An alternative protein splicing mechanism for inteins lacking an N-terminal nucleophile

Variations in the intein‐mediated protein splicing mechanism are becoming more apparent as polymorphisms in conserved catalytic residues are identified. The conserved Ser or Cys at the intein N‐terminus and the conserved intein penultimate His are absent in the KlbA family of inteins. These inteins were predicted to be inactive, since 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. Despite the presence of an N‐terminal Ala and a penultimate Ser, the KlbA inteins splice efficiently using an alternative protein splicing mechanism. In this non‐canonical pathway, the C‐extein nucleophile 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 the same: branch resolution by Asn cyclization is followed by an acyl rearrangement to form a native peptide bond between the ligated exteins.