Isochorismate synthase (PchA), the first and rate-limiting enzyme in salicylate biosynthesis of Pseudomonas aeruginosa.
In Pseudomonas aeruginosa the extracellular metabolite and siderophore pyochelin is synthesized from two major precursors, chorismate and l-cysteine via salicylate as an intermediate. The regulatory role of isochorismate synthase, the first enzyme in the pyochelin biosynthetic pathway, was studied. This enzyme is encoded by pchA, the last gene in the pchDCBA operon. The PchA protein was purified to apparent electrophoretic homogeneity from a PchA-overexpressing P. aeruginosa strain. The native enzyme was a 52-kDa monomer in solution, and its activity strictly depended on Mg(2+). At pH 7.0, the optimum, a K(m) = 4.5 microm and a k(cat) = 43.1 min(-1) were determined for chorismate. No feedback inhibitors or other allosteric effectors were found. The intracellular PchA concentration critically determined the rate of salicylate formation both in vitro and in vivo. In cultures grown in iron-limiting media to high cell densities, overexpression of the pchA gene resulted in overproduction of salicylate as well as in enhanced pyochelin formation. From this work and earlier studies, it is proposed that one important factor influencing the flux through the pyochelin biosynthetic pathway is the PchA concentration, which is determined at a transcriptional level, with pyochelin acting as a positive signal and iron as a negative signal.
Gaille
Catherine
C
Institut de Microbiologie Fondamentale, Université de Lausanne, CH-1015 Lausanne, Switzerland.
Reimmann
Cornelia
C
Haas
Dieter
D
D001426:Bacterial Proteins / Q000378:metabolism*; D019751:Intramolecular Transferases / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D012459:Salicylates / Q000378:metabolism*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D012459:Salicylates; D007501:Iron; D019751:Intramolecular Transferases; C097120:PchA protein, Pseudomonas aeruginosa