TABLE 2.

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

Compound	Efficacy as CoA ligase substratea	CoA ligase Km (μM)b	CoA ligase Ki (μM)c	PQS level (%)d
Benzoate	++	150
2-Aminobenzoate (anthranilate)	++	8
2-Amino-5-bromobenzoate	+	78		100
2-Amino-3-chlorobenzoate	−		12.9	18 ± 7
2-Amino-4-chlorobenzoate	++	480		10 ± 1
2-Amino-5-chlorobenzoate	++	703		35 ± 13
2-Amino-6-chlorobenzoate	++	122		52 ± 11
2-Amino-4-fluorobenzoate	+++	70		42 ± 12
2-Amino-5-fluorobenzoate	+++	120		36 ± 13
2-Amino-6-fluorobenzoate	++	11		0
2-Amino-3-methylbenzoate	+	2,830		100
2-Amino-5-methylbenzoate	++	980		100
2-Amino-3-methoxybenzoate	+	159		100
2-Hydroxybenzoate (salicylate)	−		18.3	76 ± 2
Anthranilonitrile	−		1,300	13 ± 3
5-Nitroanthranilonitrile	−		37.0	6 ± 1
3-Fluoro-O-anisidine	−		89.7	41 ± 12
Methylanthranilate (2-aminobenzoate methyl ester)	−		5,800	50 ± 5
N-Methylanthranilate	++

^aBased 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.

^bBased on direct assays at wavelengths determined by NanoDrop UV spectra and K_m values calculated by the direct linear plot method (25).

^cBased on direct assays with anthranilate as the substrate with K_i values calculated by the 50% inhibition method (32).

^dDetermined 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.