The deoxysugars are an important class of carbohydrates which are derived from common sugars by the replacement of one or more hydroxyl groups with hydrogens. Such a substitution generally causes a critical alteration of the biological role of the resulting sugar, and also induces a fundamental change in the metabolism of the product. Particularly notable are the 3,6-dideoxyhexoses found in the lipopolysaccharides of gram-negative bacteria and the 2,6 and 4,6- dideoxyhexoses found in many bioactive secondary metabolites. Intrigued by the great variety of the biological activities and structural features of deoxysugars, we have undertaken an investigation into the biosynthesis of this important class of molecules. In the past few years we have focused our efforts on the formation of ascarylose, a 3,6- dideoxy sugar from Yersinia pseudotuberculosis, and recently, we have also initiated a study to explore the biosynthesis of mycarose, a 2,6- dideoxyhexose from tylosin, and desosamine, a 4,6-dideoxyhexose from methymycin. Outlined in this proposal are our future plans to further establish the course of each multi-step biotransformation and to fully characterize the key enzymes involved in each pathway. The designed experiments will be used to address the following issues: the chemical nature of the radical intermediate generated during C-3 deoxygenation, the reaction mechanisms of C2 and C-4 deoxygenations, and the catalytic properties of the targeted enzymes. Overall, our efforts will require a multi-faceted approach including the expression and purification of the desired enzymes, the synthesis of substrate analogs, inhibitors, and cofactors, the characterization of products isolated from enzymatic incubations with these compounds, and the use of physical and spectroscopic methods for assessing the course and kinetics of the reactions. These mechanistic studies will not only aid in delineating how chemical transformations are affected by enzymes catalyzing these conversions, but also provide valuable information for designing strategies to control and/or mimic the catalytic roles of the target enzymes. In addition, the knowledge gained from this study will also enable us to refine our assessment concerning the potential use of the sugar biosynthetic information, both genetic and biochemical, to produce novel antibiotics. Therefore, our anticipated results should make a significant contribution to the broad field of bioorganic mechanistic enzymology and have possible impact on pharmaceutical biotechnology.
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