Kinetic and inhibition studies of dihydroxybenzoate-AMP ligase from Escherichia coli.

Inhibition of siderophore biosynthetic pathways in pathogenic bacteria represents a promising strategy for antibacterial drug development. Escherichia coli synthesize and secrete the small molecule iron chelator siderophore, enterobactin, in response to intracellular iron depletion. Here we describe a detailed kinetic analysis of EntE, one of six enzymes in the enterobactin synthetase gene cluster. EntE catalyzes the ATP-dependent condensation of 2,3-dihydroxybenzoic acid (DHB) and phosphopantetheinylated EntB (holo-EntB) to form covalently arylated EntB, a product that is vital for the final assembly of enterobactin. Initial velocity studies show that EntE proceeds via a bi-uni-uni-bi ping-pong kinetic mechanism with a k(cat) equal to 2.8 s(-1) and K(m) values of 2.5, 430, and 2.9 microM for DHB, ATP, and holo-EntB-ArCP, respectively. Inhibition and direct binding experiments suggest that, during the first half-reaction (adenylation), DHB binds first to the free enzyme, followed by ATP and the release of pyrophosphate to form the adenylate intermediate. During the second half-reaction (ligation), phosphopantetheinylated EntB binds to the enzyme followed by the release of products, AMP and arylated EntB. Two hydrolytically stable adenylate analogues, 5'-O-[N-(salicyl)sulfamoyl]adenosine (Sal-AMS) and 5'-O-[N-(2,3-dihydroxybenzoyl)sulfamoyl]adenosine (DHB-AMS), are shown to act as slow-onset tight-binding inhibitors of the enzyme with (app)K(i) values of 0.9 and 3.8 nM, respectively. Direct binding experiments, via isothermal titration calorimetry, reveal low picomolar dissociation constants for both analogues with respect to EntE. The tight binding of Sal-AMS and DHB-AMS to EntE suggests that these compounds may be developed further as effective antibiotics targeted to this enzyme.

Enterobactin synthetase-catalyzed formation of P(1),P(3)-diadenosine-5'-tetraphosphate.

The EntE enzyme, involved in the synthesis of the iron siderophore enterobactin, catalyzes the adenylation of 2,3-dihydroxybenzoic acid, followed by its transfer to the phosphopantetheine arm of holo-EntB, an aryl carrier protein. In the absence of EntB, EntE catalyzes the formation of Ap(4)A, a molecule that is implicated in regulating cell division during oxidative stress. We propose that the expression of EntE during iron starvation produces Ap(4)A to slow growth until intracellular iron stores can be restored.

Kinetic and chemical mechanism of arylamine N-acetyltransferase from Mycobacterium tuberculosis.

Arylamine N-acetyltransferases (NATs) are cytosolic enzymes that catalyze the transfer of the acetyl group from acetyl coenzyme A (AcCoA) to the free amino group of arylamines and hydrazines. Previous studies have reported that overexpression of NAT from Mycobacterium smegmatis and Mycobacterium tuberculosis may be responsible for increased resistance to the front-line antitubercular drug, isoniazid, by acetylating and hence inactivating the prodrug. We report the kinetic characterization of M. tuberculosis NAT which reveals that substituted anilines are excellent substrates but that isoniazid is a very poor substrate for this enzyme. We propose that the expression of NAT from M. tuberculosis (TBNAT) is unlikely to be a significant cause of isoniazid resistance. The kinetic parameters for a variety of TBNAT substrates were examined, including 3-amino-4-hydroxybenzoic acid and AcCoA, revealing K m values of 0.32 +/- 0.03 and 0.14 +/- 0.02 mM, respectively. Steady-state kinetic analysis of TBNAT reveals that the enzyme catalyzes the reaction via a bi-bi ping-pong kinetic mechanism. The pH dependence of the kinetic parameters reveals that one enzyme group must be deprotonated for optimal catalytic activity and that two amino acid residues at the active site of the free enzyme are involved in binding and/or catalysis. Solvent kinetic isotope effects suggest that proton transfer steps are not rate-limiting in the overall reaction for substituted aniline substrates but become rate-limiting when poor hydrazide substrates are used.