ABSTRACT Carbohydrates play a critical role in the activity of many important biological agents and pharmaceuticals, and small alterations in their structures can have a significant impact on their efficacy. Hence, it is not necessarily unexpected that naturally occurring glycans and sugar appendages are so structurally diverse. What is surprising, however, is that most of these unusual carbohydrates are biosynthesized from a relatively small pool of common sugars via a series of reactions that often include new and unusual chemical transformations. By exploiting the machinery of these pathways, it is possible to enhance or vary the biological activities of the glycosylated compounds and apply the principles learned to new systems. However, in order to fully realize the potential of such an approach, the biosynthetic pathways must be characterized and the underlying chemistry thoroughly understood. In this spirit, we have identified several systems to be investigated in the next funding period. The first specific aim is the mechanistic study of the five- to four-membered ring contraction at the heart of oxetanocin A and albucidin biosynthesis. The enzymes involved in these reactions are members of the B12- dependent radical SAM family that has only recently been discovered and remains very poorly understood. The ultimate goal of the work proposed is to determine the mechanism of the highly unusual ring contraction as well as the roles played by the B12 cofactor and the radical SAM machinery. The second specific aim is focused on understanding the mechanism by which glycosidic linkages are oxidatively cyclized to form the characteristic spirocyclic ortho-δ-lactones of the orthosomycin class of antibiotics and in particular hygromycin B. This is an unprecedented transformation in carbohydrate biochemistry that is likely to involve radical intermediates and be of value to the discovery of new orthosomycin antibiotics. The third specific aim seeks to complete the description of gentamicin biosynthesis. The final uncharacterized process in this pathway is a unique α,β-dideoxygenation that stands in contrast to all previously characterized monodeoxygenation reactions involving carbohydrates. While there is little information about how this dideoxygenation might be accomplished, genetic analyses suggest the involvement of exciting radical chemistry. Through the collective application of our expertise in deoxysugar biosynthesis, chemical synthesis and enzymology, we aim to investigate the uncharacterized enzymes in the pathway to resolve this question. These research aims have been selected on the basis of their novelty, implications for the fields of mechanistic enzymology and natural product biosynthesis as well as potential utility in biomedical and biotechnological research at the basic and translational levels.