Design of a recombinant asparaginyl ligase for site-specific modification using efficient recognition and nucleophile motifs

• DOI: 10.1038/s42004-024-01173-8
• Publication Date: 2024-04-18T11:02:03Z
• Authors: Jiabao Tang; Mengling Hao; Junxian Liu; Yaling Chen; Gulimire Wufuer; Jie Zhu; Xuejie Zhang; Tingquan Zheng; Mujin Fang; Shiyin Zhang; Tingdong Li; Shengxiang Ge; Jun Zhang; Ningshao Xia
• Keywords: Bioconjugation; Rational design; Protein Engineering

Abstract Asparaginyl ligases have been extensively utilized as valuable tools for site-specific bioconjugation or surface-modification. However, the application is hindered by laborious and poorly reproducible preparation processes, unstable activity ambiguous substrate requirements. To address these limitations, this study employed a structure-based rational approach to obtain high-yield high-activity protein ligase called OaAEP1-C247A-aa55-351. It was observed that OaAEP1-C247A-aa55-351 exhibits appreciable catalytic activities across wide pH range, addition of Fe 3+ metal ion effectively enhances power. Importantly, provides insight into recognition nucleophile peptide profiles The demonstrates higher ability “Asn-Ala-Leu” motif an N-terminus “Arg-Leu” nucleophiles, which significantly increases reaction yield. Consequently, with highly efficient motif, under buffer containing 70-fold 2-fold than previously reported OaAEP1-C247A most butelase-1, respectively. Thus, designed OaAEP1-C247A-aa55-351, its alternative options, holds promising potential applications in engineering, chemo-enzymatic modification, development drugs.