PMID:11937513

Citation	Gaille, C, Kast, P and Haas, D (2002) Salicylate biosynthesis in Pseudomonas aeruginosa. Purification and characterization of PchB, a novel bifunctional enzyme displaying isochorismate pyruvate-lyase and chorismate mutase activities. J. Biol. Chem. 277:21768-75
Abstract	Isochorismate pyruvate-lyase (IPL), the second enzyme of pyochelin biosynthesis and the product of the pchB gene, was purified to homogeneity from Pseudomonas aeruginosa. In the reaction catalyzed by this enzyme, isochorismate --> salicylate + pyruvate, no cofactors appear to be required. At the pH optimum (pH 6.8), the enzyme displayed Michaelis-Menten kinetics, with an apparent K(m) of 12.5 microm for isochorismate and a kcat of 106 min(-1), calculated per monomer. The native enzyme behaved as a homodimer, as judged by molecular sieving chromatography, electrophoresis under nondenaturing conditions, and cross-linking experiments. PchB has approximately 20% amino acid sequence identity with AroQ-class chorismate mutases (CMs). Chorismate was shown to be converted to prephenate by purified PchB in vitro, with an apparent K(m) of 150 microm and a kcat of 7.8 min(-1). An oxabicyclic diacid transition state analog and well characterized inhibitor of CMs competitively inhibited both IPL and CM activities of PchB. Moreover, a CM-deficient Escherichia coli mutant, which is auxotrophic for phenylalanine and tyrosine, was functionally complemented by the cloned P. aeruginosa pchB gene for growth in minimal medium. A mutant form of PchB, in which isoleucine 88 was changed to threonine, had no detectable IPL activity, but retained wild-type CM activity. In conclusion, the 11.5-kDa subunit of PchB appears to contain a single active site involved in both IPL and CM activity.
Links	PubMed Online version:10.1074/jbc.M202410200
Keywords	Amino Acid Sequence; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/isolation & purification; Binding, Competitive; Blotting, Western; Catalysis; Chromatography, High Pressure Liquid; Cross-Linking Reagents/pharmacology; Dimerization; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Escherichia coli/metabolism; Genetic Complementation Test; Hydrogen-Ion Concentration; Isoleucine/chemistry; Kinetics; Models, Chemical; Models, Molecular; Molecular Sequence Data; Mutation; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Pseudomonas aeruginosa/enzymology; Salicylates/metabolism; Temperature
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