In vitro conversion of propionate to pyruvate by Salmonella enterica enzymes: 2-methylcitrate dehydratase (PrpD) and aconitase enzymes catalyze the conversion of 2-methylcitrate to 2-methylisocitrate

Salmonella enterica serovar Typhimurium LT2 catabolizes propionate through the 2-methylcitric acid cycle, but the identity of the enzymes catalyzing t he conversion of 2-methylcitrate into 2-methylisocitrate is unclear. This w ork shows that the prpD gene of the prpBCDE operon of this bacterium encode s a protein with 2-methylcitrate dehydratase enzyme activity, Homogeneous P rpD enzyme did not contain an iron-sulfur center, displayed no requirements for metal cations or reducing agents for activity, and did not catalyze th e hydration of 2-methyl-cis-aconitate to 2-methylisocitrate. It was conclud ed that the gene encoding the 2-methyl-cis-aconitate hydratase enzyme is en coded outside the prpBCDE operon, Computer analysis of bacterial genome dat abases identified the presence of orthologues of the acnA gene (encodes aco nitase A) in a number of putative prp operons, Homogeneous AcnA protein of S, enterica had strong aconitase activity and catalyzed the hydration of th e 2-methyl-cis-aconitate to yield 2-methylisocitrate. The purification of t his enzyme allows the complete reconstitution of the 2-methylcitric acid cy cle in vitro using homogeneous preparations of the PrpE, PrpC, PrpD, AcnA, and PrpB enzymes, However, inactivation of the acnA gene did not block grow th of S. enterica on propionate as carbon and energy source. The existence of a redundant aconitase activity (encoded by acnB) was postulated to be re sponsible for the lack of a phenotype in acnA mutant strains. Consistent wi th 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 c atabolism, 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 r equired for the 2-methylcitric acid cycle during propionate catabolism.