Structural Insights into a Side Chain Cross-Linking Biarylitide P450 from RiPP Biosynthesis

Peptide side chain cross-linking is an important feature of many natural products, with an increasing number of examples catalyzed by cytochrome P450s being reported from ribosomal biosynthesis pathways in addition to well-known examples from nonribosomal peptide antibiotics. Despite the dramatic recent increase in the number of enzymes and reactions catalyzed, substrate bound structures of such P450s have proven elusive to date. Here, we report the structural characterization of the biarylitide cross-linking enzyme P450Blt in complex with its pentapeptide substrate MRYLH. This structure, in combination with computational and biochemical experiments, shows the importance of key I-helix residues in this P450 in coordinating to the histidine residue of the substrate and further that this appears to be central to the specificity of this enzyme for generating a C–N link between the tyrosine and histidine residues in the MRYLH substrate. The structure of the P450Blt-MRYLH complex provides the first insight into how peptide substrates can be accommodated within P450s and offers insights into how other examples of related P450s can accept the varied substrates that have recently been identified using bioinformatic methods.