Synergistic action of two radical SAM enzymes in the biosynthesis of thuricin CD, a two-component sactibiotic

Thuricin CD, composed of two ribosomally derived peptides trnα and trnβ, is a distinct two-component sactipeptide antibiotic known for its potent narrow spectrum antibacterial activity against several strains including Clostridioides difficile. Despite its early discovery, how the characteristic thioether crosslinks are installed on thuricin CD remained largely elusive. In this report, we demonstrate that neither of the two radical S-adenosylmethionine (rSAM) enzymes, TrnC and TrnD, can effectively modify the precursors individually. Instead, TrnC and TrnD form a tight complex and collaboratively catalyze thioether crosslinking on the two precursor peptides TrnA and TrnB. Although both TrnC and TrnD are active rSAM enzymes, only the rSAM activity of TrnC is strictly essential for thioether crosslinking, demonstrating a unique enzyme synergy in the biosynthesis of two-component antibiotics. We also generate an active thuricin CD variant by a procedure involving coexpression followed by in vitro proteolysis.