Portal de Periódicos da CAPES

Salicylate Biosynthesis in Pseudomonas aeruginosa

2002; Elsevier BV; Volume: 277; Issue: 24 Linguagem: Inglês

10.1074/jbc.m202410200

1083-351X

Catherine Gaille, Peter Kast, Dieter Haas,

Amino Acid Enzymes and Metabolism

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 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 μm and a k cat 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.