Cyclotide biosynthesis

Abstract

Cyclotides are bioactive macrocyclic peptides from plants that are characterized by their exceptional stability and potential applications as protein engineering or drug design frameworks. Their stability arises from their unique cyclic cystine knot structure, which combines a head-to-tail cyclic peptide backbone with three conserved disulfide bonds having a knotted topology. Cyclotides are ribosomally synthesized by plants and expressed in a wide range of tissues, including leaves, flowers, stems and roots. Here we describe recent studies that have examined the biosynthesis of cyclotides and in particular the mechanism associated with post-translational backbone cyclization.

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

“…As presegetalin A1 [27][28][29][30][31][32] binding induces closure of the PCY1 structure, addition of exogenous peptide would be expected to inhibit productive turnover of the presegetalin A1 [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] substrate. We tested this hypothesis using a 1-h end-point reaction of PCY1 incubated with 30 μM of substrate in the presence of stoichiometric and 10-fold excess of the presegetalin A1 [20][21][22][23][24][25][26][27][28][29][30][31][32] linker+follower peptide product, as well as 100-fold excess of the presegetalin A1 [27][28][29][30][31][32] follower peptide (SI Appendix, Fig.…”

“…We next interrogated the extent to which the enzyme could accommodate primary amines other than a peptide α-amino group for transamidation. First, we used a variant presegetalin A1 [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] substrate peptide that contained both the α-amino terminus and a side-chain amine (Val15→Lys). Kinetic characterization of PCY1 using the Val15→Lys presegetalin A1 [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] as a substrate demonstrates that the K M increased by 10-fold relative to the cognate presegetalin A1 [14][15][16][17][18][19][20][21][22]...…”

“…We then tested whether PCY1 could generate a macrocyclic product using a nonproteinogenic amine. To this end, we used a presegetalin A1 [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] substrate that contained an N-terminal aminohexanoic acid (Ahx) in place of Gly14. PCY1 largely produced a linear product with this substrate, although a minor amount of cyclic [AhxVPVWA] could also be detected (SI Appendix, Table S3 and Fig.…”

“…The chemical structures of orbitides consist solely of α-amide linkages and are distinguished from other macrocyclic plant peptide natural products such as cyclotides, which are further interlocked through multiple disulfide bonds (18), and amatoxins, which contain an unusual tryptathionine moiety formed by a crosslink between a Cys and Trp residue (19). The first macrocyclic orbitide, cyclolinopeptide A, was isolated in 1959 from flaxseed (Linum usitatissimum) oil and determined to be a cyclic peptide of ILVPPFFLI ( Fig.…”

The Plant Peptidome: An Expanding Repertoire of Structural Features and Biological Functions

“…They have been detected in plants of the Rubiaceae, Violaceae, Fabaceae, Solanaceae, and Cucurbitaceae. CYCLO-TIDES are embedded in preproproteins with a C-terminal prodomain and often containing several CYCLOTIDE peptides (Craik and Malik, 2013). A wide range of activities has been reported for CYCLOTIDES, including inhibitory activity on fungi, bacteria, viruses, insects, molluscs, barnacles, and nematodes, as well as inhibition of trypsin (Daly et al, 2009;Craik et al, 2010).…”