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Originally published In Press as doi:10.1074/jbc.M212324200 on March 6, 2003
J. Biol. Chem., Vol. 278, Issue 19, 16893-16898, May 9, 2003
Isochorismate Synthase (PchA), the First and Rate-limiting Enzyme
in Salicylate Biosynthesis of Pseudomonas
aeruginosa*
Catherine
Gaille,
Cornelia
Reimmann, and
Dieter
Haas
From the Institut de Microbiologie Fondamentale, Université
de Lausanne, CH-1015 Lausanne, Switzerland
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
Mg2+. At pH 7.0, the optimum, a Km = 4.5 µM and a kcat = 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.
*
This work was supported by Swiss National Foundation for
Scientific Research Grant 31-56608.99.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 41-21-692-56-31;
Fax: 41-21-692-56-35; E-mail: Dieter.Haas@imf.unil.ch.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

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Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.
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