2026-04-13T18:20:50Zhttps://recercat.cat/oai/request
oai:recercat.cat:10459.1/4642682025-09-15T18:48:36Zcom_2072_3622col_2072_479130
RECERCAT author Hemu, Xinya author Chan, Ning-Yu author Tai Liew, Heng author Hu, Side author Zhang, Xiaohong author Serra, Aida author Lescar, Julien author Liu, Chuan-Fa author Tam, James P. 2023 Peptide asparaginyl ligases (PALs) are useful tools for precision modifications of proteins and live-cell surfaces by ligating peptides after Asn/Asp (Asx). They share high sequence and structural similarity to plant legumains that are generally known as asparaginyl endopeptidases (AEPs), thus making it challenging to identify PALs from AEPs. In this study, we investigate 875 plant species from algae to seed plants with available sequence data in public databases to identify new PALs. We conducted evolutionary trace analysis on 1500 plant legumains, including eight known PALs, to identify key residues that could differentiate ligases and proteases, followed by recombinant expression and functional validation of 16 novel legumains. Previously, we showed that the substrate-binding sequences flanking the catalytic site can strongly influence the enzymatic direction of a legumain and which we named as ligase-activity determinants (LADs). Here, we show that two conserved substrate-binding Gly residues of LADs are critical, but negative determinants for ligase activity. Our results suggest that specific glycine residues are molecular determinants to identify PALs and AEPs as two different legumain subfamilies, accounting for c. 1% and 88%, respectively.This research was supported by the Academic Research Grant Tier 3 (MOE2016-T3-1-003) from the Singapore Ministry of Education and Nanyang Technological University. We thank Dr Ka Ho Wong for his pioneer contribution to the bioinformatics analysis. cc-by-nc (c) Xinya Hemu et al., 2023 Attribution-NoDerivatives 4.0 International info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc/4.0/ Asparaginyl endopeptidase Butelase Ligase-activity determinant Peptide asparaginyl ligase Plant legumain Substrate-binding glycine residues are major determinants for hydrolase and ligase activity of plant legumains info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion