title: Unraveling the structural and functional differences between purine nucleoside phosphorylase and 5'-deoxy-5'-methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus.
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.
authors.affiliation: Dipartimento di Biochimica e Biofisica F. Cedrangolo, Seconda Università di Napoli, Via Costantinopoli 16, Napoli, Italy. giovanna.cacciapuoti@unina2.it
authors.forename: Giovanna
authors.initials: G
authors.lastname: Cacciapuoti
authors.forename: Anna
authors.initials: A
authors.lastname: Marabotti
authors.forename: Francesca
authors.initials: F
authors.lastname: Fuccio
authors.forename: Marina
authors.initials: M
authors.lastname: Porcelli
mesh_terms: D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology / Q000378:metabolism; D020134:Catalytic Domain; D004795:Enzyme Stability; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D011683:Purine-Nucleoside Phosphorylase; C021784:5'-methylthioadenosine phosphorylase