TABLE 2.

Effect of substrate analogs on in vitro anthranilate-CoA ligase activity and in vivo PQS production in P. aeruginosa

CompoundEfficacy as CoA ligase substrateaCoA ligase Km (μM)bCoA ligase Ki (μM)cPQS level (%)d
Benzoate++150
2-Aminobenzoate (anthranilate)++8
2-Amino-5-bromobenzoate+78100
2-Amino-3-chlorobenzoate12.918 ± 7
2-Amino-4-chlorobenzoate++48010 ± 1
2-Amino-5-chlorobenzoate++70335 ± 13
2-Amino-6-chlorobenzoate++12252 ± 11
2-Amino-4-fluorobenzoate+++7042 ± 12
2-Amino-5-fluorobenzoate+++12036 ± 13
2-Amino-6-fluorobenzoate++110
2-Amino-3-methylbenzoate+2,830100
2-Amino-5-methylbenzoate++980100
2-Amino-3-methoxybenzoate+159100
2-Hydroxybenzoate (salicylate)18.376 ± 2
Anthranilonitrile1,30013 ± 3
5-Nitroanthranilonitrile37.06 ± 1
3-Fluoro-O-anisidine89.741 ± 12
Methylanthranilate (2-aminobenzoate methyl ester)5,80050 ± 5
N-Methylanthranilate++
  • a Based on the observation of product formation in direct assays and in NanoDrop UV spectra. Symbols refer to the reaction rate relative to that with anthranilate as the substrate under identical reaction conditions. +++, >100%; ++, 50-100%; +, <50%; −, no reaction.

  • b Based on direct assays at wavelengths determined by NanoDrop UV spectra and Km values calculated by the direct linear plot method (25).

  • c Based on direct assays with anthranilate as the substrate with Ki values calculated by the 50% inhibition method (32).

  • d Determined from triplicate TLC assays as described in Materials and Methods. Results are means ± standard deviations of PQS levels relative to that obtained with anthranilate as the substrate.