title: Pyrimidine-specific ribonucleoside hydrolase from the archaeon Sulfolobus solfataricus--biochemical characterization and homology modeling.
abstract: We report the characterization of the pyrimidine-specific ribonucleoside hydrolase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsCU-NH). The gene SSO0505 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 degrees C and is characterized by extreme thermodynamic stability (T(m) = 106 degrees C) and kinetic stability (100% residual activity after 1 h incubation at 90 degrees 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.
authors: Dipartimento di Biochimica e Biofisica F. Cedrangolo, Seconda Università di Napoli, Via Costantinopoli 16, Naples, Italy. marina.porcelli@unina2.it
authors: Marina
authors: M
authors: Porcelli
authors: Luigi
authors: L
authors: Concilio
authors: Iolanda
authors: I
authors: Peluso
authors: Anna
authors: A
authors: Marabotti
authors: Angelo
authors: A
authors: Facchiano
authors: Giovanna
authors: G
authors: Cacciapuoti
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001669:Biochemical Phenomena; D001671:Biochemistry; D015870:Gene Expression; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011743:Pyrimidines / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
chemical_list: D011743:Pyrimidines; D009699:N-Glycosyl Hydrolases; C030986:pyrimidine