ABSTRACT

Lipoic Acid and Lipoyl Carrier Proteins ........................................................... 11 Lipoic Acid Biosynthesis: Early Metabolic Feeding Studies............................ 15 Pathways of Protein Lipoylation ....................................................................... 18 Cloning of Escherichia coli lipA and lipB Genes ......................................... 20 Cloning of the Escherichia coli lplA Gene.................................................... 21 Role of the Acyl Carrier Protein in Lipoic Acid Biosynthesis ..................... 21

Characterization of Lipoate Protein Ligase A ................................................... 25 Characterization of Octanoyl-[Acyl Carrier Protein]-Protein Transferase ........ 29 Characterization of Lipoyl Synthase.................................................................. 32 Radical SAM Superfamily of Enzymes......................................................... 32 Isolation and Characterization of Lipoyl Synthase from Escherichia coli ..... 34 E. coli LipA Contains Two [4Fe-4S] Clusters per Polypeptide

in Its Active Form ..................................................................................... 36 Mechanistic Characterization of the LipA Reaction...................................... 38

Similarities between Lipoyl Synthase and Biotin Synthase .............................. 42 Conclusion ......................................................................................................... 44 Acknowledgments.............................................................................................. 46 References .......................................................................................................... 46

a-Lipoic acid (1,2-dithiolane-3-pentanoic acid, or 6,8-thioctic acid) is a sulfurcontaining cofactor found in most prokaryotic and eukaryotic microorganisms, as well as plant and animal tissue (Figure 2.1A) [1]. It is best known as a requisite component of several multienzyme complexes that function in the oxidative decarboxylation of various a-keto acids and glycine, as well as the oxidative cleavage of 3-hydroxy-2-butanone (acetoin) to acetaldehyde and acetyl-coenzyme A [2-7]. In its capacity as a cofactor, it is bound covalently in an amide linkage to the N6-amino group of a lysine residue on one of the proteins of the complex, producing a long (14 Å) tether (Figure 2.1B), which facilitates the direct

channeling of intermediates among the various active sites located on different subunits [8-10]. The key functional property of the lipoyl cofactor is its ability to undergo redox chemistry, interchanging between the cyclic disulfide (lipoamide) and reduced dithiol (dihydrolipoamide) forms (Figure 2.1B). The disulfide form of the cofactor acts as an electron acceptor in each of the multienzyme complexes that employ it, becoming reduced by two-electrons during turnover. Reoxidation of dihydrolipoamide must take place for additional rounds of turnover to occur, and is catalyzed by the flavoenzyme lipoamide dehydrogenase. The reducing equivalents generated in each overall reaction are ultimately transferred to NADþ, affording NADH and a proton.