Premium
Biochemical and structural characterization of mammalian‐like purine nucleoside phosphorylase from the Archaeon Pyrococcus furiosus
Author(s) -
Cacciapuoti Giovanna,
Gorassini Sabrina,
Mazzeo Maria Fiorella,
Siciliano Rosa Anna,
Carbone Virginia,
Zappia Vincenzo,
Porcelli Marina
Publication year - 2007
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2007.05784.x
Subject(s) - pyrococcus furiosus , purine nucleoside phosphorylase , biochemistry , inosine , thermophile , proteolysis , chemistry , phosphorolysis , guanosine , escherichia coli , purine , biology , stereochemistry , enzyme , gene , archaea
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 °C and is characterized by extreme thermodynamic stability (T m , 110 °C that increases to 120 °C in the presence of 100 m m phosphate), kinetic stability (100% residual activity after 4 h incubation at 100 °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.