25mer macrocyclization substrate: IWGIGCNPWTAEHVDQTLASGNDIC. The 25mer is the post proline product (A0A067SLB9 AAMA1_GALM3) Major toxin belonging to the bicyclic octapeptides amatoxins that acts by binding non-competitively to RNA polymerase II and greatly slowing the elongation of transcripts from target promoters
4 structures(e.g. : 5N4B, 5N4C, 5N4D... more)(less)5N4B: Prolyl oligopeptidase B from Galerina marginata bound to 25mer macrocyclization substrate - S577A mutant, 5N4C: Prolyl oligopeptidase B from Galerina marginata bound to 35mer hydrolysis and macrocyclization substrate - S577A mutant, 5N4D: Prolyl oligopeptidase B from Galerina marginata bound to 25mer macrocyclization substrate - D661A mutant, 5N4E: Prolyl oligopeptidase B from Galerina marginata bound to 35mer hydrolysis and macrocyclization substrate - H698A mutant
Title: Characterization of a dual function macrocyclase enables design and use of efficient macrocyclization substrates Czekster CM, Ludewig H, McMahon SA, Naismith JH Ref: Nat Commun, 8:1045, 2017 : PubMed
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.
