Studies of Lipoic Acid Metabolism in Escherichia Coli
Lipoic acid is a disulfide cofactor which is essential for function of several key enzymes. The studies presented in this thesis shed new light into the understanding of how lipoic acid is synthesized and incorporated into proteins in Escherichia coli. In Chapter Two, the nature of the real substrate for lipoic acid synthase was examined. It firmly established that the preferred substrate for LipA is not octanoyl-ACP but octanoyl-E2. First the means to produce octanoyl-proteins in vivo with a label specific to the octanoyl moieties was developed. Then the labeled octanoate supplement was removed and the lipA gene was subsequently introduced by transduction with phage lambda particles containing a lipA cosmid. Analysis of the isolated E2 domains by mass spectroscopy and lipoic acid bioassay revealed that the d15-labeled octanoyl-E2 domain had been converted to d13-labeled lipoyl-E2 domain species. No lipoyl domain was observed in cultures that were not transduced with the lipA cosmid. A more physiologically relevant experiment using octanoylated forms of the PDH and 2-OGDH as substrates revealed that the inactive octanoylated dehydrogenases were converted to their active forms upon restoration of LipA function. These data demonstrate an unprecedented synthetic pathway whereby a covalently bound enzyme cofactor is attached to its cognate protein in an inactive form then enzymatically converted to the active form. In Chapter Three, the reaction mechanism of LipB was studied. It demonstrated that the E. coli LipB reaction preceded through an acyl enzyme intermediate in which octanoate was first transferred from the thiol of ACP to a specific LipB thiol. The acyl-LipB intermediate was first discovered by denaturing gel electrophoresis using [14C]octanoyl-ACP as acyl donor. Then the octanoyl-LipB intermediate was purified by ion exchange chromatography. The intermediate was proved kinetically competent in that the octanoyl group of the purified octanoylated-LipB was transferred to either lipoyl domain or to ACP. The octanoylated-LipB linkage was cleaved by thiol reagents and by neutral hydroxylamine strongly suggesting a thioester bond. Peptide mapping and site-directed mutagenesis studies demonstrated that the conserved residue C169 was the acylation site and was essential for LipB function in vivo.
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University of Illinois at Urbana-Champaign