Variations in the intein-mediated protein splicing mechanism are becoming more apparent as polymorphisms in conserved catalytic residues are identified. Several families of inteins have been identified that begin with Ala rather than the consensus nucleophiles, Ser or Cys. In standard inteins, an N-terminal Ser, Cys or Thr is absolutely required for splicing. An N-terminal Ala cannot perform the initial reaction of the standard protein splicing pathway to yield the requisite N-terminal splice junction (thio)ester. However, experiments with the M. jannaschii KlbA intein demonstrated that Ala1 inteins can splice efficiently using an alternative protein splicing mechanism (Southworth 2000). In this non-canonical pathway, the C-extein nucleophile (Ser, Cys or Thr) attacks a peptide bond at the N-terminal splice junction rather than a (thio)ester bond, alleviating the need to form the initial (thio)ester at the N-terminal splice junction. The remainder of the two pathways is identical: branch resolution by Asn cyclization is followed by an acyl rearrangement to form a native peptide bond between the ligated exteins. Just like standard inteins, the Mja KlbA intein also requires the help of the conserved Thr and His in Block B to activate the N-terminal splice junction. We have also demonstrated splicing of the Mle DnaB intein (dnaB-b insertion site, E. Davis, M. Southworth & F. Perler, unpublished data) which is another Ala1 intein, suggesting that different families of naturally occurring Ala1 inteins should be capable of splicing.

The KlbA and Mle DnaB inteins have overcome the barriers to direct nucleophilic attack on the peptide bond at the N-terminal splice junction that are present in previously studied inteins with Ser or Cys at their N-terminus. It is unclear why other inteins can't perform similar reactions, since the Block B oxyanion hole is still available to facilitate direct attack on the N-terminal splice junction. Possibly, (thio)ester formation may be necessary in standard inteins to align the C-extein nucleophile, to remove steric hindrances or to induce a conformational shift that allows attack by the +1 nucleophile (Cys, Ser or Thr). The crystal structure of a S.cerevisiae VMA intein precursor has helped to resolve this question by revealing that Cys+1 is too far away to directly attack either a peptide or a thioester bond at the N-terminal splice junction, leading the authors to suggest that inteins must undergo a conformational shift to allow attack by the Cys+1 nucleophile (Poland 2000). We propose that Cys+1 (or its equivalent residue) in Ala1 inteins is already in position to attack the N-terminal splice junction amide bond in the precursors protein.

(Perler, F. B. (2002). InBase, the Intein Database. Nucleic Acids Res. 30, 383-384)