Carbohydrates constitute ~50% of the Earth's biomass where they major play roles in energy storage, signaling, molecular recognition, and cell structure. They are also required for the efficacies of many antibiotics, antifungals, anthelmintics, and antitumor agents. Indeed, the diversity of carbohydrate structures observed in nature is truly remarkable. Of particular relevance to this grant application are the di- and trideoxysugars observed in the O-antigens of Gram-negative bacteria. Studies have shown that these sugars are highly immunogenic, play roles in the virulence of the bacterium, and help the organism to evade therapeutic agents. In particular, the focus of this proposal is on the biosynthetic enzymes required for the production of three sugars: (i) colitose, (ii) 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid (D- ManNAc3NAcA) and (iii) legionaminic acid. Colitose has been observed, for example, in the O-antigen of Vibrio cholerae, the causative agent of cholera. The quite rare sugar, D-ManNAc3NAcA, has been isolated from the B-band O-antigen of the opportunistic bacterium Pseudomonas aeruginosa, a major source of nosocomial infections and in the A-band trisaccharide of the bacterium Bordetella pertussis, the causative agent of whopping cough. The third sugar, legionaminic acid, was first identified in Legionella pneumophila, the bacterium responsible for Legionnaires'disease. These three sugars are synthesized by an array of intriguing enzymes that will be investigated in this proposal by X-ray crystallography, site-directed mutagenesis experiments, and kinetic analyses. Some of the proteins in these biosynthetic pathways may ultimately serve as targets for antimicrobial drug design. In addition, the proposed investigations will reveal unprecedented chemistries and will provide fundamental contributions to mechanistic enzymology. Finally, by understanding the structures and functions of the enzymes to be investigated, it will be possible to design unique carbohydrate moieties not previously encountered in nature, and these will have important ramifications for the development of new therapeutics.

Public Health Relevance

The goal of this application is to investigate the structures and functions of enzymes involved in the biosynthesis of di- and trideoxysugars. These types of sugars are observed in the O-antigens of Gram-negative bacteria, and studies have shown that these carbohydrates are highly immunogenic, play roles in the virulence of the bacterium, and help the organism to evade therapeutic agents. The proposed investigations will reveal unprecedented chemistries and will provide fundamental contributions to mechanistic enzymology. In addition, by understanding the structures and functions of the enzymes to be investigated, it will be possible to design unique carbohydrate moieties not previously encountered in nature, and these will have important ramifications for the development of new therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK047814-18
Application #
8212025
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Sechi, Salvatore
Project Start
1994-06-01
Project End
2016-01-31
Budget Start
2012-02-01
Budget End
2013-01-31
Support Year
18
Fiscal Year
2012
Total Cost
$316,236
Indirect Cost
$98,736
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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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