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

Research output: Contribution to journalArticlepeer-review

Author(s)

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

School/Research organisations

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.
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Details

Original languageEnglish
Article number1045
JournalNature Communications
Volume8
DOIs
Publication statusPublished - 19 Oct 2017

    Research areas

  • Enzyme mechanisms, X-ray crystallography, Biocatalysis

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