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|Title:||Molecular and biochemical studies of Escherichia coli thioesterase I|
|Doctoral Committee Chair(s):||Cronan, John E.|
|Department / Program:||Biology, Molecular
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||In order to elucidate the in vivo role(s) of Escherichia coli thioesterases which currently lack an assigned function in lipid metabolism, I have cloned the structural gene for Escherichia coli thioesterase I (called tesA) by use of sequence data obtained from the purified protein. I have constructed and characterized a strain carrying a null mutation of the tesA gene and a strain carrying a plasmid that gave massive overproduction of TesA. In addition, I have studied a strain containing double null mutations in both tesA and tesB (the structural gene for E. coli thioesterase II). DNA and protein sequence data predict that thioesterase I (long believed to be a cytoplasmic protein) is exported to the cell periplasm and this prediction is supported by release of the enzyme from cells upon osmotic shock. To gain insight into the catalytic mechanism of the enzyme, I have identified the "active site serine" of thioesterase I by radiochemical modification.
The possibility of coupling between fatty acid synthesis and phospholipid synthesis has been one of the unanswered questions in E. coli lipid biosynthesis. Using a plasmid encoding a TesA protein lacking its leader peptide (generated by site-directed mutagenesis), I have shown evidence that fatty acid synthesis and phospholipid synthesis are coupled in E. coli and that the coupling is probably maintained via a negative feedback inhibition mechanism using an effector molecule(s) such as fatty acyl-ACP(s).
Finally, I have shown that the TesA protein functions not as a protease as reported by other groups but as a thioesterase in vivo. I have also discussed the significance of two open reading frames (ORFs) which overlap with the tesA gene and the possible involvement of the tesA gene and these two ORFs in uptake and/or catabolic processes of a substrate(s).
|Rights Information:||Copyright 1994 Cho, Hyeseon|
|Date Available in IDEALS:||2011-05-07|
|Identifier in Online Catalog:||AAI9503165|