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.
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.
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