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Title: A cascade enzymatic reaction scheme for irreversible transpeptidative protein ligation
Authors: Xia, Yiyin
Li, Fupeng
Zhang, Xiaohong
Balamkundu, Seetharamsing
Tang, Fan
Hu, Side
Lescar, Julien
Tam, James P.
Liu, Chuan-Fa
Keywords: Science::Biological sciences
Issue Date: 2023
Source: Xia, Y., Li, F., Zhang, X., Balamkundu, S., Tang, F., Hu, S., Lescar, J., Tam, J. P. & Liu, C. (2023). A cascade enzymatic reaction scheme for irreversible transpeptidative protein ligation. Journal of the American Chemical Society, 145(12), 6838-6844.
Project: MOE2016-T3-1-003 
Journal: Journal of the American Chemical Society 
Abstract: 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.
ISSN: 0002-7863
DOI: 10.1021/jacs.2c13628
Schools: School of Biological Sciences 
Rights: © 2023 American Chemical Society. All rights reserved.
Fulltext Permission: none
Fulltext Availability: No Fulltext
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