A Widely Distributed Biosynthetic Cassette Is Responsible for Diverse Plant Side Chain Cross‐Linked Cyclopeptides**

Abstract

Cyclopeptide alkaloids are an abundant class of plant cyclopeptides with over 200 analogs described and bioactivities ranging from analgesic to antiviral. While these natural products have been known for decades, their biosynthetic basis remains unclear. Using a transcriptome-mining approach, we link the cyclopeptide alkaloids from Ceanothus americanus to dedicated RiPP precursor peptides and identify new, widely distributed split BURP peptide cyclase containing gene clusters. Guided by our bioinformatic analysis, we identify and isolate new cyclopeptides from Coffea arabica, which we named arabipeptins. Reconstitution of the enzyme activity for the BURP found in the biosynthesis of arabipeptin A validates the activity of the newly discovered split BURP peptide cyclases. These results expand our understanding of the biosynthetic pathways responsible for diverse cyclic plant peptides and suggest that these side chain cross-link modifications are widely distributed in eudicots.

Dehydroamino acid residues in bioactive natural products

“…In these systems, the core peptides are fused to the copperdependent autocatalytic BURP domains. [127][128][129][130] These BURP domains are oxidative cyclases that install the characteristic amino acid side-chain cross-links. In this regard, dhAA residues in plant RiPPs may be derived from the action of BURP domains.…”

Functional and Promiscuity Studies of Three-Residue Cyclophane Forming Enzymes Show Nonnative C–C Cross-Linked Products and Leader-Dependent Cyclization

“…19−21 In plants, Cu-dependent BURP domain proteins have recently been shown as the macrocyclase for the plant cyclopeptide alkaloids. 15,22,23,33 In bacteria, macrocyclization can be carried out by radical SAM enzymes, specifically rSAM/SPASM enzymes, which catalyze cyclophane formation by C−C, [9][10][11]24,34 C−O, 25,35 and C−N 36 bond forming reactions. The synthetic equivalent of the post-translational C−C bond forming reaction are Pd catalyzed and employs an 8aminoquinoline auxiliary (Figure 1).…”

“…A growing number of bioactive natural products contain three or four residue cyclophanes incorporating one or two aromatic rings. − ,− These natural products occur in plants, ,,,, fungi, − and bacteria. − ,,,− The cyclophanes containing one aromatic ring are only biosynthesized from ribosomally synthesized and post-translationally modified peptide (RiPP) pathways. , For each natural product, a key post-translational modifying enzyme is responsible for cyclophane formation. In fungi, the DUF3328 proteins have been implicated in the C–O bond-forming reactions in the biosynthesis of the dikaritin natural products. − In plants, Cu-dependent BURP domain proteins have recently been shown as the macrocyclase for the plant cyclopeptide alkaloids. ,,, In bacteria, macrocyclization can be carried out by radical SAM enzymes, specifically rSAM/SPASM enzymes, which catalyze cyclophane formation by C–C, − ,, C–O, , and C–N bond forming reactions.…”

Exploring the Diverse Landscape of Biaryl-Containing Peptides Generated by Cytochrome P450 Macrocyclases

“…RiPPs exhibit diverse intramolecular linkages involving at least one aromatic ring, resulting in rigid macrocyclic structures (Figure F–M) . Although those macrocyclic linkages formed by radical SAM enzymes, − DUF3328-containing oxidases, , or BURP-domain proteins − (Figure F–H) primarily connect one aromatic ring to an aliphatic chain, P450s exclusively create the biaryl linkages in three families of RiPPsatropitides, − cittilins, and biarylitides − (Figure I–L). Recent discovery reveals that tryptorubins, the founding members of atropitides, and the cihunamides, recently isolated from a Streptomyces sp.…”