Characterization of thiamine biosynthetic enzymes and their integration in the metabolic network of Salmonella enterica
Palmer, Lauren Disterhoft
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Thiamine pyrophosphate is an essential cofactor required for function of many enzymes in central metabolism. Thiamine is made from two independently synthesized moieties, 4-amino- 5-(hydroxymethyl)-2-methylpyrimidine phosphate (HMP-P), and 4-methyl-5-(2-hydroxyethyl)- thiazole phosphate (THZ-P). Previous work in Salmonella enterica and Escherichia coli had identified all thiamine biosynthetic enzymes and reconstituted most of their activities in vitro. Gaps remained in the mechanistic understanding of thiamine biosynthetic enzymes, including the HMP-P synthase ThiC, THZ-P biosynthetic enzyme ThiI, and the fungal HMP-P synthase Thi5p. This thesis work combined physiological and biochemical approaches to better understand how thiamine biosynthetic enzymes work in the context of S. enterica metabolism. Mutational analysis of the bacterial HMP-P synthase ThiC variants found no correlation between in vivo and in vitro function, suggesting the in vivo growth phenotypes were more sensitive to changes in metabolite levels than the in vitro ThiC assay. Biochemical studies with improved ThiC in vitro assay conditions resulted in the first report of catalytic turnover, and determined that ThiC was inhibited by a number of S-adenosylmethionine metabolites. Meanwhile, genetic analysis led to a mechanistic proposal for the requirement of the sulfur trafficking enzyme ThiI in THZ-P biosynthesis. Nutritional analysis of thiI mutant strains identified an alternative sulfur trafficking pathway in S. enterica when oxidized cysteine metabolites were added to the medium. The Saccharomyces cerevisiae enzyme Thi5p was expressed heterologously in S. enterica to probe cellular factors affecting Thi5p activity and metabolic differences between S. cerevisiae and S. enterica. S. cerevisiae Thi5p functioned conditionally in S. enterica, emphasizing that metabolic modules are not always interchangeable and enzymes can be integrated into the metabolic network in unexpected ways. The findings described in this dissertation highlight the idea that enzyme activity depends on the metabolic context of the cell.