP is characterized by a broad substrate specificity with 6-aminopurine nucleosides as preferred substrates. Amino acid sequence comparison clearly shows that the hypothetical active sites of PfPNP and PfMTAP are almost identical and that, in analogy with human 5?-deoxy-5?-methylthioadenosine phosphorylase and human purine nucleoside phosphorylase, residue changes at level of the same crucial positions could be responsible for the switch of substrate specificity. To validate this hypothesis we changed the putative active site of PfPNP by site-directed mutagenesis. Substrate specificity and catalytic efficiency of PfPNP mutants were then analyzed by kinetic studies and compared with the wild-type enzyme. We carried out the molecular modeling of PfPNP and PfMTAP to obtain a picture of the overall enzyme structure and to identify structural features as well as interactions playing critical roles in thermostability. Finally, we utilized the structural models of mutant enzyme-substrate complex to rationalize the functional effects of the mutations.

Pagine da: 1358

Pagine a: 1366

Rivista:

Biochemical and biophysical research communications Elsevier [etc.]
Paese di pubblicazione: Stati Uniti d'America
Lingua: inglese
ISSN: 0006-291X

Numero volume: 1814

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