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The central theme of this dissertation explores enzyme inhibition, through the use of both synthetic and natural occurring inhibitors of three biotin-associated enzymes; the mammalian biotin ligase, holocarboxylase synthetase(HLCS), and acetyl-CoA carboxylases 1 and 2 (ACC1 and 2), whose activity is dependent on biotinylation by HLCS, and are involved in fatty acid synthesis and regulation of beta-oxidation, respectively. No null HLCS individual has ever been reported and individuals with inherited mutations in the HLCS gene, which render it incapable of biotinylating carboxylases, exhibit varying degrees of symptoms associated with multiple carboxylase deficiency. Little is known with regard to the reaction mechanism and structure of HLCS. The first chapter of this dissertation focuses on the characterization of a novel class of HLCS inhibitors, the biotin-5’-AMP analogs, b-ketophosphonate and b-hydroxyphosphonate, that could be of potential use as an analytical tool in studies of HLCS-biotinylation between the well-known carboxylase targets and novel targets in other proteins. Adapting an existing in vitro protocol in our laboratory, the inhibitors were demonstrated to be competitive inhibitors of HLCS. We extend these studies of HLCS in the next chapter by identifying resveratrol compounds as natural inhibitors of HLCS in vitro which protect Drosophila melanogaster brummer mutants from fat mass accumulation. The third chapter focuses on the effects of dual inhibition of ACC1 and ACC2 on adipocyte differentiation of 3T3-L1 cells. We report on the effects of Soraphen A, a natural occurring polyketide derived from Sorangium cellulosum, which targets the biotin carboxylase domain of the eukaryotic ACCs. We demonstrate that Soraphen A prevents adipogenic gene expression and lipid accumulation in 3T3-L1 cells suggesting dual inhibitors of ACC1 and ACC2 may be an effective strategy to prevent fat accumulation. In conclusion, we have demonstrated the inhibition of HLCS with both synthetic and natural compounds, and that ACC1 and ACC2 inhibition alters lipid metabolism in adipocytes.
Advisor: Janos Zempleni