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

Nat Commun. 2017 Oct 19;8(1):1045. doi: 10.1038/s41467-017-00862-4.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amanitins / biosynthesis*
  • Amanitins / chemistry*
  • Amanitins / metabolism
  • Basidiomycota / enzymology
  • Cyclization
  • Kinetics
  • Models, Molecular
  • Mutation
  • Peptide Hydrolases / chemistry
  • Peptide Hydrolases / metabolism


  • Amanitins
  • Peptide Hydrolases