Atropopeptides are a Novel Family of Ribosomally Synthesized and Posttranslationally Modified Peptides with a Complex Molecular Shape

Abstract

Biomacromolecules are known to feature complex three-dimensional shapes that are essential for their function. Among natural products, ambiguous molecular shapes are a rare phenomenon. The hexapeptide tryptorubin A can adopt one of two unusual atropisomeric configurations. Initially hypothesized to be a non-ribosomal peptide, we show that tryptorubin A is the first characterized member of a new family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) that we named atropopeptides. The sole modifying enzyme encoded in the gene cluster, a cytochrome P450 monooxygenase, is responsible for the atropospecific formation of one carbon-carbon and two carbon-nitrogen bonds. The characterization of two additional atropopeptide biosynthetic pathways revealed a two-step maturation process. Atropopeptides promote pro-angiogenic cell functions as indicated by an increase in endothelial cell proliferation and undirected migration. Our study expands the biochemical space of RiPP-modifying enzymes and paves the way towards the chemoenzymatic utilization of atropopeptide-modifying P450s.

Keywords: Atropisomerism; Cytochrome P450 Monooxygenase; Genome Mining; RiPPs; Stereoisomerism.

Figure 1

Example of peptide natural products with non‐trivial molecular shapes that can adopt one of two unusual atropisomeric configurations.

Figure 2

Proposed model for atropopeptide biosynthesis and structures of atropopeptides characterized in this study. a) Model for the biosynthesis of tryptorubin A and B. b) Hexa‐ and pentapeptide structures characterized in this study. Key NOE correlations that are indicative of the bridge above atropisomer are highlighted in blue. Characteristic C−C and C−N bonds are highlighted in red.

Figure 3

Bioactivity studies. a) Proliferation assay. HMECs were seeded in low‐density and treated with atropopeptides for 72 h. Cells were stained with crystal violet solution. The amount of DNA‐bound crystal violet was detected by absorbance measurements. b) Undirected migration assay. A scratch was inflicted on a confluent HMEC monolayer. The cells were incubated in growth medium or growth medium containing the indicated concentrations of atropopeptides and incubated for 4 h followed by fixation and microscopical analysis. All data are expressed as mean±standard error of the mean (SEM); (A) n=4; (B) n=3; p≤0.05 vs. ctrl was considered as statistically significant.