Phylogeny-guided discovery of a promiscuous P450 macrocyclase for the production of diverse atropopeptides

Abstract

Cyclic peptides exhibit diverse bioactivities and are distinguished by their enhanced cell permeability, improved proteolytic stability, and increased binding affinity due to their conformational rigidity. Despite significant advancements in peptide synthesis, the production of complex cyclic peptides remains a challenge. Nature has evolved diverse strategies for peptide cyclization, with an ever-growing repertoire of characterized cyclases involved in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs). These enzymes convert linear precursor peptides into complex (poly-)cyclic structures. The discovery of the atropopeptides has significantly expanded the chemical diversity of RiPPs with unique (poly-)cyclic structures. In this study, we employed a phylogeny-guided approach to identify a substrate-promiscuous cytochrome P450 macrocyclase that catalyzes the formation of cyclic peptides through atropospecific C-N or C-C bond formation between aromatic amino acid side chains. Combinatorial biosynthetic studies revealed that ScaB encoded in the scabrirubin biosynthetic gene cluster efficiently cyclizes a wide range of atropopeptide precursor peptides. Furthermore, extensive site-directed mutagenesis studies of the tetrapeptide core sequence further expanded the diversity of atropopeptides. Notably, three tested atropopeptides show antiviral activity and one of the non-natural atropopeptides displays anti-inflammatory activity. Our findings establish a broadly substrate-tolerant atropopeptide-modifying P450 as a versatile biocatalyst for the synthesis of bioactive, biaryl-bridged macrocyclic peptides.

Fig. 1. Phylogenetic tree of atropopeptide-modifying P450s. The maximum likelihood tree was rooted using P450_Blt 8U2M_B as outgroup. Bootstrap values (orange circles) are based on 1000 bootstrap replicates. Bootstrap values higher than 50 are displayed. The sca, sva and lau BGCs and their corresponding products, as well as tryptorubin A and its BGC try are displayed. Additionally, the P450s ScaB, SvaB and LauB1 used for combinatorial biosynthesis are highlighted in red and the P450 TryB is highlighted in green. The 15 tested atropopeptide precursors are labelled at the position of their corresponding P450 and their sequence identity with ScaA is shown. The triangles represent collapsed clades. The expanded tree with exact bootstrap values is depicted in Fig. S1.

Fig. 2. Combinatorial biosynthesis of the P450s ScaB, SvaB and LauB1 with each other's precursor peptides. (A) sca, sva and lau BGCs. (B) Different combinations of cytochrome P450s with different precursor peptides. (C) Extracted ion chromatogram (EIC) of compounds generated from extracts of recombinant strains.

Fig. 3. Combinatorial co-expression of genes encoding atropopeptide precursor peptides and ScaB. (A) 13 atropopeptide precursors were selected along with SvaA and LauA and co-expressed with scaB. The conserved KLSK motif and the crosslink-forming aromatic amino acids are highlighted with a grey background. Core peptides are highlighted in bold. The precursor peptides that yield the unusual Trp–Tyr crosslinks are underlined in their core peptide regions. 13 out of 15 combinations of precursor peptides with ScaB led to the generation of the corresponding cyclic peptides; (B) sequence logos of 15 selected atropopeptide precursor peptides; (C) the structures of 1, 2, 3 and 4 from the combinatorial co-expression of lauA, svaA or xaaA with scaB.

Fig. 4. Site-directed mutagenesis of ScaA core peptide. EIC of non-natural atropopeptides generated from engineered ScaA core peptides.

Fig. 5. The anti-inflammatory and antiviral activities of scabrirubin, scabrirubin CB-1 (1) and scabrirubin CB-3 (3). Mean fluorescence intensity (MFI) for ICAM1 (A) and VCAM1 (B) with each individual technical replicate (n = 6 per condition). Error bars represent the standard deviation. * = p ≤ 0.05; ** = p ≤ 0.01; **** = p ≤ 0.0001. Histograms display the fluorescence intensity distribution of representative samples of the indicated conditions. Screening of atropopeptides against H1N1pdm (C), H3N2 (D), Malaysia/B (E) and Massachusetts/B (F) in MDCK II cells.