Rational Design of Plant-Derived Protein Ligases with Altered Substrate Specificity

Asparaginyl ligases are powerful tools for peptide and protein engineering due to their ability to efficiently catalyze a variety of site-specific transpeptidation reactions. Although engineering efforts have enhanced the transpeptidation efficiency of several enzymes, attempts to modify their substrate specificity have been more limited. In a recent study, we produced the first asparaginyl ligase with engineered P2' substrate specificity by mutating Tyr188 to Ala in OaAEP1. Here, we report the engineering of two additional asparaginyl ligases from different plant families, VyPAL2 and butelase 1. We show that mutating the corresponding Tyr residue located in the S2' pocket of these enzymes also expands their substrate scope, enabling the mutant enzymes to process substrates for peptide cyclization, protein-protein ligation, and N-terminal protein labeling that their parent enzymes process poorly. These findings further establish the role of the conserved S2' Tyr residue as a general determinant of substrate specificity for asparaginyl ligases and provide a path toward more extensive engineering efforts.