Purine nucleoside phosphorylases from hyperthermophilic Archaea require a CXC motif for stability and folding

Abstract

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.

Role of N-Glycosylation Sites and CXC Motifs in Trafficking of Medicago truncatula Nod Factor Perception Protein to Plasma Membrane

“…Deletion of one AA to form a CXC motif in E. coli thioredoxin greatly increases the disulfide isomerase activity, and even a synthetic CXC tripeptide possesses such activity (52). In addition, the presence of a CXC motif at the C terminus of two enzymes from hyperthermophilic Archaea was shown to be necessary for folding of these enzymes (53), and in their absence, the addition of synthetic peptides, containing the CXC region, was able to restore the folding. These peptides were also shown to have disulfide isomerase activities (53).…”

Efficient Fludarabine‐Activating PNP From Archaea as a Guidance for Redesign the Active Site of E. Coli PNP

“…Each subunit is stabilized by two pairs of intramolecular disulfide bonds and shows in its C‐terminal region an unusual CXC motif as a typical feature [Cacciapuoti et al, ; Zhang et al, ]. By enzymatic activity measurements, it has been demonstrated that this unusual disulfide plays a specific stabilizing role by acting as a redox reagent in restoring the native state of reduced or scrambled structural disulfide of the enzyme [Cacciapuoti et al, , ]. The hexamer of SsMTAPII, consisting of a dimer‐of‐trimers with one active site per monomer, is considerably different from the hexamer of SsMTAP and fits more closely with members of the trimeric class of PNPs [Zhang et al, ].…”