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-Methylisoc

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• 作者：Alexander R. Horswill and Jorge C. Escalante-Semerena
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：April 17, 2001
• 年：2001
• 卷：40
• 期：15
• 页码：4703 - 4713
• 全文大小：127K
• 年卷期：v.40,no.15(April 17, 2001)
• ISSN：1520-4995

文摘

Salmonella enterica serovar Typhimurium LT2 catabolizes propionate through the 2-methylcitricacid 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 bacteriumencodes a protein with 2-methylcitrate dehydratase enzyme activity. Homogeneous PrpD enzyme did notcontain 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 thatthe 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. entericahad strong aconitase activity and catalyzed the hydration of the 2-methyl-cis-aconitate to yield2-methylisocitrate. The purification of this enzyme allows the complete reconstitution of the 2-methylcitricacid 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 andenergy source. The existence of a redundant aconitase activity (encoded by acnB) was postulated to beresponsible for the lack of a phenotype in acnA mutant strains. Consistent with this hypothesis, homogeneousAcnB 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, anacnA and acnB double mutant was constructed, and this mutant strain cannot grow on propionate evenwhen supplemented with glutamate. The phenotype of this double mutant indicates that the aconitaseenzymes are required for the 2-methylcitric acid cycle during propionate catabolism.