Creating an Unusual Glycine-Rich Motif in a Peptide Amidase Leads to Versatile Protein C-Terminal Traceless Functionalization

The rising demand for regioselective protein modifications in chemical biology and pharmaceutical manufacturing has fueled efforts to develop diverse techniques that functionalize native amino acid residues. Although many powerful strategies have provided elegant solutions for functionalizing N-termini and side chains, sequence-unconstrained versatile C-terminal functionalization remains a challenge. Here, we report an engineered peptide amidase (PAM) for C-terminal traceless functionalization in aqueous solution with a broad spectrum of both nucleophiles and protein sequences, excellent yields (up to 98%), and good compatibility with the click reaction. Computational analysis suggested an expanded nucleophile pocket induced by the introduction of an unusual glycine-rich motif, which may enrich the structural diversity in protein design. We anticipate that the successfully engineered PAM holds great potential in the applications of protein chemistry and proteomics, and highlights the employment of serine hydrolases in catalyzing acyl shift reactions that compete with hydrolysis under aqueous conditions.