Date of Award
Doctor of Philosophy (PhD)
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.