Purine nucleoside phosphorylases from hyperthermophilic Archaea require a CXC motif for stability and folding.
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.
Cacciapuoti
Giovanna
G
Department of Biochemistry and Biophysics 'F. Cedrangolo', Second University of Naples, Italy. giovanna.cacciapuoti@unina2.it
Peluso
Iolanda
I
Fuccio
Francesca
F
Porcelli
Marina
M
D020816:Amino Acid Motifs / Q000235:genetics / Q000502:physiology; D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology*; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017510:Protein Folding; D055550:Protein Stability; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D019843:Archaeal Proteins; D011683:Purine-Nucleoside Phosphorylase