Enzymatic evidence for a revised congocidine biosynthetic pathway

Naturally produced pyrrolamides, such as congocidine, are nonribosomal peptides that bind to the minor groove of DNA. Efforts to delineate the biosynthetic machinery responsible for their assembly have mainly employed genetic methods, and the enzymes responsible for their biosynthesis remain largely uncharacterized. We report the biochemical characterization of four proteins involved in congocidine formation: the adenylation-thiolation (A-T) di-domain Cgc18(1-610), its MbtH-like partner SAMR0548, the AMP-binding enzyme Cgc3, and the T domain Cgc19. We assayed the ATP-dependent activation of various commercially available and chemically synthesized compounds with Cgc18(1-610) and Cgc3. We report the revised substrate specificities of Cgc18(1-610) and Cgc3, and loading of 4-acetamidopyrrole-2-carboxylic acid onto Cgc19. Based on these biochemical studies, we suggest a revised congocidine biosynthetic pathway. Validation of novel congocidine biosynthetic pathway. By biochemical characterization of the Cgc18 minimal NRPS module, the AMP-binding Cgc3, and the free-standing thiolation domain Cgc19, we demonstrate that the substrates originally proposed for these enzymes were reversed, and we propose a revised biosynthesis of this pyrrolamide natural product.