Unraveling the structural and functional differences between purine nucleoside phosphorylase and 5′-deoxy-5′-methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus

Giovanna Cacciapuoti, Anna Marabotti, Francesca Fuccio, Marina Porcelli

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)1358-1366
Number of pages9
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1814
Issue number10
DOIs
Publication statusPublished - Oct 2011

Keywords

  • 5′-deoxy-5′-methylthioadenosine phosphorylase
  • Purine nucleoside phosphorylase
  • Pyrococcus furiosus
  • Site-directed mutagenesis
  • Substrate specificity

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Analytical Chemistry
  • Molecular Biology

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