Catalysis of individual steps of protein splicing of the
            <i>Pyrococcus abyssi</i>
            PolII intein

Dorval, Deirdre M.; Connor, Katherine R.; Mills, Kenneth V.

doi:10.1096/fasebj.20.4.a40-d

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“…The bond cleavage is also temperature- and time-dependent (Figure B). The traditional N-terminal cleavage mediated by thioester formation is known to be temperature and time dependent as well . In that case, it is likely that the thermophilic intein, which we have shown by NMR analysis to be highly conformationally rigid at room temperature, requires higher temperatures to have sufficient flexibility in the active site to promote the amide-to-thioester rearrangement.…”

Section: Resultsmentioning

confidence: 95%

Intein-Promoted Cyclization of Aspartic Acid Flanking the Intein Leads to Atypical N-Terminal Cleavage

et al. 2017

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Protein splicing is a post-translational reaction facilitated by an intein, or intervening protein, which involves the removal of the intein and the ligation of the flanking polypeptides, or exteins. A DNA polymerase II intein from Pyrococcus abyssi (Pab PolII intein) can promote protein splicing in vitro on incubation at high temperature. Mutation of active site residues Cys1, Gln185, and Cys+1 to Ala results in an inactive intein precursor, which cannot promote the steps of splicing, including cleavage of the peptide bond linking the N-extein and intein (N-terminal cleavage). Surprisingly, coupling the inactivating mutations to a change of the residue at the C-terminus of the N-extein (N-1 residue) from the native Asn to Asp reactivates N-terminal cleavage at pH 5. Similar "aspartic acid effects" have been observed in other proteins and peptides but usually only occur at lower pH values. In this case, however, the unusual N-terminal cleavage is abolished by mutations to catalytic active site residues and unfolding of the intein, indicating that this cleavage effect is mediated by the intein active site and the intein fold. We show via mass spectrometry that the reaction proceeds through cyclization of Asp resulting in anhydride formation coupled to peptide bond cleavage. Our results add to the richness of the understanding of the mechanism of protein splicing and provide insight into the stability of proteins at moderately low pH. The results also explain, and may help practitioners avoid, a side reaction that may complicate intein applications in biotechnology.

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Section: Resultsmentioning

confidence: 95%