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

Clarissa Melo Czekster; Hannes Ludewig; Stephen A. McMahon; James H. Naismith

doi:10.1038/s41467-017-00862-4

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

Clarissa Melo Czekster, Hannes Ludewig, Stephen A. McMahon, James H. Naismith

Research output: Contribution to journal › Article › peer-review

30 Citations (Scopus)

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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.

Original language	English
Article number	1045
Journal	Nature Communications
Volume	8
DOIs	https://doi.org/10.1038/s41467-017-00862-4
Publication status	Published - 19 Oct 2017

Keywords

Enzyme mechanisms
X-ray crystallography
Biocatalysis

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10.1038/s41467-017-00862-4Licence: CC BY

Naismith-2017_NatComm_DualFunctionMacro_CCBY-VoR
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CRITICAT CDT: Critical Resource Catalysis - CRITICAT
Smith, A. D. (PI), Nolan, S. P. (CoI) & Westwood, N. J. (CoI)
EPSRC
1/05/14 → 31/10/22
Project: Standard
NBC-TNT: NCB-TNT
Naismith, J. (PI)
European Research Council
1/03/14 → 28/02/19
Project: Standard
Exploring the mechanism: Exploring the mechanism and scope of the enzymatic formation of five membered ring
Naismith, J. (PI), Botting, C. H. (CoI), Koehnke, J. A. J. G. (CoI) & Schwarz-Linek, U. (CoI)
BBSRC
1/10/13 → 30/09/17
Project: Standard

Clarissa Melo Czekster
- cmc27st-andrews.acuk
- School of Biology - Reader
- Biomedical Sciences Research Complex
Person: Academic, Academic - Research

Characterization of a dual function macrocyclase enables design and use of efficient 2 macrocyclization substrates (dataset 2)
Melo Czekster, C. (Creator), Ludewig, H. (Creator), McMahon, S. A. (Creator) & Naismith, J. H. (Creator), Addgene, 2017
https://www.addgene.org/92234/ and 6 more links, https://www.addgene.org/92235/, https://www.addgene.org/92236/, https://www.addgene.org/92237/, https://www.addgene.org/92238/, https://www.addgene.org/92240/, https://www.addgene.org/92241/ (show fewer)
Dataset
Characterization of a dual function macrocyclase enables design and use of efficient 2 macrocyclization substrates (dataset)
Melo Czekster, C. (Creator), Ludewig, H. (Creator), McMahon, S. A. (Creator) & Naismith, J. H. (Creator), Protein Data Bank (PDB), 1 Nov 2017
http://dx.doi.org/10.2210/pdb5n4b/pdb and 4 more links, http://dx.doi.org/10.2210/pdb5n4c/pdb, http://dx.doi.org/10.2210/pdb5n4d/pdb, http://dx.doi.org/10.2210/pdb5n4e/pdb, http://dx.doi.org/10.2210/pdb5n4f/pdb (show fewer)
Dataset

Cite this

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title = "Characterization of a dual function macrocyclase enables design and use of efficient macrocyclization substrates",

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. ",

keywords = "Enzyme mechanisms, X-ray crystallography, Biocatalysis",

author = "{Melo Czekster}, Clarissa and Hannes Ludewig and McMahon, {Stephen A.} and Naismith, {James H.}",

note = "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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AU - Melo Czekster, Clarissa

AU - Ludewig, Hannes

AU - McMahon, Stephen A.

AU - Naismith, James H.

N1 - 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.

PY - 2017/10/19

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N2 - 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.

AB - 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.

KW - Enzyme mechanisms

KW - X-ray crystallography

KW - Biocatalysis

U2 - 10.1038/s41467-017-00862-4

DO - 10.1038/s41467-017-00862-4

M3 - Article

SN - 2041-1723

VL - 8

JO - Nature Communications

JF - Nature Communications

M1 - 1045

ER -

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

Abstract

Keywords

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Projects

CRITICAT CDT: Critical Resource Catalysis - CRITICAT

NBC-TNT: NCB-TNT

Exploring the mechanism: Exploring the mechanism and scope of the enzymatic formation of five membered ring

Profiles

Clarissa Melo Czekster

Datasets

Characterization of a dual function macrocyclase enables design and use of efficient 2 macrocyclization substrates (dataset 2)

Characterization of a dual function macrocyclase enables design and use of efficient 2 macrocyclization substrates (dataset)

Cite this