The emergence and loss of cyclic peptides in Nicotiana illuminate dynamics and mechanisms of plant metabolic evolution

Specialized metabolism plays a central role in how plants cope with both biotic and abiotic stresses in order to survive and reproduce within dynamic and challenging environments. One recently circumscribed class of plant-specific, ribosomally synthesized and post-translationally modified peptides are the burpitides, which are characterized by the installation of distinct sidechain macrocycles by enzymes known as burpitide cyclases. While they are found across many plant families and exhibit diverse bioactivities, little is known about their evolution or how new variants arise. Here we present the discovery of a new burpitide cyclase, resurrected from a defunct pseudogene from the model organism Nicotiana attenuata, the coyote tobacco. By repairing the pseudogene{Psi} NatBURP2 and expressing it heterologously in Nicotiana benthamiana, we successfully reconstituted its original enzymatic activity. As an autocatalytic peptide cyclase, it installs a unique C-C bond between the tyrosine side chain and a specific backbone a-carbon of a heptapeptide core motif, resulting in the burpitide dubbed "nanamin." Despite its pseudogenization in N. attenuata, we found that the closely related species, N. clevelandii, retains the wild-type gene and produces nanamins. Phylogenetic analyses and targeted mutagenesis experiments reveal that this chemotype must have evolved from the duplication and neofunctionalization of a more promiscuous ancestral gene. This work highlights how novel peptide chemotypes may rapidly emerge and disappear in plants, while expanding the molecular toolkit for engineering novel peptides with applications in agriculture and drug discovery. SignificanceWhile RiPPs represent a major source of antibiotics and bioactive compounds, much research has focused on microbial sources even as plant RiPPs go understudied. Here, we resurrect an extinct peptide cyclase from the coyote tobacco through analysis of its functional relatives in other species. This newly identified cyclase installs a novel carbon-carbon macrocycle into heptapeptides, expanding the diversity of plant-derived cyclic peptides. By interconverting two distinct cyclases through targeted mutations, we illuminate how these enzymes evolve new functions. This work highlights the evolutionary dynamics of plant peptide natural products and their potential applications in drug discovery and biotech crop development, while illustrating how genomic archaeology can reveal lost biosynthetic capabilities.