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dc.contributor.authorChen, Yuen_US
dc.contributor.authorZhang, Dingpengen_US
dc.contributor.authorZhang, Xiaohongen_US
dc.contributor.authorWang, Zhenen_US
dc.contributor.authorLiu, Chuan-Faen_US
dc.contributor.authorTam, James P.en_US
dc.identifier.citationChen, Y., Zhang, D., Zhang, X., Wang, Z., Liu, C. & Tam, J. P. (2021). Site-specific protein modifications by an engineered asparaginyl endopeptidase from Viola canadensis. Frontiers in Chemistry, 9, 768854-.
dc.description.abstractAsparaginyl endopeptidases (AEPs) or legumains are Asn/Asp (Asx)-specific proteases that break peptide bonds, but also function as peptide asparaginyl ligases (PALs) that make peptide bonds. This ligase activity can be used for site-specific protein modifications in biochemical and biotechnological applications. Although AEPs are common, PALs are rare. We previously proposed ligase activity determinants (LADs) of these enzymes that could determine whether they catalyze formation or breakage of peptide bonds. LADs are key residues forming the S2 and S1' substrate-binding pockets flanking the S1 active site. Here, we build on the LAD hypothesis with the engineering of ligases from proteases by mutating the S2 and S1' pockets of VcAEP, an AEP from Viola canadensis. Wild type VcAEP yields <5% cyclic product from a linear substrate at pH 6.5, whereas the single mutants VcAEP-V238A (Vc1a) and VcAEP-Y168A (Vc1b) targeting the S2 and S1' substrate-binding pockets yielded 34 and 61% cyclic products, respectively. The double mutant VcAEP-V238A/Y168A (Vc1c) targeting both the S2 and S1' substrate-binding pockets yielded >90% cyclic products. Vc1c had cyclization efficiency of 917,759 M-1s-1, which is one of the fastest rates for ligases yet reported. Vc1c is useful for protein engineering applications, including labeling of DARPins and cell surface MCF-7, as well as producing cyclic protein sfGFP. Together, our work validates the importance of LADs for AEP ligase activity and provides valuable tools for site-specific modification of proteins and biologics.en_US
dc.description.sponsorshipMinistry of Education (MOE)en_US
dc.relationSYNC, 001304-00001en_US
dc.relation.ispartofFrontiers in Chemistryen_US
dc.rights© 2021 Chen, Zhang, Zhang, Wang, Liu and Tam. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.en_US
dc.subjectScience::Biological sciencesen_US
dc.titleSite-specific protein modifications by an engineered asparaginyl endopeptidase from Viola canadensisen_US
dc.typeJournal Articleen
dc.contributor.schoolSchool of Biological Sciencesen_US
dc.contributor.researchNanyang Institute of Structural Biologyen_US
dc.contributor.researchSynzymes and Natural Products Centeren_US
dc.description.versionPublished versionen_US
dc.subject.keywordsAsparaginyl Endopeptidasesen_US
dc.subject.keywordsPeptide Asparaginyl Ligasesen_US
dc.description.acknowledgementThis research was supported by Academic Research Grant Tier 3 (MOE2016-T3-1-003) from Singapore Ministry of Education (MOE), and Synzyme and Natural Products Center (SYNC, 001304-00001) at Nanyang Technological University.en_US
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