Peptide Macrocyclization Catalyzed by a Prolyl Oligopeptidase Involved in α-Amanitin Biosynthesis

Abstract

SUMMARY Amatoxins are ribosomally-encoded and post-translationally modified peptides (RiPPs) that account for the majority of fatal mushroom poisonings of humans. A representative amatoxin is the bicyclic octapeptide α-amanitin formed via head-to-tail macrocyclization, which is ribosomally biosynthesized as a 35-amino acid propeptide in Amanita bisporigera and in the distantly related mushroom Galerina marginata. Although members of the prolyl oligopeptidase (POP) family of serine proteases were proposed to play a role in α- amanitin post-translational processing the exact mechanistic details are not known. Here, we show that a specific prolyl oligopeptidase (GmPOPB) is required for toxin maturation in G. marginata. Recombinant GmPOPB catalyzed two nonprocessive reactions: hydrolysis at an internal Pro to release the C-terminal 25mer from the 35mer propeptide, and transpeptidation at the second Pro to produce the cyclic octamer. On the other hand, we show that GmPOPA, the putative housekeeping POP of G. marginata, behaves like a conventional POP.

Characterization of the macrocyclase involved in the biosynthesis of RiPP cyclic peptides in plants

“…Similar patterns of conservation are observed for the precursors of other cyclic peptides including cyanobactins (31) and amatoxins (19,32). Alanine scanning mutational analysis of presegetalin A1 demonstrates that the necessary elements for substrate recognition by PCY1 reside solely in the 12-residue follower sequence (hereafter, presegetalin A1 [20][21][22][23][24][25][26][27][28][29][30][31][32]) that is excised during the formation of the macrocyclic product (27). Notably, PCY1 can accept a range of substrates leading to rings of different sizes as this single enzyme is capable of processing almost all of the orbitides that are observed in S. vaccaria.…”

“…Competitive binding between this labeled peptide and various unlabeled peptides was used to determine the K i values for the unlabeled peptides. These competition experiments demonstrate that unlabeled presegetalin A1 [27][28][29][30][31][32] bound to PCY1 with a K i of 31.0 μM (Table 1 and SI Appendix, Fig. S5B).…”

“…S5B). The longer, full-length linear by-product of the cyclization reaction, presegetalin A1 [20][21][22][23][24][25][26][27][28][29][30][31][32], binds with a much higher affinity with a K i of 0.40 μM (Table 1 and SI Appendix, Fig. S5C).…”

“…To determine the affinity of PCY1 for various follower peptides, we carried out competition binding measurements (Table 1). A six-residue presegetalin A1 [27][28][29][30][31][32] peptide conjugated to fluorescein isothiocyanate bound to PCY1 with a dissociation constant (K d ) of 4.83 μM as determined by fluorescence polarization (SI Appendix, Fig. S5A).…”

“…1B). The biosynthesis of cyclotides (28,29), amatoxins (30), and cyanobactins (31) uses similar biosynthetic strategies in which a sequence-specific peptidase catalyzes the transamidation of a linear peptide substrate to produce a macrocyclic product.…”