Regulation of heme biosynthesis targets the key enzyme HemA by a mechanism of protein stabilization in Salmonella typhimurium [electronic resource] /
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
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.
School:West Virginia University
School Location:USA - West Virginia
Source Type:Master's Thesis
Keywords:heme salmonella typhimurium
Date of Publication: