Structural determinants for peptide-bond formation by asparaginyl ligases

Asparaginyl endopeptidases (AEPs) are cysteine proteases which break Asx (Asn/Asp)-Xaa bonds in acidic conditions. Despite sharing a conserved overall structure with AEPs, certain plant enzymes such as butelase 1 act as a peptide asparaginyl ligase (PAL) and catalyze Asx-Xaa bond formation in near-n...

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Main Authors: Hemu, Xinya, El Sahili, Abbas, Hu, Side, Wong, Kaho, Chen, Yu, Wong, Yee Hwa, Zhang, Xiaohong, Serra, Aida, Goh, Boon Chong, Darwis, Dina Amallia, Chen, Ming Wei, Sze, Siu Kwan, Liu, Chuan-Fa, Lescar, Julien, 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/10356/148284
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spelling sg-ntu-dr.10356-1482842023-02-28T16:59:04Z Structural determinants for peptide-bond formation by asparaginyl ligases Hemu, Xinya El Sahili, Abbas Hu, Side Wong, Kaho Chen, Yu Wong, Yee Hwa Zhang, Xiaohong Serra, Aida Goh, Boon Chong Darwis, Dina Amallia Chen, Ming Wei Sze, Siu Kwan Liu, Chuan-Fa Lescar, Julien Tam, James P. School of Biological Sciences NTU Institute of Structural Biology Science Asparaginyl Ligase Cyclopeptide Asparaginyl endopeptidases (AEPs) are cysteine proteases which break Asx (Asn/Asp)-Xaa bonds in acidic conditions. Despite sharing a conserved overall structure with AEPs, certain plant enzymes such as butelase 1 act as a peptide asparaginyl ligase (PAL) and catalyze Asx-Xaa bond formation in near-neutral conditions. PALs also serve as macrocyclases in the biosynthesis of cyclic peptides. Here, we address the question of how a PAL can function as a ligase rather than a protease. Based on sequence homology of butelase 1, we identified AEPs and PALs from the cyclic peptide-producing plants Viola yedoensis (Vy) and Viola canadensis (Vc) of the Violaceae family. Using a crystal structure of a PAL obtained at 2.4-Å resolution coupled to mutagenesis studies, we discovered ligase-activity determinants flanking the S1 site, namely LAD1 and LAD2 located around the S2 and S1' sites, respectively, which modulate ligase activity by controlling the accessibility of water or amine nucleophile to the S-ester intermediate. Recombinantly expressed VyPAL1-3, predicted to be PALs, were confirmed to be ligases by functional studies. In addition, mutagenesis studies on VyPAL1-3, VyAEP1, and VcAEP supported our prediction that LAD1 and LAD2 are important for ligase activity. In particular, mutagenesis targeting LAD2 selectively enhanced the ligase activity of VyPAL3 and converted the protease VcAEP into a ligase. The definition of structural determinants required for ligation activity of the asparaginyl ligases presented here will facilitate genomic identification of PALs and engineering of AEPs into PALs. Ministry of Education (MOE) Accepted version This research was supported by Academic Research Grant Tier 3 (MOE2016-T3-1-003) from the Singapore Ministry of Education (MOE) to the J.P.T., J.L., and C.-F.L. laboratories, and NMRC Grants CBRG/0028/2014 and NRF2016NRF-CRP001-063. 2021-04-22T08:48:09Z 2021-04-22T08:48:09Z 2019 Journal Article Hemu, X., El Sahili, A., Hu, S., Wong, K., Chen, Y., Wong, Y. H., Zhang, X., Serra, A., Goh, B. C., Darwis, D. A., Chen, M. W., Sze, S. K., Liu, C., Lescar, J. & Tam, J. P. (2019). Structural determinants for peptide-bond formation by asparaginyl ligases. Proceedings of the National Academy of Sciences of the United States of America, 116(24), 11737-11746. https://dx.doi.org/10.1073/pnas.1818568116 1091-6490 https://hdl.handle.net/10356/148284 10.1073/pnas.1818568116 24 116 11737 11746 en MOE2016-T3-1-003 NRF2016NRF-CRP001-063 CBRG/0028/2014 Proceedings of the National Academy of Sciences of the United States of America © 2019 The Author(s) (Published by National Academy of Sciences). All rights reserved. This paper was published in Proceedings of the National Academy of Sciences of the United States of America and is made available with permission of The Author(s) (Published by National Academy of Sciences). 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
Asparaginyl Ligase
Cyclopeptide
spellingShingle Science
Asparaginyl Ligase
Cyclopeptide
Hemu, Xinya
El Sahili, Abbas
Hu, Side
Wong, Kaho
Chen, Yu
Wong, Yee Hwa
Zhang, Xiaohong
Serra, Aida
Goh, Boon Chong
Darwis, Dina Amallia
Chen, Ming Wei
Sze, Siu Kwan
Liu, Chuan-Fa
Lescar, Julien
Tam, James P.
Structural determinants for peptide-bond formation by asparaginyl ligases
description Asparaginyl endopeptidases (AEPs) are cysteine proteases which break Asx (Asn/Asp)-Xaa bonds in acidic conditions. Despite sharing a conserved overall structure with AEPs, certain plant enzymes such as butelase 1 act as a peptide asparaginyl ligase (PAL) and catalyze Asx-Xaa bond formation in near-neutral conditions. PALs also serve as macrocyclases in the biosynthesis of cyclic peptides. Here, we address the question of how a PAL can function as a ligase rather than a protease. Based on sequence homology of butelase 1, we identified AEPs and PALs from the cyclic peptide-producing plants Viola yedoensis (Vy) and Viola canadensis (Vc) of the Violaceae family. Using a crystal structure of a PAL obtained at 2.4-Å resolution coupled to mutagenesis studies, we discovered ligase-activity determinants flanking the S1 site, namely LAD1 and LAD2 located around the S2 and S1' sites, respectively, which modulate ligase activity by controlling the accessibility of water or amine nucleophile to the S-ester intermediate. Recombinantly expressed VyPAL1-3, predicted to be PALs, were confirmed to be ligases by functional studies. In addition, mutagenesis studies on VyPAL1-3, VyAEP1, and VcAEP supported our prediction that LAD1 and LAD2 are important for ligase activity. In particular, mutagenesis targeting LAD2 selectively enhanced the ligase activity of VyPAL3 and converted the protease VcAEP into a ligase. The definition of structural determinants required for ligation activity of the asparaginyl ligases presented here will facilitate genomic identification of PALs and engineering of AEPs into PALs.
author2 School of Biological Sciences
author_facet School of Biological Sciences
Hemu, Xinya
El Sahili, Abbas
Hu, Side
Wong, Kaho
Chen, Yu
Wong, Yee Hwa
Zhang, Xiaohong
Serra, Aida
Goh, Boon Chong
Darwis, Dina Amallia
Chen, Ming Wei
Sze, Siu Kwan
Liu, Chuan-Fa
Lescar, Julien
Tam, James P.
format Article
author Hemu, Xinya
El Sahili, Abbas
Hu, Side
Wong, Kaho
Chen, Yu
Wong, Yee Hwa
Zhang, Xiaohong
Serra, Aida
Goh, Boon Chong
Darwis, Dina Amallia
Chen, Ming Wei
Sze, Siu Kwan
Liu, Chuan-Fa
Lescar, Julien
Tam, James P.
author_sort Hemu, Xinya
title Structural determinants for peptide-bond formation by asparaginyl ligases
title_short Structural determinants for peptide-bond formation by asparaginyl ligases
title_full Structural determinants for peptide-bond formation by asparaginyl ligases
title_fullStr Structural determinants for peptide-bond formation by asparaginyl ligases
title_full_unstemmed Structural determinants for peptide-bond formation by asparaginyl ligases
title_sort structural determinants for peptide-bond formation by asparaginyl ligases
publishDate 2021
url https://hdl.handle.net/10356/148284
_version_ 1759856153390481408