P450-Catalyzed Biaryl Macrocyclization of Leaderless Ribosomal Peptides

Macrocyclic peptides, an intriguing class of molecules, embody promising frameworks for the development of chemical probes and prospective therapeutic agents. However, the lack of an established wide-substrate scope synthesis methodology has made it challenging to achieve both efficiency and selectivity simultaneously. P450-catalyzed ribosomal synthesized and post-translational modification peptides (RiPPs) are a new family of macrocyclic peptides with typical biaryl crosslinks. In this study, we report the discovery of a RiPP-modified P450 macrocyclase, GpeC, which natively catalyzes the biaryl C(sp ² )-C(sp ² ) crosslink of tryptophan-tyrosine in a tetrapeptide module. Unlike most previously documented P450-RiPPs that possess a substantial leader peptide region, hindering biocatalytic applications, our P450 GpeC exhibits catalytic potential with leaderless precursor peptides and a wide substrate tolerance, highlighting its adaptability and potential for creating diverse macrocyclic structures. The crystal structure of GpeC, combined with in silico calculations and site-directed mutagenesis, provides strong evidence for the extreme flexibility of intra-cyclic amino acids within the biaryl-cyclized tetrapeptide. This flexibility enables the substitution of any of the other 19 amino acids, indicating that GpeC is a robust and adaptable catalyst for biaryl cyclization, providing a valuable resource for the biocatalytic synthesis of complex macrocyclic compounds.