Date of Award
Summer 2014
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Biological Sciences
First Advisor
Noel, Dale
Second Advisor
Stuart, Rosemary
Third Advisor
Yang, Pinfen
Abstract
All organisms synthesize amidosugars, such as N-acetylglucosamine (GlcNAc), and deoxysugars, such as fucose. They are found in important polysaccharides and glycoconjugates such as glycoproteins. N-acetylquinovosamine (QuiNAc) is both an amido- and a deoxy-sugar. It is found in many examples of an important prokaryotic glycoconjugate, the lipopolysaccharide (LPS) that coats the surface of Gram-negative bacteria. Like N-glycosylation of glycoproteins, LPS has a portion that is synthesized first on a polyprenyl lipid carrier and then transferred to the rest of the molecule. QuiNAc is believed to initiate the O-antigen portion of LPS of Rhizobium etli CE3. Genetic studies identified three genes, wreV, wreU and wreQ, required for the initial steps of O-antigen synthesis in R. etli CE3. Based on the predicted roles of the gene products and the theory of polysaccharide biosynthesis, there was a very straightforward prediction of the initial events: WreV catalyzes conversion of UDP-GlcNAc to its 4-keto-6-deoxy derivative, which WreQ reduces to UDP-QuiNAc, followed by transfer of QuiNAc-1-phosphate to the lipid carrier by WreU. However, the LPS structure of R. etli wreQ mutants was also consistent with a second, novel possibility. These two hypotheses were tested by developing assays in vitro for each of the predicted enzymes. Two key findings were 1) that WreU catalysis is 30-fold faster with the 4-keto intermediate as substrate than with UDP-QuiNAc, and 2) WreQ catalyzes the reduction the 4-keto sugar to QuiNAc orders of magnitude faster when it is linked to lipid rather than to UDP. The results strongly support the second hypothesis, outlined as follows: After 4,6-dehydration of UDP-GlcNAc, the 4-keto-6-deoxysugar-1-phosphate moiety is transferred to the lipid carrier, thereby providing the sugar stem for the rest of O-antigen synthesis. Only then is the 4-keto sugar reduced to QuiNAc. The order of enzyme reactions suggests an interesting chemical coordination between the initiation of the O antigen and QuiNAc synthesis. It also includes the unorthodox completion of deoxysugar synthesis on a lipid carrier. Because the presence of QuiNAc and WreQ homologs are highly correlated in bacterial species, this may be the normal mechanism by which QuiNAc becomes part of bacterial polysaccharides.