(27ag) Discovery and Characterization of a Structurally Unique Heterocyclic Peptide Enterolysin S

Linear azol(in)e-containing peptides (LAPs) are a unique subgroup of ribosomally synthesized and post-translationally modified peptides (RiPPs) and exhibit either antibacterial or hemolytic activity. The incorporation of an azol(in)e moiety into the peptide backbone is thought to enhance structural rigidity, resulting in better binding to the targets. The earliest work on LAPs dates back to early 20th century with the discovery of the β-hemolytic factor Streptolysin S (SLS), produced by Streptococcus pyogenes. However, due to its problematic physicochemical properties, the chemical structure of SLS remains elusive even after 128 years since its discovery. Here we report the discovery and characterization of a structurally unique and hemolytic SLS-like peptide from Enterococcus caccae, which we have named Enterolysin S. Despite the difficulties in purifying Enterolysin S due to its troublesome physiological properties similar to that of SLS, we were able to determine that Enterolysin S contained an unprecedented catenated octathiazole through heterologous expression, site-directed mutagenesis, chemoselective modification, and high-resolution mass spectrometry. We also conceived of chemical synthesis routes to access the catenated polythiazole motif, which could aid in deciphering the mechanism of action of SLS-mediated hemolysis in the future. Overall, this study represents the first structural characterization of a SLS-like peptide, which expands our knowledge of this important group of hemolytic LAPs.