Peptide ligases, natural enzymes that form new amide bonds to join peptide backbones, contribute to the diversity of peptidyl natural products. While many function as oligopeptide cyclases, they are infrequently found. Among these, the post-translational cyclases of plant cyclotides, represented by the first identified, butelase-1, are Asn/Asp-specific peptide ligases [1]. Collectively referred to as peptide asparaginyl ligases (PALs), they demonstrate impressive catalytic efficiency (kcat/KM = 105-106 M-1/s-1), semi-traceless action (leaving only a single residue at the ligation site), and broad activity in pH 3-9. Furthermore, they can process a wide range of peptidyl substrates of varied sizes and compositions. Practically, PALs are invaluable tools in peptide and protein engineering, finding application in peptide and protein cyclization, site-specific labeling, conjugation, oligomer formation, and live-cell surface precision modifications.[2] PALs are Structurally homologous to asparaginyl endopeptidases (AEPs), thus understanding the factors that differentiate PALs from AEPs is crucial for the discovery of new PALs and for converting the readily available AEPs into ligases. By analyzing the eight known PALs and performing a universal trace analysis of 1,500 plant legumain sequences from public database, we discovered that the key differentiators, or Ligase Activity Determinants (LADs), are three conserved Gly residues found in the substrate-binding pockets. [3-4] Our study, based on the LAD hypothesis, has led to the discovery of seven new PALs and the successful engineering of consensus PALs with a 20-fold higher expression yield and enhanced ligase efficiency. [5] This expands the pool of available peptide ligases, enhancing their utility in peptide and protein engineering and extending their potential applications.