Phylogeny-guided discovery of a promiscuous P450 macrocyclase for the production of diverse atropopeptides

Cyclic peptides exhibit diverse bioactivities and are distinguished by their enhanced cell permeability, improved proteolytic stability, and increased binding affinity due to their conformational rigidity. Despite significant advancements in peptide synthesis, the production of complex cyclic peptides remains a challenge. Nature has evolved diverse strategies for peptide cyclization, with an ever-growing repertoire of characterized cyclases involved in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs). These enzymes convert linear precursor peptides into complex (poly-)cyclic structures. The discovery of the atropopeptides has significantly expanded the chemical diversity of RiPPs with unique (poly-)cyclic structures. In this study, we employed a phylogeny-guided approach to identify a substrate-promiscuous cytochrome P450 macrocyclase that catalyzes the formation of cyclic peptides through atropospecific C-N or C-C bond formation between aromatic amino acid side chains. Combinatorial biosynthetic studies revealed that ScaB encoded in the scabrirubin biosynthetic gene cluster efficiently cyclizes a wide range of atropopeptide precursor peptides. Furthermore, extensive site-directed mutagenesis studies of the tetrapeptide core sequence further expanded the diversity of atropopeptides. Notably, three tested atropopeptides show antiviral activity and one of the non-natural atropopeptides displays anti-inflammatory activity. Our findings establish a broadly substrate-tolerant atropopeptide-modifying P450 as a versatile biocatalyst for the synthesis of bioactive, biaryl-bridged macrocyclic peptides.