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

Abstract

Cytochrome P450 enzymes (P450s) catalyze diverse oxidative cross-coupling reactions between aromatic substrates in the natural product biosynthesis. Specifically, P450s install distinct biaryl macrocyclic linkages in three families of ribosomally synthesized and post-translationally modified peptides (RiPPs). However, the chemical diversity of biaryl-containing macrocyclic RiPPs remains largely unexplored. Here, we demonstrate that P450s have the capability to generate diverse biaryl linkages on RiPPs, collectively named “cyptides”. Homology-based genome mining for P450 macrocyclases revealed 19 novel groups of homologous biosynthetic gene clusters (BGCs) with distinct aromatic residue patterns in the precursor peptides. Using the P450-modified precursor peptides heterologously coexpressed with corresponding P450s in Escherichia coli, we determined the NMR structures of three novel biaryl-containing peptidesthe enzymatic products, roseovertin (1), rubrin (2), and lapparbin (3)and confirmed the formation of three unprecedented or rare biaryl linkages: Trp C-7′-to-His N-τ in 1, Trp C-7′-to-Tyr C-6 in 2, and Tyr C-6-to-Trp N-1′ in 3. Biochemical characterization indicated that certain P450s in these pathways have a relaxed substrate specificity. Overall, our studies suggest that P450 macrocyclases have evolved to create diverse biaryl linkages in RiPPs, promoting the exploration of a broader chemical space for biaryl-containing peptides encoded in bacterial genomes.

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

“…Comparing the sequences of recently reported His-His RiPP cross-linking P450s , that are closely related to the biarylitide Tyr-His cross-linking enzymes is also highly revealing when interrogated through the lens of the P450 Blt /MRYLH complex. Such an analysis shows that the only major difference in the active site of His-His cross-linking P450s compared to Tyr-His cross-linking P450s is the C-terminal loop residue Leu-382, which is replaced by a Glu in His-His P450s and likely plays a role in coordinating to the His-3 residue present in the altered substrate of these enzymes (MRHxH). , While the I-helix residues Ala-231/His-234 from P450 Blt are replaced by Val and Gln in His-His cross-linking enzymes, these in fact match those found in the initially reported biarylitide C–C bond forming enzyme from Planomonospora and are therefore also in agreement with the lack of an N-cross-link in the reported product from such His-His cross-linking enzymes .…”

“…The importance of Arg residues in coordinating the C-terminus of the peptide substrate, previously implied by biochemical experiments, is clarified in the structure of P450 Blt . Indeed, comparison of this structure with the sequence alignments of this enzyme and those accepting C-terminally extended biarylitide peptides shows that these Arg residues present in P450 Blt are replaced by residues with far smaller side chains in enzymes with extended substrates, thus allowing them to accept longer heptapeptide sequences.…”

“…Turning to other residues within the I-helix, His-234 appears to play a central role in controlling the C–N cross-linking outcome mediated by P450 Blt , which otherwise would be likely to favor formation of a C–C bond as has been shown for other biarylitide P450 enzymes . Comparison of these central residues across biarylitide cross-linking P450s more broadly generally shows limited diversity in these positions (Ala-231 to Val, Ser, Thr, Met or Leu; His-234 to Leu, Ile, Asn or Gln), although further studies are required to better understand how the control of cross-linking outcomes can be controlled through such interactions. The acid/alcohol pair Asp-238/Ser-239 found in P450 Blt suggests that oxygen activation proceeds via the canonical P450 catalytic cycle in these enzymes, and furthermore that despite the prevalence of hydrogen bonding in such peptides that the replacement of these crucial interactions via those mediated by the substrate are not sufficient for these P450s. ,, The conservation of residues in these acid/alcohol positions that can form hydrogen bond interactions in biarylitide P450s also suggests that recent reports of P450 cross-linking enzymes using a superoxide anion intermediate for catalysis are unlikely to be realized for such P450s, although definitive experiments to address this question remain to be performed.…”

Atroposelective Total Synthesis of Cihunamide B

“…Biaryl linkages within cyclic peptides act as linkages between aromatic compounds, resulting in exceptionally robust linkages that confer stability and rigidity beyond that of conventional cyclic peptides, thereby contributing to the preservation of their steric structure. 8 Furthermore, cyclic peptides with biaryl linkages have been observed to exhibit a wide range of significant biological activities. 8 However, despite the structural and bioactive attractiveness of cihunamide B (1), a synthetic method for this compound has not yet been reported.…”

“…8 Furthermore, cyclic peptides with biaryl linkages have been observed to exhibit a wide range of significant biological activities. 8 However, despite the structural and bioactive attractiveness of cihunamide B (1), a synthetic method for this compound has not yet been reported. Therefore, this Letter presents the development of an atroposelective synthesis of cihunamide B (1).…”