A cascade enzymatic reaction scheme for irreversible transpeptidative protein ligation

• Main Authors: Xia, Yiyin, Li, Fupeng, Zhang, Xiaohong, Balamkundu, Seetharamsing, Tang, Fan, Hu, Side, Lescar, Julien, Tam, James P., Liu, Chuan-Fa
• Other Authors: School of Biological Sciences
• Format: Article
• Language: English
• Published: 2023
• Subjects: Science::Biological sciences; Enzymes; Molar Ratio
• Online Access: https://hdl.handle.net/10356/169065
• Institution: Nanyang Technological University

Enzymatic peptide ligation holds great promise in the study of protein functions and development of protein therapeutics. Owing to their high catalytic efficiency and a minimal tripeptide recognition motif, peptidyl asparaginyl ligases (PALs) are particularly useful tools for bioconjugation. However, as an inherent limitation of transpeptidases, PAL-mediated ligation is reversible, requiring a large excess of one of the ligation partners to shift the reaction equilibrium in the forward direction. Herein, we report a method to make PAL-mediated intermolecular ligation irreversible by coupling it to glutaminyl cyclase (QC)-catalyzed pyroglutamyl formation. In this method, the acyl donor substrate of PALs is designed to have glutamine at the P1' position of the Asn-P1'-P2' tripeptide PAL recognition motif. Upon ligation with an acyl acceptor substrate, the acyl donor substrate releases a leaving group in which the exposed N-terminal glutamine is cyclized by QC, quenching the Gln Nα-amine in a lactam. Using this method, PAL-mediated ligation can achieve near-quantitative yields even at an equal molar ratio between the two ligation partners. We have demonstrated this method for a wide range of applications, including protein-to-protein ligations. We anticipate that this cascade enzymatic reaction scheme will make PAL enzymes well suited for numerous new uses in biotechnology.