The biological importance of carbohydrates cannot be overstated for they are essential elements in nearly every physiological process and represent the most abundant bio-molecules in living systems. Apart from their role in providing metabolic energy, carbohydrates are involved in a wide range of biological processes including the immune response, cell-cell interactions, fertilization, and cell adhesion, among others. This grant has supported over the last 11 years both x-ray structural and biochemical analyses of the four enzymes that constitute the Leloir pathway for galactose metabolism. Defects in the genes encoding these enzymes can lead to diseased states referred to collectively as galactosemia. Clinical severity of these disorders varies widely, depending upon the gene affected, and the resulting degree of enzyme impairment. Through the efforts of this laboratory, the structures of all of these enzymes are now known, two of which were solved during the current funding period. Additionally, in recent years, this grant has supported the structural analyses of enzymes involved in the synthesis of the unusual deoxysugars: tyvelose, colitose, and desosamine. Tyvelose and colitose are 3,6-dideoxyhexoses that occur in the O-antigens of some Gram- negative bacteria and have been speculated to play a role in the pathogenicity of these organisms. Desosamine is a 3-(dimethylamino)-3,4,6-trideoxyhexose found in certain macrolide antibiotics such as the commonly prescribed erythromycin. The addition of unusual deoxysugars such as D-desosamine to such polyketide antibiotics provides or enhances their biological activity. The goals of this competitive renewal are twofold in nature: (i) to continue our research on the structure and function of enzymes involved in deoxysugar biosynthesis, and (ii) to initiate structural studies on the proteins in Saccharomyces cerevisiae that regulate transcription of the genes encoded by the GAL genetic switch. This new study is a natural extension to our past research on the Leloir pathway. Specifically, the GAL genetic switch encodes the genes for the enzymes of the Leloir pathway. Thus far three key components have been identified in controlling transcription of these genes: GalSp, GalSOp, and Gal4p and these will be targeted for structural/functional studies. For all of the planned investigations, a combination of x-ray crystallographic, site-directed mutagenesis, and biochemical techniques will be employed. Taken together, these studies will provide new information with respect to both sugar metabolism and eukaryotic transcriptional regulation.
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