The genetic origin of evolidine, the first cyclopeptide discovered in plants, and related orbitides.

Cyclic peptides are reported to have antibacterial, antifungal, and other bioactivities. Orbitides are a class of cyclic peptides that are small, head-to-tail cyclized, composed of proteinogenic amino acids and lack disulfide bonds; they are also known in several genera of the plant family Rutaceae. is the Australian rain forest tree of the Rutaceae family in which evolidine, the first plant cyclic peptide, was discovered. Evolidine (cyclo-SFLPVNL) has subsequently been all but forgotten in the academic literature, so to redress this we used tandem MS and transcriptomics to rediscover evolidine and decipher its biosynthetic origin from a short precursor just 48 residues in length. We also identified another six orbitides using the same techniques. These peptides have atypically diverse C termini consisting of residues not recognized by either of the known proteases plants use to macrocyclize peptides, suggesting new cyclizing enzymes await discovery. We examined the structure of two of the novel orbitides by NMR, finding one had a definable structure, whereas the other did not. Mining RNA-seq and whole genome sequencing data from other species of the Rutaceae family revealed that a large and diverse family of peptides is encoded by similar sequences across the family and demonstrates how powerful transcriptomics can be at accelerating the discovery of new peptide families.