Ribosomally synthesised and post‐translationally modified peptides (RiPPs) represent a diverse superfamily of natural products. RiPPs are produced by biosynthetic gene clusters (BGCs), which are groups of open reading frames that typically encode a precursor peptide and the biosynthetic enzymes responsible for its successive post‑translational modifications. These modifications often result in significant structural change in the mature peptides, such as macrocyclisation. Macrocyclic linkages in RiPPs serve as powerful constraints, limiting the peptides’ conformational flexibility and are often essential for their biological function.
In this project, we applied genome mining tools and bioinformatic analyses to identify a previously uncharacterised RiPP produced by a minimal BGC. This BGC contains a novel, functionally divergent biosynthetic enzyme from the YcaO superfamily that catalyses macrocycle formation both in vivo and in vitro. High‑resolution mass spectrometry and peptide fragmentation analysis revealed the formation of two novel macrocyclic linkages. In addition, the crystal structures of this enzymes in complex with the peptide and reaction substrates demonstrate a unique mode of substrate recognition. Investigating functionally uncharacterised RiPPs and their biosynthetic enzymes could expand the RiPP chemical space and unlock their potential in biomedical and biotechnological applications.