The deoxy sugars are found ubiquitously in nature and are formally 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. The conversion of ribonucleotide to deoxyribonucleotide is a well documented example, however, a similar disparity of metabolic fate and function exists between hexoses and deoxyhexoses. Particularly notable are the 3,6-dideoxyhexoses found in the lipopolysaccharides (LPS) of gram- negative bacteria. Since LPS is the major surface antigen of the gram- negative cell envelope, the immunological heterogeneity among gram- negative species is often attributed to the O antigen, of which the 3,6- dideoxy-hexoses play an indispensable role in the cell's immunological determination. It has also been shown that the 2,6- and 4,6- dideoxyhexoses, found commonly in antibiotics, play crucial roles in conferring optimal biological activity on these natural products. Although the biological importance of deoxyhexoses is well recognized, little is known about the biosynthetic formation of these unusual sugars. Inspired by the uniqueness of their occurrence in nature and the intriguing properties of their immunological effects, we have begun a study to explore the formation of ascarylose, a 3,6-dideoxyhexoses, in Yersinia pseudotuberculosis. Our emphasis has been placed on the mechanistic studies of the C-3 deoxygenation which seems to proceed via a radical mechanism and is fundamentally distinct from that catalyzed by ribonucleotide reductase. As a continuation of our ongoing efforts to study the biosynthesis of ascarylose, this proposal outlines our future plans to fully characterize the course of this multi-step bio- transformation. The results of these experiments will be used to address the following issues: a) the catalytic and redox properties of the target enzymes, b) the interaction between enzymes catalyzing consecutive steps, and c) the reaction mechanism of each enzymatic conversion. With these experiments well under way for the 3,6-dideoxyhexose pathway, our efforts will then be shifted to study the mechanism of the biosynthesis of deoxyhexoses found in antibiotics. An understanding of the molecular basis of the biosynthetic formation of these deoxy sugars will not only aid in delineating how chemical transformations are affected by enzymes catalyzing these conversions, but will also provide invaluable knowledge for designing approaches to control and/or mimic their production and biological activities. Since the significance of sugar residues in determining the biological activity of the antibiotics has been well established, and some of the glycosyl transferases involved in the biosynthesis of antibiotics have been shown to have somewhat relaxed substrate specificity, it is expected that the new insights gained from these studies will also lay groundwork for both gene transfer experiments and for site-directed mutagenesis to produce novel or hybrid antibiotics.
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