Date of Graduation
School of Medicine
Microbiology, Immunology, and Cell Biology
Heme serves as a cofactor of cytochromes and catalases. It is essential for energy generation and in defense against toxic hydrogen peroxide in nearly all cells including Salmonella typhimurium and Escherichia coli. Indirect evidence has suggested that heme synthesis is a regulated process. Little is known about how heme synthesis is regulated in enteric bacteria even though the heme synthetic pathway is genetically well-defined. This research represents the first report that heme synthetic regulation affects the first committed heme pathway enzyme, glutamyl-tRNA reductase (HemA), by an unusual mechanism.;HemA, encoded by hemA gene, catalyzes the rate-limiting step of heme biosynthesis. This project demonstrated that when these bacteria are starved for heme, HemA enzyme activity and protein abundance increase 10--25 fold, while gene expression is not affected much (less than 2-fold induction). These results provide the first direct evidence that heme synthetic regulation targets HemA and suggest that the HemA regulation occurs at the post-transcriptional level.;The results of this project revealed a unique mechanism of HemA regulation by a conditional stability of the HemA protein. The half-life of HemA is about 20 min in unrestricted cells, but increases to >300 min in heme-limited cells. The ATP-dependent proteases responsible for HemA turnover were discovered by testing E. coli mutants. HemA turnover is completely blocked in a lon clpP double mutant, but not in either single mutant, indicating that both Lon and ClpP are involved in HemA proteolysis. ClpA, but not ClpX was further determined to have a role in HemA degradation as the chaperone of ClpP.;The amino acids of HemA that signal degradation were determined in this project. A hybrid HemA-lacZ protein containing the first 18 amino acids of the HemA N-terminal region, is also stabilized in a lon clpP mutant. Insertion of two lysines after the second N-terminal amino acid of HemA completely stabilizes this protein while not impairing enzyme function. This finding confirms the hypothesis that HemA degradation tag lies in the N-terminus. Several models are discussed in this dissertation for the signals and regulatory components of the HemA regulation pathway.
Wang, Liying, "Regulation of heme biosynthesis targets the key enzyme HemA by a mechanism of protein stabilization in Salmonella typhimurium" (1999). Graduate Theses, Dissertations, and Problem Reports. 3140.