The molecular diversity of toxin gene families in lethal Amanita mushrooms

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

“…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).…”

“…Analysis of the predicted primary sequences of dozens of putative orbitide precursor peptides reveals that residues within the core sequence that is cyclized are highly divergent, whereas residues in the preceding leader sequence (removed by OLP1) and the trailing follower sequence (excised as a linear peptide by PCY1) are highly conserved. 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).…”

“…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).…”

“…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).…”

“…The nearly 80-fold greater binding affinity for the longer presegetalin A1 [20][21][22][23][24][25][26][27][28][29][30][31][32] peptide is curious given that only the last six residues of the peptide are involved in contacts with PCY1 in the cocrystal structure. The additional binding affinity, relative to presegetalin A1 [27][28][29][30][31][32], may be due to solvent exclusion or another nonsequence-dependent mechanism. The amino acids that make up the final cyclic product do not seem to contribute to binding as substrate presegetalin A1 [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] was measured to bind to the catalytically inhibited PCY1 H695A with a K i of 1.10 μM, very similar to that of presegetalin A1 [20][21][22][23][24][25][26][27]...…”