Enzymatic C‐to‐C Protein Ligation

Transpeptidase-catalyzed protein and peptide modifications have been widely utilized for generating conjugates of interest biological investigation or therapeutic applications. However, all known transpeptidases are constrained to ligating in the N-to-C orientation, limiting scope attainable products. Here, we report that an engineered asparaginyl ligase accepts diverse incoming nucleophile substrate mimetics, particularly when a means selectively quenching reactivity byproducts released from recognition sequence is employed. In addition directly catalyzing formation l-/d- α-/β-amino acid junctions, find C-terminal Leu-ethylenediamine (Leu-Eda) motifs be bona fide mimetics native N-terminal Gly-Leu sequences. Appending Leu-Eda synthetic peptides or, via intein-splicing approach, recombinant proteins enables direct transpeptidase-catalyzed C-to-C ligations. This work significantly expands enzyme-catalyzed transpeptidation reactions.