Peptide macrocyclization by a bifunctional endoprotease

Chem Biol. 2015 May 21;22(5):571-82. doi: 10.1016/j.chembiol.2015.04.010. Epub 2015 May 7.


Proteases usually cleave peptides, but under some conditions, they can ligate them. Seeds of the common sunflower contain the 14-residue, backbone-macrocyclic peptide sunflower trypsin inhibitor 1 (SFTI-1) whose maturation from its precursor has a genetic requirement for asparaginyl endopeptidase (AEP). To provide more direct evidence, we developed an in situ assay and used (18)O-water to demonstrate that SFTI-1 is excised and simultaneously macrocyclized from its linear precursor. The reaction is inefficient in situ, but a newfound breakdown pathway can mask this inefficiency by reducing the internal disulfide bridge of any acyclic-SFTI to thiols before degrading it. To confirm AEP can directly perform the excision/ligation, we produced several recombinant plant AEPs in E. coli, and one from jack bean could catalyze both a typical cleavage reaction and cleavage-dependent, intramolecular transpeptidation to create SFTI-1. We propose that the evolution of ligating endoproteases enables plants like sunflower and jack bean to stabilize bioactive peptides.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Arabidopsis / enzymology
  • Canavalia / enzymology
  • Cyclization
  • Cysteine Endopeptidases / genetics
  • Cysteine Endopeptidases / metabolism*
  • Disulfides / chemistry
  • Escherichia coli / metabolism
  • Helianthus / enzymology
  • Helianthus / metabolism
  • Kinetics
  • Molecular Sequence Data
  • Oxygen Isotopes / chemistry
  • Peptides, Cyclic / chemistry
  • Peptides, Cyclic / metabolism*
  • Plant Proteins / genetics
  • Plant Proteins / metabolism*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Seeds / metabolism
  • Sequence Alignment
  • Tandem Mass Spectrometry
  • Water / chemistry


  • Disulfides
  • Oxygen Isotopes
  • Peptides, Cyclic
  • Plant Proteins
  • Recombinant Proteins
  • SFTI-1 peptide, sunflower
  • Water
  • Cysteine Endopeptidases
  • asparaginylendopeptidase