Highly Efficient Transpeptidase-Catalyzed Isopeptide Ligation

Transpeptidases are specialized enzymes that have evolved for site-selective modification of peptides and proteins at their backbone termini. Approaches for adapting transpeptidases to catalyze side chain modifications are substantially more restricted, and typically rely on large recognition tags or require specific reaction conditions that are not easily compatible with broader applications. Here we show that the engineered asparaginyl ligase OaAEP1 catalyzes direct isopeptide ligation by accepting an internal 2,3-diaminopropionic acid (Dap) residue adjacent to Leu, a motif that mimics the canonical N-terminal Gly-Leu substrate. These reactions proceed efficiently at near-neutral pH without any required additives, enabling straightforward formation of diverse isopeptide-linked products under simple reaction conditions. We demonstrate that OaAEP1-catalyzed isopeptide ligation can be utilized for site-selective side chain labeling at an introduced Dap residue with minimal off-target labeling of Lys residues. Additionally, we generate engineered peptide topologies via intramolecular side chain-to-tail cross-links and produce direct protein-cyclic peptide fusions via efficient intermolecular ligation. We also show that OaAEP1-catalyzed isopeptide ligation extends to d-peptide acceptors containing a retro-inverso d-Leu-d-Dap motif. This capability further expands the range and complexity of isopeptide-linked products that can be accessed with OaAEP1, which we exemplify by forming a hybrid d-/l- bicyclic peptide topology where both termini are linked to internal side chains.