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J. Biol. Chem., Vol. 277, Issue 24, 21768-21775, June 14, 2002

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Salicylate Biosynthesis in Pseudomonas aeruginosa
PURIFICATION AND CHARACTERIZATION OF PchB, A NOVEL BIFUNCTIONAL ENZYME DISPLAYING ISOCHORISMATE PYRUVATE-LYASE AND CHORISMATE MUTASE ACTIVITIES^*

Catherine Gaille, Peter Kast^§, and Dieter Haas^¶

From the  Laboratoire de Biologie Microbienne, Université de Lausanne, CH-1015 Lausanne and the ^§ Laboratorium für Organische Chemie, Swiss Federal Institute of Technology, CH-8093 Zürich, Switzerland

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 µM for isochorismate and a k_cat of 106 min¹, 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 µM and a k_cat of 7.8 min¹. 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.

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