The deoxy sugars, found ubiquitously in plants, fungi, bacteria and mammals, are an important class of carbohydrates. The widespread occurrence of deoxy sugars in different organisms suggests a broad spectrum of possible roles for these unusual carbohydrates. For example, it has been well established that the 3,6-dideoxyhexoses, present only in the lipopolysaccharides of gram-negative bacteria, are immuno-dominant sugars that contribute to the serological specificity of many immunologically active polysaccharides. It has also been shown that the 2,6-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 3,6-dideoxyhexoses, this proposal outlines our future plans to fully characterize the course of this bio-transformation. Specifically, the following experiments will be carried out: 1) purification of enzymes involved in these biosynthetic sequences and cloning of the enzymes' genes; 2) synthesis of a variety of alternative substrates and cofactors containing mechanistically informative functionalities at key sites of the molecules; 3) incubation of these compounds with the target enzymes followed by appropriate product analysis to assess the results; 4) site-directed mutagenesis to determine the catalytic role of key amino acids in the active-site of target enzymes. The results of these experiments will be used to address the following issues: a) the stereochemical course of these enzymatic steps; b) the chemical nature of the reaction intermediates, products,and an unknown cofactor, c) the locus of the catalytic centers and the potential roles of the redox active thiols at the active-sites; d) a likely prerequisite for a proximate interaction between enzymes for catalysis; e) the possible involvement of pyridoxamine phosphate in both dehydration and reduction; f) the radical mechanism of the deoxygenation, etc. With these experiments well under way for the 3,6-dideoxy sugar enzymes, part of our efforts will then be shifted to study the mechanism of the biosynthesis of 2,6-dideoxyhexoses. Isolation of the oxidoreductase involved in the formation of chromose from Streptomyces griseus will be our initial goal. Characterization of each of the possible intermediates and the purification of other enzymes involved in this process will also be attempted. 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 the enzymes catalyzing these conversions but will also provide invaluable knowledge for designing approaches to control and/or mimic their production and biological activities.
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