UNCOVERING NEW METALLOENZYME-MEDIATED CHEMISTRY FROM NATURE’S TOOLKIT: RADICAL S-ADENOSYLMETHIONINE REACTIONS IN NATURAL PRODUCT BIOSYNTHESIS

Abstract

Currently, 50% of all therapeutic agents are linked to a natural product. Recent advancements in DNA sequencing technologies, however, reveal that the vast majority of encoded natural products are undiscovered. Our efforts to identify structurally and functionally new natural products have guided us to Ribosomally-synthesized and post-translationally-modified peptides (RiPPs), an emerging class of natural products that harbor diverse chemical motifs introduced via the action of tailoring enzymes. Specifically, we focus on RiPPs that are modified by unusual metalloenzymes, as these may install yet unknown alterations. Using a novel bioinformatic search strategy, we uncovered an array of unexplored RiPP biosynthetic gene clusters that are quorum sensing-regulated and contain uncharacterized radical S-adenosylmethionine (RaS) metalloenzymes, a diverse protein superfamily capable of catalyzing chemically difficult transformations. Further, our identified RiPP subfamilies are produced by streptococci, a genus of bacteria prevalent in mammalian microbiomes that includes mutualistic, commensal, and pathogenic members.Herein, we investigated several of the identified RiPP subfamilies and found novel transformations which add to the already impressive reaction repertoire of RaS enzymes. In the NxxC subfamily, we found that the RaS enzyme NxxcB installs an intramolecular β-thioether bond onto its substrate peptide, a novel reaction for RaS enzymes. We went on to identify the mature RiPP product of the nxxc operon in Streptococcus equi, which contains two sequential β-thioether macrocycles. We next elucidated the key modification of the QMP subfamily as two sequential α-thioether crosslinks, generating a sactipeptide of the rare type-2 ring topology. Subsequently, we characterized a member of the RRR subfamily and showed the RaS enzyme RrrB installs a macrocyclic carbon-carbon bond. Moreover, we used a combination of computational tools and circular dichroism spectroscopy to identify the chirality of the RaS enzyme-generated stereocenter. Finally, we clarified the detailed mechanism of carbon-carbon bond formation in lysine-tryptophan crosslinking RaS enzymes using isotope labeling studies in conjunction with electron paramagnetic resonance spectroscopy. Our studies revealed an unprecedented crosslinked Lys–Trp radical intermediate, defining a mechanistic paradigm for enzyme-catalyzed carbon-carbon bond-forming reactions. Future investigations of the remaining RaS-RiPP gene clusters and their mature natural products are sure to reveal new chemistries and biological activities.