RESUMO
5'-Deoxy-5'-methylthioadenosine phosphorylase II from Sulfolobus solfataricus (SsMTAPII) and purine nucleoside phosphorylase from Pyrococcus furiosus (PfPNP) are hyperthermophilic purine nucleoside phosphorylases stabilized by intrasubunit disulfide bonds. In their C-terminus, both enzymes harbour a CXC motif analogous to the CXXC motif present at the active site of eukaryotic protein disulfide isomerase. By monitoring the refolding of SsMTAPII, PfPNP and their mutants lacking the C-terminal cysteine pair after guanidine-induced unfolding, we demonstrated that the CXC motif is required for the folding of these enzymes. Moreover, two synthesized CXC-containing peptides with the same amino acid sequences present in the C-terminal regions of SsMTAPII and PfPNP were found to act as in vitro catalysts of oxidative folding. These small peptides are involved in the folding of partially refolded SsMTAPII, PfPNP and their CXC-lacking mutants, with a concomitant recovery of their catalytic activity, thus indicating that the CXC motif is necessary to obtain complete reversibility from the unfolded state of the two proteins. The two CXC-containing peptides are also able to reactivate scrambled RNaseA. The data obtained in the present study represent the first example of how the CXC motif improves both stability and folding in hyperthermophilic proteins with disulfide bonds.
Assuntos
Archaea/enzimologia , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Purina-Núcleosídeo Fosforilase/química , Purina-Núcleosídeo Fosforilase/metabolismo , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/fisiologia , Sequência de Aminoácidos , Proteínas Arqueais/genética , Dados de Sequência Molecular , Dobramento de Proteína , Estabilidade Proteica , Purina-Núcleosídeo Fosforilase/genética , Homologia de Sequência de AminoácidosRESUMO
We report the biochemical and structural characterization of the purine-specific ribonucleoside hydrolase from the archaeon Sulfolobus solfataricus (SsIAG-NH). SsIAG-NH is a homodimer of 70kDa specific for adenosine, guanosine and inosine. SsIAG-NH is highly thermophilic and is characterized by extreme thermodynamic stability (T(m), 107 degrees C), kinetic stability and remarkable resistance to guanidinium chloride-induced unfolding. A disulfide bond that, on the basis of SDS-PAGE is positioned intersubunits, plays an important role in thermal stability. SsIAG-NH shares 43% sequence identity with the homologous pyrimidine-specific nucleoside hydrolase from S. solfataricus (SsCU-NH). The comparative sequence alignment of SsIAG-NH, SsCU-NH, purine non-specific nucleoside hydrolase from Crithidia fasciculata and purine-specific nucleoside hydrolase from Trypanosoma vivax shows that, only few changes in the base pocket are responsible for different substrate specificity of two S. solfataricus enzymes. The structure of SsIAG-NH predicted by homology modeling allows us to infer the role of specific residues in substrate specificity and thermostability.
Assuntos
N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/metabolismo , Sulfolobus solfataricus/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Domínio Catalítico , Clonagem Molecular , Primers do DNA/genética , Estabilidade Enzimática , Genes Arqueais , Cinética , Modelos Moleculares , Dados de Sequência Molecular , N-Glicosil Hidrolases/genética , Nucleosídeos de Purina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Sulfolobus solfataricus/genética , TermodinâmicaRESUMO
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.
