| A Salmonella enterica locus required for the metabolism of propionate was discovered in the Escalante laboratory and found to contain an operon, prpBCDE, consisting of genes encoding structural proteins of the 2-methylcitrate cycle. Subsequently, propionate metabolism in many prokaryotes has been shown to proceed via the 2-methylcitrate cycle which involves the oxidation of propionate to pyruvate through seven-carbon tricarboxylate intermediates.; The primary focus of this dissertation was to genetically and biochemically characterize the conversion of 2-methylcitrate to pyruvate and succinate in prokaryotes utilizing the 2-methylcitrate cycle. The first part of this dissertation describes the enzymatic function encoded by the acnD gene which is not found in S. enterica, but is present in the propionate utilization operons of several other bacteria. It was found through this work that AcnD is a Fe/S cluster-dependent 2-methylcitrate dehydratase. This is the first example of a Fe/S-dependent dehydratase enzyme that can utilize 2-methylcitrate as a substrate. The second half of this dissertation focuses on the biochemical characterization of PrpB, the 2-methylisocitrate lyase of S. enterica which cleaves 2-methylisocitrate into pyruvate and succinate. Mutations were constructed in PrpB to determine that two of these residues are crucial for 2-methylisocitrate lyase activity. |
