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.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Macromolecular Structure and Function B Study Section (MSFB)
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Sechi, Salvatore
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University of Wisconsin Madison
Schools of Earth Sciences/Natur
United States
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Thoden, James B; Vinogradov, Evgeny; Gilbert, Michel et al. (2015) Bacterial Sugar 3,4-Ketoisomerases: Structural Insight into Product Stereochemistry. Biochemistry 54:4495-506
Salinger, Ari J; Brown, Haley A; Thoden, James B et al. (2015) Biochemical studies on WbcA, a sugar epimerase from Yersinia enterocolitica. Protein Sci 24:1633-9
Riegert, Alexander S; Young, N Martin; Watson, David C et al. (2015) Structure of the external aldimine form of PglE, an aminotransferase required for N,N'-diacetylbacillosamine biosynthesis. Protein Sci 24:1609-16
Woodford, Colin R; Thoden, James B; Holden, Hazel M (2015) New role for the ankyrin repeat revealed by a study of the N-formyltransferase from Providencia alcalifaciens. Biochemistry 54:631-8
Delvaux, Nathan A; Thoden, James B; Holden, Hazel M (2015) Molecular architecture of KedS8, a sugar N-methyltransferase from Streptoalloteichus sp. ATCC 53650. Protein Sci 24:1593-9
Genthe, Nicholas A; Thoden, James B; Benning, Matthew M et al. (2015) Molecular structure of an N-formyltransferase from Providencia alcalifaciens O30. Protein Sci 24:976-86
Thoden, James B; Holden, Hazel M (2014) Production of a novel N-monomethylated dideoxysugar. Biochemistry 53:1105-7
Thoden, James B; Holden, Hazel M (2014) The molecular architecture of QdtA, a sugar 3,4-ketoisomerase from Thermoanaerobacterium thermosaccharolyticum. Protein Sci 23:683-92
Zimmer, Alex L; Thoden, James B; Holden, Hazel M (2014) Three-dimensional structure of a sugar N-formyltransferase from Francisella tularensis. Protein Sci 23:273-83
Thoden, James B; Holden, Hazel M; Grant, Gregory A (2014) Structure of L-serine dehydratase from Legionella pneumophila: novel use of the C-terminal cysteine as an intrinsic competitive inhibitor. Biochemistry 53:7615-24

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