Design of a Recombinant Asparaginyl Ligase for Site-Specific Modification Using Efficient Recognition and Nucleophile Motifs

Asparaginyl ligases have been extensively utilized as valuable tools for site-specific bioconjugation or surface-modification. However, the application is hindered by the laborious and poorly reproducible preparation processes, unstable activity and ambiguous substrate requirements. To address these limitations, this study employed a structure-based rational approach to obtain a high-yield and high-activity protein ligase called OaAEP1-C247A-aa55-351. It was observed that OaAEP1-C247A-aa55-351 exhibits appreciable catalytic activities across a wide pH range, and the addition of the Fe3+ metal ion effectively enhances the catalytic power. Importantly, this study provides insight into the recognition and nucleophile peptide profiles of OaAEP1-C247A-aa55-351. The ligase demonstrates a higher recognition ability for the "Asn-Ala-Leu" motif and an N-terminus "Arg-Leu" as nucleophiles, which significantly increases the reaction yield. Consequently, the catalytic activity of OaAEP1-C247A-aa55-351 with highly efficient recognition and nucleophile motif, "Asn-Ala-Leu" and "Arg-Leu" under the buffer containing Fe3+ is 70-fold and 2-fold higher than previously reported OaAEP1-C247A and the most efficient butelase-1, respectively. Thus, the designed OaAEP1-C247A-aa55-351, with its highly efficient recognition and alternative nucleophile options, holds promising potential for applications in protein engineering, chemo-enzymatic modification, and the development of drugs. Asparaginyl ligases have been utilized as valuable tools for protein engineering, such as through site-specific bioconjugation or surface modification, however, their application is limited due to time-consuming preparation processes and unstable activities. Here, the authors develop a truncated protein ligase OaAEP1-C247A-aa55-351 from an OaAEP1-C247A mutant, which simplifies the preparation steps, tolerates a wider pH range, and enhances the catalytic activities by using efficient recognition and nucleophile motifs.