Noncanonical Inteins and Their Mechanisms

Significant insight into the canonical mechanism of protein splicing has been afforded by the study of inteins whose activities have been modulated by mutation. In the sections that follow, we will describe the splicing of inteins or intein-like domains that lack conserved intein elements yet still promote protein splicing or splicing side reactions, and how the study of non-canonical inteins broadens our understanding of how inteins promote the individual steps of splicing.

3.1 Substitution of the N-Terminal Nucleophile

Fourteen non-canonical inteins are known that lack an N-terminal nucleophile, of which 13 have N-terminal Ala (Perler 2002). Four of these inteins are known to promote protein splicing (Southworth et al. 2000; Yamamoto et al. 2001; Southworth and Perler 2002), suggesting that they have evolved to splice by bypassing the initial NIS acyl shift (Fig. 2) through a direct attack by the downstream nucleophile on the upstream scissile peptide bond (Fig. 4A). In two such inteins, mutation of the N-terminal Ala to Gly blocks protein splicing, suggesting that the local conformation of the active site is important for coordinating the direct attack of the downstream nucleophile on the peptide bond (Southworth et al. 2000; Yamamoto et al. 2001). In the Mja KlbA intein, mutation of the N-terminal Ala to Cys or Ser yields significant amounts of spliced product, and a double mutant (Ala1Cys and Cys+1Ala) promotes an NIS acyl rearrangement (Southworth et al. 2000; Yamamoto et al. 2001), implying that the Cys1Ala substitution is relatively recent. Mutation of the conserved Thr and His residues of motif N3 greatly reduces the extent of splicing, implying a role for these residues in the attack of the C-terminal nucleophile on the upstream scissile peptide bond.

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