Cytochrome P450_BltEnables Versatile Peptide Cyclisation to Generate Histidine‐ and Tyrosine‐Containing Crosslinked Tripeptide Building Blocks

Abstract

We report our investigation of the utility of peptide crosslinking cytochrome P450 enzymes from biarylitide biosynthesis to generate a range of cyclic tripeptides from simple synthons. The crosslinked tripeptides produced by this P450 include both tyrosine-histidine (AÀ NÀ B) and tyrosine-tryptophan (AÀ OÀ B) crosslinked tripeptides, the latter a rare example of a phenolic crosslink to an indole moiety. Tripeptides are easily isolated following proteolytic removal of the leader peptide and can incorporate a wide range of amino acids in the residue inside the crosslinked tripeptide. Given the utility of peptide crosslinks in important natural products and the synthetic challenge that these can represent, P450 enzymes have the potential to play roles as important tools in the generation of high-value cyclic tripeptides for incorporation in synthesis, which can be yet further diversified using selective chemical techniques through specific handles contained within these tripeptides.

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

“…One-residue shorter or longer intervals between conserved Trp and His also led to, albeit less efficient, precursor modification (Figures C and S52B). The formation of diverse biaryl linkages by a single P450 was also previously reported with those for biarylitides and DKPs . Collectively, these results suggest that certain P450s have substrate tolerance, facilitating the evolution of diverse biaryl-containing RiPPs.…”

Cytochromes P450 involved in bacterial RiPP biosyntheses

“…The different connectivity of the crosslink bridges in biarylitides and myxarylin led to further investigations into the versatility and substrate tolerance of BytO-like enzymes from bacterial families (Zhao et al., 2022 ). The BytO-like enzyme from Micromonospora sp.…”

Cytochromes P450 Associated with the Biosyntheses of Ribosomally Synthesized and Post-translationally Modified Peptides

“…The Arg(−1) residue also shows some effect on the peptide binding to P450 Blt , as Ser and Asn replacements noticeably affect the cyclization of precursor peptide. The substitutions of Leu2 to amino acids with limited side chain size like Val, Ile, Nle, Ser, Cys, and even the non-natural amino acids acetamidomethyl protected Cys (AcmCys) and 2-amino-4-pentynoic acid are considerable tolerance, while substitutions to Asp, Trp, and diaminopropanoic acid (Dap) considerably hinder this process …”