Site-specific protein modifications by an engineered asparaginyl endopeptidase from Viola canadensis

Asparaginyl 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...

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Main Authors: Chen, Yu, Zhang, Dingpeng, Zhang, Xiaohong, Wang, Zhen, Liu, Chuan-Fa, Tam, James P.
Other Authors: School of Biological Sciences
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Language:English
Published: 2021
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Online Access:https://hdl.handle.net/10356/154024
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spelling sg-ntu-dr.10356-1540242023-02-28T17:10:07Z Site-specific protein modifications by an engineered asparaginyl endopeptidase from Viola canadensis Chen, Yu Zhang, Dingpeng Zhang, Xiaohong Wang, Zhen Liu, Chuan-Fa Tam, James P. School of Biological Sciences Nanyang Institute of Structural Biology Synzymes and Natural Products Center Science::Biological sciences Asparaginyl Endopeptidases Peptide Asparaginyl Ligases Asparaginyl 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. Ministry of Education (MOE) Published version This 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. 2021-12-19T06:53:02Z 2021-12-19T06:53:02Z 2021 Journal Article Chen, 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-. https://dx.doi.org/10.3389/fchem.2021.768854 2296-2646 https://hdl.handle.net/10356/154024 10.3389/fchem.2021.768854 34746098 2-s2.0-85118759578 9 768854 en MOE2016-T3-1-003 SYNC, 001304-00001 Frontiers in Chemistry © 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. application/pdf
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic Science::Biological sciences
Asparaginyl Endopeptidases
Peptide Asparaginyl Ligases
spellingShingle Science::Biological sciences
Asparaginyl Endopeptidases
Peptide Asparaginyl Ligases
Chen, Yu
Zhang, Dingpeng
Zhang, Xiaohong
Wang, Zhen
Liu, Chuan-Fa
Tam, James P.
Site-specific protein modifications by an engineered asparaginyl endopeptidase from Viola canadensis
description Asparaginyl 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.
author2 School of Biological Sciences
author_facet School of Biological Sciences
Chen, Yu
Zhang, Dingpeng
Zhang, Xiaohong
Wang, Zhen
Liu, Chuan-Fa
Tam, James P.
format Article
author Chen, Yu
Zhang, Dingpeng
Zhang, Xiaohong
Wang, Zhen
Liu, Chuan-Fa
Tam, James P.
author_sort Chen, Yu
title Site-specific protein modifications by an engineered asparaginyl endopeptidase from Viola canadensis
title_short Site-specific protein modifications by an engineered asparaginyl endopeptidase from Viola canadensis
title_full Site-specific protein modifications by an engineered asparaginyl endopeptidase from Viola canadensis
title_fullStr Site-specific protein modifications by an engineered asparaginyl endopeptidase from Viola canadensis
title_full_unstemmed Site-specific protein modifications by an engineered asparaginyl endopeptidase from Viola canadensis
title_sort site-specific protein modifications by an engineered asparaginyl endopeptidase from viola canadensis
publishDate 2021
url https://hdl.handle.net/10356/154024
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