This study addresses limitations of asparaginyl ligases in site-specific bioconjugation by employing a structure-based approach to create a high-yield, high-activity protein ligase, OaAEP1-C247A-aa55-351. This ligase shows significant catalytic activity across a wide pH range, enhanced by Fe³⁺. It exhibits high recognition for the "Asn-Ala-Leu" motif and uses an N-terminus "Arg-Leu" as a nucleophile, increasing reaction yield. Its activity is 70-fold higher than previous OaAEP1-C247A and 2-fold higher than butelase-1. This improved ligase shows great potential in protein engineering and drug development.
