P450‐Modified Multicyclic Cyclophane‐Containing Ribosomally Synthesized and Post‐Translationally Modified Peptides

Abstract

AbstractCyclic peptides with cyclophane linkers are an attractive compound type owing to the fine‐tuned rigid three‐dimensional structures and unusual biophysical features. Cytochrome P450 enzymes are capable of catalyzing not only the C−C and C−O oxidative coupling reactions found in vancomycin and other nonribosomal peptides (NRPs), but they also exhibit novel catalytic activities to generate cyclic ribosomally synthesized and post‐translationally modified peptides (RiPPs) through cyclophane linkage. To discover more P450‐modified multicyclic RiPPs, we set out to find cryptic and unknown P450‐modified RiPP biosynthetic gene clusters (BGCs) through genome mining. Synergized bioinformatic analysis reveals that P450‐modified RiPP BGCs are broadly distributed in bacteria and can be classified into 11 classes. Focusing on two classes of P450‐modified RiPP BGCs where precursor peptides contain multiple conserved aromatic amino acid residues, we characterized 11 novel P450‐modified multicyclic RiPPs with different cyclophane linkers through heterologous expression. Further mutation of the key ring‐forming residues and combinatorial biosynthesis study revealed the order of bond formation and the specificity of P450s. This study reveals the functional diversity of P450 enzymes involved in the cyclophane‐containing RiPPs and indicates that P450 enzymes are promising tools for rapidly obtaining structurally diverse cyclic peptide derivatives.