Uncovering the Peptide Language of Streptococci: New Metalloenzyme-Catalyzed Transformations in Natural Product Biosynthesis

Abstract

Natural products remain a tremendous source of therapeutic agents and chemical tools with which to investigate biological phenomena. For decades, the discovery of natural products relied on screens of biological activity or chemical novelty. Recent advances in DNA sequencing technologies and bioinformatics have enabled alternative gene-first approaches. Ribosomally synthesized and post-translationally modified peptides (RiPPs) have emerged as particularly attractive targets for genome mining studies.Herein, we devised a bioinformatic search strategy to investigate the underexplored biosynthetic potential of streptococci, a genus of bacteria prevalent in mammalian microbiomes that includes mutualistic, commensal, and pathogenic members. To prioritize chemical novelty as well as functional relevance, we searched for peptide-modifying radical S-adenosylmethionine (RaS) enzymes in the genomic neighborhood of quorum sensing (QS) operons. RaS enzymes are known to catalyze some of the most unusual reactions in biology, while the chosen QS system is known to regulate community behaviors, such as virulence and biofilm formation. The search identified ~600 RiPP gene clusters that grouped into 16 subfamilies. Focusing on specific subfamilies, we found a variety of new natural products. With the WGK subfamily, we identified an unprecedented posttranslational modification, consisting of a tetrahydro[5,6]benzindole in which four unactivated positions are linked by two C−C bonds in a regio- and stereospecific manner by a single RaS enzyme. We also characterized the final product and observed potent antibiotic activity against select streptococci. With the GGG subfamily, we demonstrated that the RaS enzyme installs a duo of C–S bonds onto its substrate, which enabled discovery of the natural product, streptosactin. Streptosactin is the first streptococcal sactipeptide; it contains two sequential four residue sactionine macrocycles, an unusual topology for this compound family. Bioactivity assays revealed bactericidal activity only against the producing strain, suggesting, along with other phenotypes, that streptosactin may be a fratricidal agent in Streptococcus thermophilus. Finally, we investigated the HGH subfamily and identified a bicyclic motif with two unprecedented crosslinks, a β-ether C–O bond and a backbone peptide N–C bond in the natural product. Future examination of the remaining RaS-RiPP gene clusters is sure to deliver new chemical, enzymatic, and biological insights.