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.

Bernath-Levin K¹, Nelson C², Elliott AG³, Jayasena AS¹, Millar AH², Craik DJ³, Mylne JS⁴.

Abstract

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.