Microbes produce natural products to thrive in unique environments where they face a wide range of conditions and competing organisms. These diverse molecules are often unique to individual species and play roles in cellular communication and signaling, interspecies competition, nutrient acquisition, and virulence. The ability of natural products to function in the biological setting make them attractive candidates for the discovery of new pharmaceuticals. Indeed, roughly two-thirds of small molecule drugs are derived from natural products. Understanding the biosynthesis of unusual natural products will facilitate the analysis of the thousands of biosynthetic gene clusters with no known product and may lead to the discovery of new pharmaceutically active molecules. One family of peptide-based natural products is produced by the multidomain nonribosomal peptide synthetases (NRPSs). Among the hundreds of NRPS products are the important peptide antibiotics vancomycin and teixobactin, β-lactam antibiotics like some penicillins and cephalosporins, the genotoxin colibactin, the immunosuppressant cyclosporin, as well as bacterial siderophores like enterobactin, pyoverdine, and mycobactin. NRPSs use a remarkable assembly line architecture; multiple protein domains are joined in a single protein that can be thousands or tens of thousands of residues in length. During biosynthesis, amino acid building blocks are covalently loaded onto a carrier domain and delivered to neighboring catalytic domains for peptide extension and modification. The use of nonproteinogenic amino acids in a ribosome- and mRNA-independent synthesis, as well as subsequent chemical modification by auxiliary domains, enables the striking diversity of NRPS products. We propose to continue our biochemical and structural studies to understand the molecular basis of NRPS function. We will identify the features that govern the efficient progression through the NRPS structural cycle to allow the delivery of bound substrates in a coordinated, efficient process. These studies will determine structures of large, multidomain enzymes in catalytically relevant conformations to understand features that enable this structural cycle. We will also examine unusual NRPSs to identify the features that endow NRPS domains with the ability to perform unexpected chemical reactions. We will examine NRPSs with undetermined biosynthetic mechanism with a particular focus on unusual catalytic properties of condensation and thioester domains, and will determine the products of uncharacterized NRPSs from human pathogens.