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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM115921-25
Application #
9623957
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Barski, Oleg
Project Start
1994-06-01
Project End
2020-01-31
Budget Start
2019-02-01
Budget End
2020-01-31
Support Year
25
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Earth Sciences/Resources
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Delvaux, Nathan A; Thoden, James B; Holden, Hazel M (2018) Molecular architectures of Pen and Pal: Key enzymes required for CMP-pseudaminic acid biosynthesis in Bacillus thuringiensis. Protein Sci 27:738-749
Dow, Garrett T; Thoden, James B; Holden, Hazel M (2018) The three-dimensional structure of NeoB: An aminotransferase involved in the biosynthesis of neomycin. Protein Sci 27:945-956
Brown, Haley A; Thoden, James B; Tipton, Peter A et al. (2018) The structure of glucose-1-phosphate thymidylyltransferase from Mycobacterium tuberculosis reveals the location of an essential magnesium ion in the RmlA-type enzymes. Protein Sci 27:441-450
Brown, Haley A; Vinogradov, Evgeny; Gilbert, Michel et al. (2018) The Mycobacterium tuberculosis complex has a pathway for the biosynthesis of 4-formamido-4,6-dideoxy-d-glucose. Protein Sci 27:1491-1497
Taylor, Zane W; Brown, Haley A; Narindoshvili, Tamari et al. (2017) Discovery of a Glutamine Kinase Required for the Biosynthesis of the O-Methyl Phosphoramidate Modifications Found in the Capsular Polysaccharides of Campylobacter jejuni. J Am Chem Soc 139:9463-9466
Riegert, Alexander S; Chantigian, Daniel P; Thoden, James B et al. (2017) Biochemical Characterization of WbkC, an N-Formyltransferase from Brucella melitensis. Biochemistry 56:3657-3668
Dunsirn, Murray M; Thoden, James B; Gilbert, Michel et al. (2017) Biochemical Investigation of Rv3404c from Mycobacterium tuberculosis. Biochemistry 56:3818-3825
Taylor, Zane W; Brown, Haley A; Holden, Hazel M et al. (2017) Biosynthesis of Nucleoside Diphosphoramidates in Campylobacter jejuni. Biochemistry 56:6079-6082
Riegert, Alexander S; Thoden, James B; Schoenhofen, Ian C et al. (2017) Structural and Biochemical Investigation of PglF from Campylobacter jejuni Reveals a New Mechanism for a Member of the Short Chain Dehydrogenase/Reductase Superfamily. Biochemistry 56:6030-6040
Li, Zack Z; Riegert, Alexander S; Goneau, Marie-France et al. (2017) Characterization of the dTDP-Fuc3N and dTDP-Qui3N biosynthetic pathways in Campylobacter jejuni 81116. Glycobiology 27:358-369

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