ABSTRACT Natural products have been and continue to be a rich source of lead compounds for the treatment and study of human diseases. Many of these compounds have unusual cyclic skeletons on which their biological properties depend and often require equally unusual enzyme-catalyzed reactions for their construction. By mapping the biosynthetic pathways of these natural products and elucidating the chemical mechanisms of the reactions therein, we aim to enrich the repertoire of tools available to natural product chemists and synthetic biologists in their efforts to develop and engineer new technologies and pharmaceuticals for the benefit of human health. However, in order to fully realize the potential of natural product biosynthesis, the pathways must be characterized, and the underlying chemistry thoroughly understood. In this spirit, we have identified three principal systems for study in the next funding period. Thus, the first specific aim is to explore the unprecedented biosynthetic pathway of ladderane lipids. The cis-fused cyclobutane ring systems of the ladderanes have long been of interest to scientists given their importance in anammox bacteria, their impact on the global nitrogen cycle and their potential as biofuels. However, their biosynthesis remains enigmatic as the necessary enzyme transformations are essentially unknown and may very well involve a number of radical- mediated reactions catalyzed by radical SAM enzymes. The second specific aim seeks to understand the biological origin of two unique peptidyl nucleoside antibiotics (PNAs). Polyketide and carbohydrate biosynthesis have traditionally been considered two separate paradigms in secondary metabolism. However, recent biosynthetic investigations of amipurimycin and miharamycins have suggested that the high-carbon sugar cores of these PNAs are likely biosynthesized as polyketides. We aim to rigorously test this hypothesis by reconstituting the biosynthetic pathways in vitro. We will determine the origin of the sugar cores in these compounds and establish the sequence and nature of the reactions involved in their construction. The third specific aim is to elucidate the pathway and reactions that are responsible for pyrazole ring formation in formycin A and pyrazofurin. The pyrazole moieties in these C-nucleoside antibiotics are notable for their N–N linkage that may require formation of an organohydrazine intermediate. However, the biological transformations underlying N–N bond formation and cyclization are presently almost entirely speculative. Thorough investigation of these hypotheses will require the collective application of our expertise in molecular biology...