The Two-step Biosynthesis of Cyclic Peptides from Linear Precursors in a Member of the Plant Family Caryophyllaceae Involves Cyclization by a Serine Protease-like Enzyme

Abstract

Background: In the Caryophyllaceae, cyclic peptides (CP) are biosynthesized from linear precursors via an unknown pathway. Results: Two protease-like enzymes are involved in precursor processing. Conclusion: A serine protease-like enzyme was recruited for the cyclization step in CP biosynthesis. Significance: This represents a very significant advance in our understanding of the mode and evolution of CP biosynthesis in plants.

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

“…The binding of the carboxyl terminus of the peptide is of particular interest, as it forms hydrogen bonds with PCY1 residues Ser495 and Ser493. This observation explains prior alanine-scanning mutational analysis of presegetalin A1 [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32], which demonstrates that PCY1 is tolerant of mutation along the entirety of the primary sequence of the substrate but cannot process a substrate with the deletion of the C-terminal Val32, indicating the importance of the follower peptide C-terminal carboxylate (27).…”

“…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).…”

“…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).…”

“…Subsequent biochemical assays with seed extracts, using synthetic peptide substrates, revealed the order of posttranslational modifications that generate the orbitide segetalin A ( Fig. 1 A and B) (27). Briefly, segetalin A is first produced ribosomally as a 32-aa peptide termed presegetalin A1.…”