Kinetic and inhibition studies of dihydroxybenzoate-AMP ligase from escherichia coli

Alison L. Sikora, Daniel J. Wilson, Courtney C. Aldrich, John S. Blanchard

Research output: Contribution to journalArticlepeer-review

29 Scopus citations


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 kcat equal to 2.8 s-1 and Km values of 2.5, 430, and 2.9 μM 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 appKi 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.

Original languageEnglish (US)
Pages (from-to)3648-3657
Number of pages10
Issue number17
StatePublished - May 4 2010


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