Lipoic acid, 6,8,-thioctic acid, is a key component of many multienzyme complexes that are essential in primary metabolism. It is found in most prokaryotes and eukaryotes as well as plant and animal tissue. Because of its antioxidant properties, lipoic acid has been shown to prevent diabetic neuropathy and to successfully treat heavy metal poisoning. Although lipoic acid is a relatively simple cofactor in structure, the mechanism by which it is biosynthesized is presently unknown. Recent studies from several laboratories indicate that the product of the lipA gene in E. coli (lipoic acid synthase or LAS) mediates insertion of two sulfur atoms into the 6 and 8 positions of octanoic acid. Characterization of the protein product of the lipA gene suggests that LAS is a member of an emerging class of enzymes that use AdoMet synergistically with iron- sulfur clusters to initiate radical dependent enzymatic reactions, cleaving AdoMet to 5'-deoxyadenosine and methionine in the process. Well- characterized members of this family include lysine 2,3-aminomutase, pyruvate-formate lyase activase, and anaerobic ribonucleotide reductase activase. The goal of the researched proposed herein is to show experimentally that AdoMet functions similarly in LAS. This will be carried out by determining whether AdoMet binds to LAS and by assessing whether tritium or deuterium is transferred to 5'- deoxyadenosine from various isotopically labeled forts of octanoic acid. Lastly, analogues of AdoMet with the potential to stabilize a radical at the 5'-carbon of 5'-deoxyadenosine will be used to establish the involvement of radical intermediates in the LAS reaction.
| Nesbitt, Natasha M; Baleanu-Gogonea, Camelia; Cicchillo, Robert M et al. (2005) Expression, purification, and physical characterization of Escherichia coli lipoyl(octanoyl)transferase. Protein Expr Purif 39:269-82 |
| Cicchillo, Robert M; Iwig, David F; Jones, A Daniel et al. (2004) Lipoyl synthase requires two equivalents of S-adenosyl-L-methionine to synthesize one equivalent of lipoic acid. Biochemistry 43:6378-86 |
| Cicchillo, Robert M; Lee, Kyung-Hoon; Baleanu-Gogonea, Camelia et al. (2004) Escherichia coli lipoyl synthase binds two distinct [4Fe-4S] clusters per polypeptide. Biochemistry 43:11770-81 |
