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

DOI: 10.1038/s41467-017-00862-4 DOI Help

Authors: Clarissa M. Melo Czekster (The University of St Andrews) , Hannes Ludewig (The University of St Andrews) , Stephen Mcmahon (The University of St Andrews) , James Naismith (The University of St Andrews; Sichuan University; RCaH, Rutherford Appleton Laboratory; University of Oxford)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Nature Communications , VOL 8

State: Published (Approved)
Published: October 2017

Open Access Open Access

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 25 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 eight 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.

Journal Keywords: Biocatalysis; Enzyme mechanisms; X-ray crystallography

Subject Areas: Biology and Bio-materials, Medicine

Instruments: I02-Macromolecular Crystallography , I03-Macromolecular Crystallography , I04-1-Macromolecular Crystallography (fixed wavelength)

Other Facilities: European Synchrotron Radiation Facility (ESRF)