Chemical Bypass of Intein-Catalyzed N-S Acyl Shift in Protein Splicing

摘要

Inteins are self-processing protein domains that excise themselves out of a precursor polypeptide chain in a multi-step pathway termed protein splicing. In the course of this reaction, the sequences flanking the intein, termed N- and C-terminal exteins, are linked with a peptide bond (Scheme 1 A). Inteins perform only a single turn-over, however, they employ catalytic strategies similar to those of enzymes. While inteins have found widespread use in many applications in biotechnology and protein chemistry, important aspects of the mechanism of protein splicing are still not understood. In particular, the N-S (or N-O) acyl shift of the upstream scissile peptide bond into a thioester (or oxoester) remains intriguing (Scheme 1B). This rearrangement represents the first step of protein splicing in standard inteins and is widely exploited for the generation of protein thioesters. Although thermodynamically unfavored, it does not require any cofactors or energy sources. Several catalytic mechanisms have been proposed for this reaction, including destabiliza-tion of the ground state of the scissile peptide bond, general acid-base catalysis to increase the nucleophilicity of the cysteine (or serine) side chain at position 1 of the intein, and stabilization of the tetrahedral intermediate by means of an oxyanion hole (Scheme 1B). Some of these mechanisms, if not all, can likely be used in combination and may contribute to catalysis to different degrees in different inteins. Here, we pursued a novel chemical approach to directly probe the importance of ground-state destabilization by introducing an alkyl substituent at the amide nitrogen of the scissile peptide bond (Scheme 1B); a strategy inspired by the recent development of N-S switch devices for the chemical synthesis of peptide thioesters. This chemical manipulation indeed supported the N-S acyl shift, even to the extent that it could complement an otherwise essential part of the catalytic framework of the intein, the highly conserved block B histidine. Together, our findings reveal the role of the histidine in a ground-state destabilization mechanism and rule out other roles previously proposed.