Bacteria produce an astonishingly diverse array of carbohydrate--??based macromolecules that serve important physiological roles. The lipopolysaccharide or LPS, for example, is a complex glycoconjugate attached to the outer membranes of Gram?negative bacteria. Conceptually, the LPS can be thought of in terms of three regions: the lipid A component, the core oligosaccharide, and the O?antigen. It is the O?antigen that displays the most variation from species to species and that, in addition to the lipid A moiety, plays a role in virulence. Likewise, the capsular polysaccharides, which surround both pathogenic Gram?positive and Gram?negative bacteria, serve as the first lines of defense against the host immune system. These high molecular weight polysaccharides function by camouflaging cell surface components that would normally elicit the immune response. Often the sugars in the capsular polysaccharides are modified by the attachment of a variety of moieties including an O?methyl phosphoramidate group, which has been shown to be involved in host invasion and bacteriophage recognition. The intellectual goals of this competitive renewal are twofold: (1) to expand upon our current knowledge on the structures and activities of the enzymes involved in the biosynthesis of O?antigen sugars and (2) to provide a molecular framework for understanding the biosynthesis of the O?methyl phosphoramidate group. Techniques to be utilized include X?ray crystallography, site?directed mutagenesis, and kinetic analyses. Enzymes required for these studies have been purified, preliminary crystals have been obtained for some of the proteins, and three?dimensional models of various apoenzymes have already been determined to high resolution. The proposed investigations will reveal unprecedented chemistries and provide fundamental contributions to mechanistic enzymology. More importantly, given that the LPS and capsular polysaccharides play critical roles in bacterial virulence, some of the enzymes to be investigated may ultimately serve as targets for antimicrobial drug design.
The first goal of the application is to investigate the enzymes involved in the biosythesis of unusual dideoxysugars found on the lipopolysaccharides of Gram-negative bacteria. The second goal is to study the enzymes involved in the biosynthesi of the O-methyl phosphoramidate modification found in bacterial capsular polysaccharides. The two components of this application are linked together in that both unusual sugars and odifications thereof play key roles in bacterial invasion and serum resistance.
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