Isochorismate Synthase (PchA), the First and Rate-limiting Enzyme in Salicylate Biosynthesis of Pseudomonas aeruginosa

Abstract

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 M 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.

Ferripyochelin uptake genes are involved in pyochelin-mediated signalling in Pseudomonas aeruginosa

“…This is likely due to the fact that under the experimental conditions used, gene expression is not the only parameter which determines the amount of product formed. Indeed, we have shown previously that pyochelin formation could be significantly increased when the growth medium was supplemented with cysteine (Gaille et al, 2003), indicating that the intracellular cysteine pool is another important factor.…”

Arabidopsis Isochorismate Synthase Functional in Pathogen-induced Salicylate Biosynthesis Exhibits Properties Consistent with a Role in Diverse Stress Responses

“…SDS-PAGE performed on eluted fractions indicated that the ICS monomer (estimated to be ϳ59 kDa, supplemental data) was exclusively enriched in the fractions with ICS activity. Therefore, similar to the bacterial ICS proteins Pae PchA (48) and Eco EntC (4), AtICS1 appears to function as a monomer, whereas the SAS Yec Irp9 appears to function as a dimer (9).…”

“…Reactions containing no added metal ion were also included. IPL activity is reported to be unaffected by divalent cations (48). However, lactic dehydrogenase (Sigma L-1254, rabbit muscle) activity may be inhibited by heavy metals such as Hg 2ϩ and Pb 2ϩ (Sigma-Aldrich Technical Service).…”

“…Yec Irp9 (9)) or an enzyme complex consisting of ICS and IPL genes coexpressed and present in cis (e.g. Pae PchBA (6,48)). ICS and SAS enzymes share similar global structure and highly conserved active sites (9, 10); therefore, it was important for us to determine whether AtICS1 exhibits monofunctional ICS activity or bifunctional SAS activity.…”

“…Coupled HPLC Assay for ICS Activity-This assay was modified from a previous study (48). 437.5 l of a solution of AtICS1 (3.8 g/ml) and PchB in excess (218 g/ml) in buffer E (100 mM potassium phosphate buffer, pH 7.0, 15 mM MgCl 2 , 10% glycerol, and 1 mM DTT) was mixed with 62.5 l of substrate solution (4 mM chorismic acid in buffer E) and incubated for 60 min at 30°C.…”