Structure and function of a peptide crosslinking P450 from biarylitide biosynthesis Max Cryle Monash University, Australia Cytochrome P450s are a superfamily of oxidative hemoproteins that display a wide range of oxidative transformations across diverse biosynthetic processes. 1 Whilst the archetypal P450-mediated transformation is hydroxylation of non-activated C-H bonds, these enzymes are capable of a wide range of transformations including epoxidation, heteroatom oxidation, C-C bond cleavage, carbon skeleton rearrangements and the crosslinking of aromatic groups. Given this synthetic utility and combination of oxidative power and regiochemical precision, it is little surprise that this family of enzymes have been widely implicated as potential biocatalysts. The application of P450s as tools for generation of specific, high-value synthetic precursors has been anticipated, but few systems have been found that display the combination of substrate promiscuity with the importance of the potential synthon of the P450-catalysed transformation. In this study, we now report our investigation of the utility of peptide crosslinking P450 enzymes from biarylitide biosynthesis 2 to generate a range of cyclic tripeptide species. We demonstrate that such enzymes can generate a range of crosslinked tripeptides YxH/YxW in minimal pentapeptide substrates, which can be easily isolated following proteolytic digestion of the parent pentapeptides. We have further determined the structure of a P450 from a biarylitide crosslinking pathway both in isolation and in complex with the peptide substrate, which provides the first molecular insights into the crosslinking of linear peptide substrates by Cytochrome P450 enzymes. Given the utility of peptide crosslinks in important natural products and the synthetic challenge that these can represent, these P450 enzymes have the potential to play roles as important synthetic tools in the generate of high-value cyclic tripeptides for incorporation in synthesis pathways, which can be further diversified using selective chemical techniques through specific handles contained within these tripeptides. References 1. A. Greule, J. E. Stok, J. J. De Voss, M. J. Cryle, Nat Prod Rep 2018 , 35 , 757-791. 2. M. M. Zdouc, M. M. Alanjary, G. S. Zarazúa, S. I. Maffioli, M. Crüsemann, M. H. Medema, S. Donadio, M. Sosio, Cell Chem Biol 2021 , 28 , 733-739.e734.
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