A Single Computationally Redesigned Enzyme Enables Traceless Protein C-Terminal Functionalization

Protein C-terminal functionalization is pivotal for the development of therapeutic peptides and in chemical protein synthesis. Conventional traceless functionalization strategies rely on the use of inteins, which are often prone to premature hydrolysis. Herein, we repurposed a computationally redesigned peptide amidase variant (termed PAM27) into a glycine-specific carboxypeptidase, enabling traceless protein C-terminal functionalization via cleavage of a glycine residue and conjugation with amines. Twenty computationally designed stabilizing mutations were incorporated for enhancing enzyme stability, while seven substitutions aimed at reducing steric hindrance expanded the acyl acceptor channel, enabling the transit of glycine. PAM27 was found to exhibit stringent specificity for P1′ glycine and broad tolerance for diverse P1 residues as well as nucleophilic agents, facilitating its integration with peptiligase-catalyzed ligation and bio-orthogonal chemical conjugation. PAM27 serves as a promising platform for developing versatile enzymes capable of catalyzing traceless C-terminal modification of various protein or peptide targets with diverse functional moieties.