Salmonella enterica serovar Typhimurium LT2 catabolizes propionate through the 2-methylcitric acid cycle, but the identity of the enzymes catalyzing the conversion of 2-methylcitrate into 2-methylisocitrate is unclear. This work shows that the prpD gene of the prpBCDE operon of this bacterium encodes a protein with 2-methylcitrate dehydratase enzyme activity. Homogeneous PrpD enzyme did not contain an iron-sulfur center, displayed no requirements for metal cations or reducing agents for activity, and did not catalyze the hydration of 2-methyl-cis-aconitate to 2-methylisocitrate. It was concluded that the gene encoding the 2-methyl-cis-aconitate hydratase enzyme is encoded outside the prpBCDE operon. Computer analysis of bacterial genome databases identified the presence of orthologues of the acnA gene (encodes aconitase A) in a number of putative prp operons. Homogeneous AcnA protein of S. enterica had strong aconitase activity and catalyzed the hydration of the 2-methyl-cis-aconitate to yield 2-methylisocitrate. The purification of this enzyme allows the complete reconstitution of the 2-methylcitric acid cycle in vitro using homogeneous preparations of the PrpE, PrpC, PrpD, AcnA, and PrpB enzymes. However, inactivation of the acnA gene did not block growth of S. enterica on propionate as carbon and energy source. The existence of a redundant aconitase activity (encoded by acnB) was postulated to be responsible for the lack of a phenotype in acnA mutant strains. Consistent with this hypothesis, homogeneous AcnB protein of S. enterica also had strong aconitase activity and catalyzed the conversion of 2-methyl-cis-aconitate into 2-methylisocitrate. To address the involvement of AcnB in propionate catabolism, an acnA and acnB double mutant was constructed, and this mutant strain cannot grow on propionate even when supplemented with glutamate. The phenotype of this double mutant indicates that the aconitase enzymes are required for the 2-methylcitric acid cycle during propionate catabolism.