Pseudomonas aeruginosa PqsA is an anthranilate-coenzyme A ligase.
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.
Coleman
James P
JP
Department of Microbiology and Immunology, The Brody School of Medicine at East Carolina University, 600 Moye Blvd., Greenville, North Carolina 27834, USA.
Hudson
L Lynn
LL
McKnight
Susan L
SL
Farrow
John M
JM
Calfee
M Worth
MW
Lindsey
Claire A
CA
Pesci
Everett C
EC
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D003065:Coenzyme A / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007700:Kinetics; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D015363:Quinolones / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D062367:ortho-Aminobenzoates / Q000378:metabolism*
C407944:2-heptyl-3-hydroxy-4-quinolone; D001426:Bacterial Proteins; D015363:Quinolones; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; D000255:Adenosine Triphosphate; D003066:Coenzyme A Ligases; D003065:Coenzyme A