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

• Published in: Journal of the American Chemical Society 2023-10, Vol.145 (40), p.22047-22057
• Main Authors: Nam, Hyunsung, An, Joon Soo, Lee, Jaepil, Yun, Yonghwan, Lee, Hyunbin, Park, Hyungou, Jung, Yousung, Oh, Ki-Bong, Oh, Dong-Chan, Kim, Seokhee
• Format: Article
• Language: English
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/jacs.3c07140

Cytochrome P450 enzymes (P450s) catalyze diverse oxidative cross-coupling reactions between aromatic substrates in the natural product biosynthesis. Specifically, P450s install distinct biaryl macrocyclic linkages in three families of ribosomally synthesized and post-translationally modified peptides (RiPPs). However, the chemical diversity of biaryl-containing macrocyclic RiPPs remains largely unexplored. Here, we demonstrate that P450s have the capability to generate diverse biaryl linkages on RiPPs, collectively named “cyptides”. Homology-based genome mining for P450 macrocyclases revealed 19 novel groups of homologous biosynthetic gene clusters (BGCs) with distinct aromatic residue patterns in the precursor peptides. Using the P450-modified precursor peptides heterologously coexpressed with corresponding P450s in Escherichia coli, we determined the NMR structures of three novel biaryl-containing peptidesthe enzymatic products, roseovertin (1), rubrin (2), and lapparbin (3)and confirmed the formation of three unprecedented or rare biaryl linkages: Trp C-7′-to-His N-τ in 1, Trp C-7′-to-Tyr C-6 in 2, and Tyr C-6-to-Trp N-1′ in 3. Biochemical characterization indicated that certain P450s in these pathways have a relaxed substrate specificity. Overall, our studies suggest that P450 macrocyclases have evolved to create diverse biaryl linkages in RiPPs, promoting the exploration of a broader chemical space for biaryl-containing peptides encoded in bacterial genomes.