Highly Efficient Transpeptidase-Catalyzed\nIsopeptide\nLigation

Transpeptidases are specialized enzymes that have evolved\nfor site-selective\nmodification of peptides and proteins at their backbone termini. Approaches\nfor adapting transpeptidases to catalyze side chain modifications\nare substantially more restricted, and typically rely on large recognition\ntags or require specific reaction conditions that are not easily compatible\nwith broader applications. Here we show that the engineered asparaginyl\nligase <i>Oa</i>AEP1 catalyzes direct isopeptide ligation\nby accepting an internal 2,3-diaminopropionic acid (Dap) residue adjacent\nto Leu, a motif that mimics the canonical N-terminal Gly-Leu substrate.\nThese reactions proceed efficiently at near-neutral pH without any\nrequired additives, enabling straightforward formation of diverse\nisopeptide-linked products under simple reaction conditions. We demonstrate\nthat <i>Oa</i>AEP1-catalyzed isopeptide ligation can be\nutilized for site-selective side chain labeling at an introduced Dap\nresidue with minimal off-target labeling of Lys residues. Additionally,\nwe generate engineered peptide topologies via intramolecular side\nchain-to-tail cross-links and produce direct protein–cyclic\npeptide fusions via efficient intermolecular ligation. We also show\nthat <i>Oa</i>AEP1-catalyzed isopeptide ligation extends\nto d-peptide acceptors containing a retro-inverso d-Leu-d-Dap motif. This capability further expands the range\nand complexity of isopeptide-linked products that can be accessed\nwith <i>Oa</i>AEP1, which we exemplify by forming a hybrid d-/l- bicyclic peptide topology where both termini\nare linked to internal side chains.