Non-ribosomal peptide synthetases (NRPSs) are megaenzymes responsible for the biosynthesis of many clinically important natural products, from early modern medicines (penicillin, bacitracin) to current blockbuster drugs (cubicin, vancomycin) and newly approved therapeutics (rezafungin)1,2. The key chemical step in these biosyntheses is amide bond formation between aminoacyl building blocks, catalysed by the condensation (C) domain3. There has been much debate over the mechanism of this reaction3–12. NRPS condensation has been difficult to fully characterize because it is one of many successive reactions in the NRPS synthetic cycle and because the canonical substrates are each attached transiently as thioesters to mobile carrier domains, which are often both contained in the same very flexible protein as the C domain. Here we have produced a dimodular NRPS protein in two parts, modified each with appropriate non-hydrolysable substrate analogues13,14, assembled the two parts with protein ligation15, and solved the structures of the substrate- and product-bound states. The structures show the precise orientation of the megaenzyme preparing the nucleophilic attack of its key chemical step, and enable biochemical assays and quantum mechanical simulations to precisely interrogate the reaction. These data suggest that NRPS C domains use a concerted reaction mechanism, whereby the active-site histidine likely functions not as a general base, but as a crucial stabilizing hydrogen bond acceptor for the developing ammonium.
