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

Abstract

Cyclic peptides are reported to have antibacterial, antifungal and other bioactivities. Orbitides are a class of cyclic peptide 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. Melicope xanthoxyloides 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 mass spectrometry and de novo transcriptomics to rediscover evolidine and decipher its biosynthetic origin from a short precursor just 48 residues in length. We also identify another six M. xanthoxyloides 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 a large and diverse family of peptides is encoded by similar sequences across the family and demonstrates how powerful de novo transcriptomics can be at accelerating the discovery of new peptide families.

Plant derived cyclic peptides

“…Orbitides from other plant families have also been found recently, including a cyclic peptide from Pseudostellaria heterophylla [ 14 ] and a novel orbitide derived from a genetic deletion was detected from flaxseed [ 15 ]. Furthermore, a study that revisited the discovery of evolidine from the Rutaceae species M. xanthoxyloides also resulted in the discovery of six novel orbitides (xanthoxycyclin A–F) encoded by similar transcripts [ 3 ]. The structures of synthetic versions of two of these orbitides were studied using NMR spectroscopy, with the 8-residue peptide xanthoxycyclin D showing a well-defined structure in contrast with the 9-residue peptide xanthoxycyclin F, which appeared to have conformational heterogeneity [ 3 ].…”

“…Furthermore, a study that revisited the discovery of evolidine from the Rutaceae species M. xanthoxyloides also resulted in the discovery of six novel orbitides (xanthoxycyclin A–F) encoded by similar transcripts [ 3 ]. The structures of synthetic versions of two of these orbitides were studied using NMR spectroscopy, with the 8-residue peptide xanthoxycyclin D showing a well-defined structure in contrast with the 9-residue peptide xanthoxycyclin F, which appeared to have conformational heterogeneity [ 3 ]. The sequences of these peptides differ significantly with the latter containing two proline residues, which might be responsible for the structural distinctions.…”

“…Interestingly, when the recombinant precursor was incubated with a recombinant sunflower AEP (HaAEP), the formation of the cyclic peptide was inefficient [ 34 ]. This observation coupled with the range of C-terminal residues found in the orbitides from M. xanthoxyloides , which are not consistent with the known plant cyclizing enzymes, suggests that there are cyclizing enzymes yet to be discovered [ 3 ].…”

“…Orbitides are plant cyclic peptides that do not contain any disulfide bonds or non-natural amino acids. The first orbitide, evolidine, was isolated in the 1950s from the rainforest tree Melicope xanthoxyloides and subsequent studies have confirmed its sequence, as summarised by Fisher et al [ 3 ]. These peptides, which contain between 5 and 16 residues, were originally referred to as Caryophyllaceae-type cyclic peptides, but have since been termed orbitides [ 4–6 ].…”

Synthesis of [1‐8‐NαC]‐zanriorb A1, alanine‐containing analogues, and their cytotoxic and anti‐inflammatory activity

“…The homodetic orbitides have been isolated from at least nine plant families, and many have bioactivities such as antioxidative, antimalarial, anticancer and immunomodulation. [5][6][7] For example, cycloleonurinin exhibited a significant immunosuppressive activity comparable with the clinical drug cyclosporin A. 8 Phytochemical investigation of Zanthoxylum riedelianum Engl.…”