Bioinspired peptide cyclisation enables exploration of cytochrome P450 catalysed sequential oxidation in rufomycin biosynthesis.

Natural product biosynthetic cytochrome P450s (CYPs) are of increasing interest for their incredible ability to selectively activate inert C-H bonds on highly complex substrates. However, these enzymes are understudied due to difficulties accessing substrates. Here, we use our recently developed peptide cyclisation chemistry to produce a library of natural product peptide analogues to study the rufomycin tailoring CYP, RufM. Rufomycins are antimicrobial non-ribosomal cyclic peptides that undergo sequential alkyl oxidation catalysed by RufM. This transformation is essential for rufomycin antimycobacterial activity but results in several naturally produced derivatives, of which one intermediate has the greatest activity. Due to current interest in engineering the rufomycin pathway to generate derivatives for improved properties, we use our peptide library to explore the complex interplay of enzyme catalysed and spontaneous transformations involved in RufM oxidation, suprising RufM substrate promiscuity and the role of a mechanistically important residue in sequential oxidation. Our work demonstrates the power of our chemistry both as a tool to study biosynthetic tailoring CYPs as potential late-stage functionalizing biocatalysts and to enable antibiotic development.