Pyrimidine-specific ribonucleoside hydrolase from the archaeon Sulfolobus solfataricus- biochemical characterization and homology modeling

Marina Porcelli, Luigi Concilio, Iolanda Peluso, Anna Marabotti, Angelo Facchiano, Giovanna Cacciapuoti

Research output: Contribution to journalArticle

Abstract

We report the characterization of the pyrimidine-specific ribonucleoside hydrolase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsCU-NH). The gene encoding SsCU-NH was cloned and expressed in Escherichia coli and the recombinant protein was purified to homogeneity. SsCU-NH is a homotetramer of 140 kDa that recognizes uridine and cytidine as substrates. SsCU-NH shares 34% sequence identity with pyrimidine-specific nucleoside hydrolase from E. coli YeiK. The alignment of the amino acid sequences of SsCU-NH with nucleoside hydrolases whose 3D structures have been solved indicates that the amino acid residues involved in the calcium- and ribose-binding sites are preserved. SsCU-NH is highly thermophilic with an optimum temperature of 100°C and is characterized by extreme thermodynamic stability (Tm = 106°C) and kinetic stability (100% residual activity after 1 h incubation at 90°C). Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is necessary for the integrity of the active site. The structure of the enzyme determined by homology modeling provides insight into the proteolytic analyses as well as into mechanisms of thermal stability. This is the first nucleoside hydrolase from Archaea.

Original languageEnglish
Pages (from-to)1900-1914
Number of pages15
JournalFEBS Journal
Volume275
Issue number8
DOIs
Publication statusPublished - Apr 2008

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Keywords

  • Homology modeling
  • Hyperthermostability
  • Nucleoside hydrolase
  • Nucleoside metabolism
  • Sulfolobus solfataricus

ASJC Scopus subject areas

  • Biochemistry

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