Protein modification and ligation are crucial in biochemistry, protein engineering, and drug development. Chemical methods, while widely used, depend on specific amino acid residues, requiring case-by-case optimization and potentially masking epitopes or inactivating proteins. Enzyme-catalytic ligation offers advantages like mild conditions, site-specific modification, and bioactivity preservation. Existing ligases, such as Sortase A and peptide asparaginyl ligases (PALs), have limitations including long recognition sequences (Sortase A), low yields, and complex purification (PALs like butelase-1 and OaAEP1). Butelase-1, although efficient, suffers from low yields and complicated processes. OaAEP1, while showing promise with its OaAEP1-C247A mutant, still has limitations in purification and activation. This research aims to develop a highly efficient, easily produced ligase for improved site-specific protein conjugation.

The literature review focuses on existing protein modification techniques, comparing chemical methods with enzyme-catalytic ligation. Sortase A, while useful, has limitations related to its long recognition sequence and reversible reactions. PALs offer the advantage of shorter recognition sequences, but existing PALs like butelase-1 and OaAEP1 have been limited by low yields and complex purification procedures. The researchers highlight the need for a more efficient and easily produced ligase. The crystal structure of OaAEP1 has provided insights, leading to the OaAEP1-C247A mutant with improved activity. However, challenges related to acid activation and purification remain.

The study used a structure-based rational approach to optimize OaAEP1. Four truncations of OaAEP1-C247A (OaAEP1-C247A-aa24-351, OaAEP1-C247A-aa55-351, OaAEP1-C247A-aa24-325, OaAEP1-C247A-aa55-325) were designed and expressed in *E. coli*. The proteins were purified using a cobalt-based IMAC column. Ligation activity was evaluated using a model substrate (Pep133-NGL) and a nucleophile peptide (Gly-Leu). ELISA was used to analyze ligation products. The optimal truncation, OaAEP1-C247A-aa55-351, was further investigated. pH scanning assays and metal ion studies were conducted to optimize ligation conditions. Substrate and nucleophile specificity were determined by substituting amino acids at various positions in the substrate and nucleophile peptides. A Förster resonance energy transfer (FRET) assay was used to study the kinetics of OaAEP1-C247A-aa55-351. The ability to label a well-folded protein was also tested using a truncated nucleocapsid protein of SARS-CoV-2.

OaAEP1-C247A-aa55-351 showed superior ligation activity compared to other truncations. It exhibited activity across a wide pH range (4.0-8.0), with optimal activity at pH 7.0-7.2. Fe³⁺ significantly enhanced its activity. The ligase demonstrated high recognition for the "Asn-Ala-Leu" motif and the N-terminal "Arg-Leu" as a nucleophile. The combination of "Asn-Ala-Leu" and "Arg-Leu" under Fe³⁺ conditions resulted in a 70-fold increase in catalytic activity compared to OaAEP1-C247A and a 2-fold increase compared to butelase-1. Kinetic analysis using the FRET assay showed a high Kcat/Km value. OaAEP1-C247A-aa55-351 successfully labeled a well-folded recombinant protein (SARS-CoV-2 nucleocapsid protein). Over 90% yield of ligation product was achieved in a peptide ligation reaction.

The results demonstrate the successful design of a highly efficient asparaginyl ligase, overcoming the limitations of previous ligases. The improved activity and simplified purification process make OaAEP1-C247A-aa55-351 a valuable tool for various applications. The identification of optimal recognition and nucleophile motifs enhances the specificity and efficiency of the ligation reaction. The effect of Fe³⁺ further improves the catalytic efficiency. The successful labeling of a well-folded protein demonstrates its potential for use in protein engineering and drug development.

This study successfully engineered OaAEP1-C247A-aa55-351, a highly efficient asparaginyl ligase with a simplified purification process. The optimized recognition motif ("Asn-Ala-Leu") and nucleophile ("Arg-Leu"), along with Fe³⁺ enhancement, significantly improved its catalytic activity. This improved ligase has promising applications in protein engineering, chemo-enzymatic modification, and drug development. Future research could explore further optimization of the ligase, broader substrate specificity studies, and applications in various biotechnological areas.

The study focused on a specific set of substrates and nucleophiles. While OaAEP1-C247A-aa55-351 showed high activity with "Asn-Ala-Leu" and "Arg-Leu", further investigation is needed to determine its broader substrate compatibility. The role of Fe³⁺ in the mechanism of the ligation reaction needs further investigation. The study primarily used in vitro assays. Future research could validate the findings in in vivo systems.