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Characterization of a dual function macrocyclase enables design and use of efficient macrocyclization substrates

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Date
19/10/2017
Author
Melo Czekster, Clarissa
Ludewig, Hannes
McMahon, Stephen A.
Naismith, James H.
Funder
BBSRC
EPSRC
European Research Council
Grant ID
BB/K015508/1
EP/L016419/1
NCB-TNT
Keywords
Enzyme mechanisms
X-ray crystallography
Biocatalysis
QD Chemistry
DAS
BDC
R2C
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Abstract
Peptide macrocycles are promising therapeutic molecules because they are protease resistant, structurally rigid, membrane permeable and capable of modulating protein-protein interactions. Here, we report the characterization of the dual function macrocyclase-peptidase enzyme involved in the biosynthesis of the highly toxic Amanitin toxin family of macrocycles. The enzyme first removes 10 residues from the N-terminus of a 35-residue substrate. Conformational trapping of the amino acid peptide forces the enzyme to release this intermediate rather than proceed to macrocyclization. The enzyme rebinds the 25 amino acid peptide in a different conformation and catalyzes macrocyclization of the N-terminal 8 residues. Structures of the enzyme bound to both substrates and biophysical analysis characterize the different binding modes rationalizing the mechanism. Using these insights simpler substrates with only five C-terminal residues were designed, allowing the enzyme to be more effectively exploited in biotechnology.
Citation
Melo Czekster , C , Ludewig , H , McMahon , S A & Naismith , J H 2017 , ' Characterization of a dual function macrocyclase enables design and use of efficient macrocyclization substrates ' , Nature Communications , vol. 8 , 1045 . https://doi.org/10.1038/s41467-017-00862-4
Publication
Nature Communications
Status
Peer reviewed
DOI
https://doi.org/10.1038/s41467-017-00862-4
ISSN
2041-1723
Type
Journal article
Rights
© 2017 the Authors. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Description
H.L. is funded by the George & Stella Lee Scholarship and Criticat EPSRC. This project was also funded by the European Research Council project 339367 NCB-TNT and by the BBSRC (K015508/1). JHN is 1000 talent scholar of the Chinese Academy of Sciences at the University of Sichuan.
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  • University of St Andrews Research
URI
http://hdl.handle.net/10023/11887

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