| Summary: | Lipoic acid is a cofactor used during oxidative metabolism reactions by several enzymes, including branched chain keto acid dehydrogenases, the glycine cleavage system, pyruvate dehydrogenase and a-ketoglutarate dehydrogenase. The lipoic acid is attached to the e-amino group of a specific lysine residue via an amide bond and acts as a carrier of acyl groups between active sites of these large multienzyme complexes. The biosynthesis of lipoyl product requires as a cofactor the protein lipoyl synthase (LipA), which has two [4Fe-4S]1+/2+ clusters, and uses S-adenosylmethionine as a substrate. LipA is the product of the lipA gene and has 36 % sequence homology to biotin synthase in E.coli, another protein involved in a sulfur insertion reaction. The mechanism of sulfur insertion during lipoic acid biosynthesis is thought to be related to that for the BioB catalysed synthesis of biotin. LipA and BioB belong to a group of enzymes known to as the “radical SAM” superfamily. All members of this group reductively cleave AdoMet to give methionine and the highly reactive 5′-deoxyadenosyl radical (5′- Ado•) which abstracts hydrogen atoms from appropriate substrates forming the side product 5’-deoxyadenosine and a substrate radical. The work in this thesis describes experiments that were carried out to study the mechanism of the LipA mediated reaction. LipA was expressed and purified in E.coli and two types of octanoylated substrates were synthesized; fluorescent and non-fluorescent substrates. These substrates were used in assays to probe the mechanism of the LipA mediated reaction. The binding constant of the co-substrate SAM to LipA was determined using fluorescence polarization assays. Experiments were also carried out to try and crystallize the LipA in the presence of octanoyl substrates and the co-substrate SAM.
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