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 #
5R01DK047814-20
Application #
8606739
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
2014-02-01
Budget End
2015-01-31
Support Year
20
Fiscal Year
2014
Total Cost
$284,613
Indirect Cost
$88,863
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; 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
Reinhardt, Laurie A; Thoden, James B; Peters, Greg S et al. (2013) pH-rate profiles support a general base mechanism for galactokinase (Lactococcus lactis). FEBS Lett 587:2876-81
Thoden, James B; Goneau, Marie-France; Gilbert, Michel et al. (2013) Structure of a sugar N-formyltransferase from Campylobacter jejuni. Biochemistry 52:6114-26
Chantigian, Daniel P; Thoden, James B; Holden, Hazel M (2013) Structural and biochemical characterization of a bifunctional ketoisomerase/N-acetyltransferase from Shewanella denitrificans. Biochemistry 52:8374-85
Carney, Amanda E; Holden, Hazel M (2011) Molecular architecture of TylM1 from Streptomyces fradiae: an N,N-dimethyltransferase involved in the production of dTDP-D-mycaminose. Biochemistry 50:780-7
Cook, Paul D; Kubiak, Rachel L; Toomey, Daniel P et al. (2009) Two site-directed mutations are required for the conversion of a sugar dehydratase into an aminotransferase. Biochemistry 48:5246-53
Thoden, James B; Cook, Paul D; Schaffer, Christina et al. (2009) Structural and functional studies of QdtC: an N-acetyltransferase required for the biosynthesis of dTDP-3-acetamido-3,6-dideoxy-alpha-D-glucose. Biochemistry 48:2699-709
Firestine, Steven M; Paritala, Hanumantharao; McDonnell, Jane E et al. (2009) Identification of inhibitors of N5-carboxyaminoimidazole ribonucleotide synthetase by high-throughput screening. Bioorg Med Chem 17:3317-23
Thoden, James B; Schaffer, Christina; Messner, Paul et al. (2009) Structural analysis of QdtB, an aminotransferase required for the biosynthesis of dTDP-3-acetamido-3,6-dideoxy-alpha-D-glucose. Biochemistry 48:1553-61
Thoden, James B; Holden, Hazel M; Firestine, Steven M (2008) Structural analysis of the active site geometry of N5-carboxyaminoimidazole ribonucleotide synthetase from Escherichia coli. Biochemistry 47:13346-53

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