X-ray Studies of Sugar-Modifying Enzymes

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Nucleotide-linked sugars are found in all organisms where they fulfill a variety of important biological roles. In galactose metabolism, which has been one of the main focuses of this laboratory for twelve years, the sugar is attached to UDP. Interestingly, many of the deoxysugars, which display wide ranges of biological activities from mediating cell-cell interactions to serving as components in various antibiotics, are synthesized via biochemical pathways starting with the attachment of alpha-D-glucose-1 phosphate to either CDP or TDP. The overall goal of this grant renewal is to understand by x-ray crystallographic and site-directed mutagenesis techniques, the structures of enzymes that specific modify nucleotide-linked sugars. The systems that will be investigated include human UDP-galactose 4-epimerase, CDP-D-tyvelose 2-epimerase from Y. pseudotuberculosis IVA, 2,3-d4ehydratase from S. fradiae, and seven enzymes isolated from S. venezuelae that are involved in the biosynthesis of desosamine. UDP-galactose 4-epimerase functions in galactose metabolism by catalyzing the interconversion of UDP-galactose and UDP-glucose. CDP-tyvelose. Tyvelose occurs in the O-antigens of some types of gram-negative bacteria. These 2,3-dehydratase to be studied catalyzes the first step in mycarose biosynthesis. Both mycarose and desosamine are deoxysugars found in some macrolide antibiotics such as erythromycin. The research on UDP-galactose 4-epimerase is in its final stage. The proposed studies on enzymes involved deoxysugars biosynthesis are completely new. Ultimately new structural analysis will yield detailed three-dimensional descriptions of protein: ligand interactions and may eventually provide a molecular foundation upon which to base the design of new antimicrobial agents.