title: AcnC of Escherichia coli is a 2-methylcitrate dehydratase (PrpD) that can use citrate and isocitrate as substrates.
abstract: Escherichia coli possesses two well-characterized aconitases (AcnA and AcnB) and a minor activity (designated AcnC) that is retained by acnAB double mutants and represents no more than 5% of total wild-type aconitase activity. Here it is shown that a 2-methylcitrate dehydratase (PrpD) encoded by the prpD gene of the propionate catabolic operon (prpRBCDE) is identical to AcnC. Inactivation of prpD abolished the residual aconitase activity of an AcnAB-null strain, whereas inactivation of ybhJ, an unidentified acnA paralogue, had no significant effect on AcnC activity. Purified PrpD catalysed the dehydration of citrate and isocitrate but was most active with 2-methylcitrate. PrpD also catalysed the dehydration of several other hydroxy acids but failed to hydrate cis-aconitate and related substrates containing double bonds, indicating that PrpD is not a typical aconitase but a dehydratase. Purified PrpD was shown to be a monomeric iron-sulphur protein (M(r) 54000) having one unstable [2Fe-2S] cluster per monomer, which is needed for maximum catalytic activity and can be reconstituted by treatment with Fe(2+) under reducing conditions.
authors.affiliation: The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK1.
authors.forename: Lindsay
authors.initials: L
authors.lastname: Blank
authors.affiliation: The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK1.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Green
authors.affiliation: The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK1.
authors.forename: John R
authors.initials: JR
authors.lastname: Guest
mesh_terms: D000154:Aconitate Hydratase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D002951:Citrates / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D017353:Gene Deletion; D006836:Hydro-Lyases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D007523:Isocitrates / Q000378:metabolism*; D008969:Molecular Sequence Data; D011422:Propionates / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D002951:Citrates; D007506:Iron-Sulfur Proteins; D007523:Isocitrates; D011422:Propionates; C031605:2-methylcitric acid; C034219:isocitric acid; D006836:Hydro-Lyases; D000154:Aconitate Hydratase