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. 2008 Feb;190(4):1247-55.
doi: 10.1128/JB.01140-07. Epub 2007 Dec 14.

Pseudomonas aeruginosa PqsA is an anthranilate-coenzyme A ligase

James P Coleman  1 , L Lynn Hudson, Susan L McKnight, John M Farrow 3rd, M Worth Calfee, Claire A Lindsey, Everett C Pesci
Affiliations

Pseudomonas aeruginosa PqsA is an anthranilate-coenzyme A ligase

James P Coleman et al. J Bacteriol. 2008 Feb.

Abstract

Pseudomonas aeruginosa is an opportunistic human pathogen which relies on several intercellular signaling systems for optimum population density-dependent regulation of virulence genes. The Pseudomonas quinolone signal (PQS) is a 3-hydroxy-4-quinolone with a 2-alkyl substitution which is synthesized by the condensation of anthranilic acid with a 3-keto-fatty acid. The pqsABCDE operon has been identified as being necessary for PQS production, and the pqsA gene encodes a predicted protein with homology to acyl coenzyme A (acyl-CoA) ligases. In order to elucidate the first step of the 4-quinolone synthesis pathway in P. aeruginosa, we have characterized the function of the pqsA gene product. Extracts prepared from Escherichia coli expressing PqsA were shown to catalyze the formation of anthraniloyl-CoA from anthranilate, ATP, and CoA. The PqsA protein was purified as a recombinant His-tagged polypeptide, and this protein was shown to have anthranilate-CoA ligase activity. The enzyme was active on a variety of aromatic substrates, including benzoate and chloro and fluoro derivatives of anthranilate. Inhibition of PQS formation in vivo was observed for the chloro- and fluoroanthranilate derivatives, as well as for several analogs which were not PqsA enzymatic substrates. These results indicate that the PqsA protein is responsible for priming anthranilate for entry into the PQS biosynthetic pathway and that this enzyme may serve as a useful in vitro indicator for potential agents to disrupt quinolone signaling in P. aeruginosa.

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Figures

FIG. 1.
FIG. 1.
Proposed pathway for PQS biosynthesis. Anthranilate is activated by PqsA to form anthraniloyl-CoA, and this is condensed with 2-oxo-decanoyl-ACP which is recruited from the fatty acid biosynthetic pathway. The recruitment and condensation are postulated to be catalyzed by PqsB, PqsC, and PqsD. The resulting 2-heptyl-4-quinolone is then hydroxylated at the third carbon by the PqsH monooxygenase to produce PQS.
FIG. 2.
FIG. 2.
Dependence of CoA thioester formation on the presence of PqsA. The indicated amounts of crude extract protein prepared from E. coli Rosetta 2(DE3) cells containing either pSLM20 (▪) or the control vector pEX1.8 (▴) were assayed as described in Materials and Methods by using the direct spectrophotometric assay with potassium anthranilate as the substrate. CoA thioester formation was monitored at 365 nm, the maximum λ of anthraniloyl-CoA.
FIG. 3.
FIG. 3.
SDS-PAGE analysis of steps of purification of His-tagged anthranilate-CoA ligase. Samples from stages of purification (described in Materials and Methods) of His-tagged anthranilate-CoA ligase were subjected to SDS-PAGE on an 8% polyacrylamide gel. Lanes: 1, crude extract, 20 μg; 2, ammonium sulfate pellet, 20 μg; 3, His-Select column peak fractions, 2 μg; 4, butyl-Sepharose peak fractions, 2 μg; 5, HiTrap Q column peak fractions, 2 μg; and 6, molecular mass markers, with corresponding kilodalton values indicated on the right.
FIG. 4.
FIG. 4.
TLC analysis of anthranilate-CoA ligase reaction products. (A) A 2-ml CoA ligase reaction mixture was sampled at time zero (before enzyme addition) and at 5, 15, 30, and 60 min. After the addition of the enzyme, the progress of the reaction was monitored in a spectrophotometer at 365 nm. Samples (0.4 ml) were removed at various times, added to 50 μl of ice-cold formic acid, and then centrifuged to remove precipitated proteins. A 50-μl aliquot of each sample was evaporated to dryness, redissolved in 5 μl of water, and spotted onto a silica gel TLC plate. The plate was developed in butanol-acetic acid-water (60:35:25) and photographed under long-wave UV light. Lanes: 1 and 9, synthetic anthraniloyl-CoA; 2 and 8, anthranilate; 3, 0 min (A365 = 0); 4, 5 min (A365 = 0.07); 5, 15 min (A365 = 0.17); 6, 30 min (A365 = 0.28); and 7, 60 min (A365 = 0.39). (B) Autoradiograph of polyethyleneimine-cellulose-separated reaction samples containing [8-14C]ATP in place of unlabeled ATP. Samples were treated and subjected to chromatography as described in Materials and Methods, and TLC plates were then exposed to X-ray film. Unlabeled ATP, ADP, and AMP were spotted alongside samples, and their positions were determined using a handheld UV lamp.
FIG. 5.
FIG. 5.
Hanes plots for anthranilate, CoA, and ATP. Each substrate was varied independently in reaction mixes containing saturating amounts of the other two substrates. The substrate concentration ([S]) is plotted on the abscissa, and the reaction velocity (v; micromoles per minute per milligram of protein) is plotted on the ordinate. Values represent the average of results from three assays. Km value estimates were determined from the x intercept (x intercept = −Km), and Vmax values were determined from the slope (slope = 1/Vmax) as described by Hanes (33).
FIG. 6.
FIG. 6.
Sequence comparisons of PqsA with other functionally verified CoA ligases. The region containing the AMP-binding domain is shown (15, 18). Gray and black shading designates similar and identical residues, respectively. Alignments were performed using AlignX (Vector NTI Advance; Invitrogen Corp.), and the figure was produced using Boxshade 3.21 (http://www.ch.embnet.org/software/BOX_form.html). The sequences used were as follows: PQSA, anthranilate-CoA ligase (this work); AEACL, 2-aminobenzoate-CoA ligase from Azoarcus evansii (55); AEBCL, benzoate-CoA ligase from Azoarcus evansii (30); BURXE, benzoate-CoA ligase from Burkholderia xenovorans LB400 (4); CBACL, 4-chlorobenzoate-CoA ligase from Pseudomonas sp. strain CBS-3 (14); MAGSP, benzoate-CoA ligase from Magnetospirillum sp. strain TS-6 (34); RHOPA, benzoate-CoA ligase from Rhodopseudomonas palustris (24); STACS, acetate-CoA ligase from Salmonella enterica serovar Typhimurium LT2 (31); THAAR, benzoate-CoA ligase from Thauera aromatica (54); XNALB, benzoate-CoA ligase from Xanthomonas albilineans (GenBank accession no. EF117322) (S. M. Hashimi and R. G. Birch, unpublished results). The numbers at the left indicate the starting residue for each sequence. Consensus residues are shown on the bottom line, with lowercase letters indicating conserved residues (present in >6 of 10 sequences) and bold uppercase letters indicating residues identical in all sequences. The location of the AMP-binding motif described previously (15, 18) is shown.
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References

    1. Altenschmidt, U., and G. Fuchs. 1992. Novel aerobic 2-aminobenzoate metabolism. Purification and characterization of 2-aminobenzoate-CoA ligase, localisation of the gene on a 8-kbp plasmid, and cloning and sequencing of the gene from a denitrifying Pseudomonas sp. Eur. J. Biochem. 205721-727. - PubMed
    1. Auburger, G., and J. Winter. 1992. Purification and characterization of benzoyl-CoA ligase from a syntrophic, benzoate-degrading, anaerobic mixed culture. Appl. Microbiol. Biotechnol. 37789-795. - PubMed
    1. Babbitt, P. C., G. L. Kenyon, B. M. Martin, H. Charest, M. Slyvestre, J. D. Scholten, K. H. Chang, P. H. Liang, and D. Dunaway-Mariano. 1992. Ancestry of the 4-chlorobenzoate dehalogenase: analysis of amino acid sequence identities among families of acyl:adenyl ligases, enoyl-CoA hydratases/isomerases, and acyl-CoA thioesterases. Biochemistry 315594-5604. - PubMed
    1. Bains, J., and M. J. Boulanger. 2007. Biochemical and structural characterization of the paralogous benzoate CoA ligases from Burkholderia xenovorans LB400: defining the entry point into the novel benzoate oxidation (box) pathway. J. Mol. Biol. 373965-977. - PubMed
    1. Bandara, M. B., H. Zhu, P. R. Sankaridurg, and M. D. Willcox. 2006. Salicylic acid reduces the production of several potential virulence factors of Pseudomonas aeruginosa associated with microbial keratitis. Investig. Ophthalmol. Vis. Sci. 474453-4460. - PubMed

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