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

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.