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Istituto di scienza dell'alimentazione

Torna all'elenco Contributi in rivista anno 2007

Contributo in rivista

Tipo: Articolo in rivista

Titolo: Biochemical and structural characterization of mammalian-like purine nucleoside phosphorylase from the Archaeon Pyrococcus furiosus

Anno di pubblicazione: 2007

Autori: Cacciapuoti G, Gorassini S, Mazzeo MF, Siciliano RA, Carbone V, Zappia V, Porcelli M.

Affiliazioni autori: Dipartimento di Biochimica e Biofisica ‘F. Cedrangolo’, Seconda Universita` di Napoli, Italy Centro di Spettrometria di Massa Proteomica e Biomolecolare, Istituto di Scienze dell’Alimentazione del CNR, Avellino, Italy

Autori CNR:

  • VIRGINIA CARBONE
  • MARIA FIORELLA MAZZEO
  • ROSA ANNA SICILIANO

Lingua: inglese

Abstract: 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 P. furiosus (PfMTAP). The alignment of the protein sequences of PfPNP and PfMTAP 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 degrees C and is characterized by extreme thermodynamic stability (T(m), 110 degrees C that increases to 120 degrees C in the presence of 100 mm phosphate), kinetic stability (100% residual activity after 4 h incubation at 100 degrees C), and remarkable SDS-resistance. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. By integrating biochemical methodologies with mass spectrometry we assigned three pairs of intrasubunit disulfide bridges that play a role in the stability of the enzyme against thermal inactivation. The characterization of the thermal properties of the C254S/C256S mutant suggests that the CXC motif in the C-terminal region may also account for the extreme enzyme thermostability.

Lingua abstract: inglese

Pagine da: 2482

Pagine a: 2495

Rivista:

The FEBS journal Blackwell,
Paese di pubblicazione: Regno Unito
Lingua: inglese
ISSN: 1742-464X

Numero volume: 274

DOI: 10.1111/j.1742-4658.2007.05784.x

Referee: Sì: Internazionale

Indicizzato da:

  • ISI Web of Science (WOS) [000246029800005]
  • PubMed [17419725]

Parole chiave:

  • CXC motif
  • 5'-deoxy-5'-methylthioadenosine phosphorylase
  • disulfide bonds
  • hyperthermostability
  • purine nucleoside

Strutture CNR:

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