Post-Translational Modification With Lipoic Acid in Escherichia Coli
Jordan, Sean Wesley
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Permalink
https://hdl.handle.net/2142/86637
Description
Title
Post-Translational Modification With Lipoic Acid in Escherichia Coli
Author(s)
Jordan, Sean Wesley
Issue Date
2002
Doctoral Committee Chair(s)
Cronan, John E., Jr
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Microbiology
Language
eng
Abstract
Lipoic acid is a cofactor essential for the function of key metabolic pathways in eukaryotes and most prokaryotes. Physiological function of lipoic acid is dependent on covalent attachment of the cofactor to highly specific lysine residues of enzymes complex subunits. E. coli encodes two distinct systems for lipoylating the specific lysine residues of the apoprotein subunits of these complexes. The first pathway identified involves the lplA (lipoate-protein ligase) gene product, which utilizes free lipoic acid found in the environment to modify apoproteins. A second pathway was known to be dependent on lipB. I have identified lipB as the structural gene involved in the second, lplA independent pathway. I have shown that this LipB pathway utilizes lipoyl-ACP (lipoyl-acyl-carrier protein) as the lipoyl donor, establishing a link between fatty acid synthesis and lipoic acid synthesis. Additionally, I have shown that LplA will also utilize lipoyl-ACP as well as free lipoic acid, albeit at a much slower rate. ACP is the carrier of growing fatty acyl groups in fatty acid biosynthesis, suggesting that the likely precursor for lipoic acid biosynthesis in E. coli is octanoyl-ACP. Recent results show that octanoyl-ACP indeed acts as a precursor for lipoyl-ACP biosynthesis in vitro. I isolated a temperature sensitive (ts) mutant of lipB and used this is mutant to isolate suppressor mutants. This allowed me to identify, clone and construct mutants in a gene involved in lipoate metabolism I designate as lipC. I made a null mutant of lipC and determined that LipC is required for function in vivo of the endogenous lipoylation pathway. However, experiments with purified LipB and LipC show that only LipB is required for the transfer of lipoyl moieties from lipoyl-ACP to apo-proteins in vitro. LipC is highly homologous to Lpd (lipoamide dehydrogenase), the E3 subunit of PDC and ODC. The physiological role of Lpd is to oxidize protein-bound dihydrolipoamide and pass electrons to NAD. I hypothesize that the role of LipC is to perform similar redox chemistry using lipoyl-ACP as a substrate. Perhaps in vivo, the activity of LipB is dependent on lipoyl-ACP being presented in a given redox state.
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