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.