PMID:20359185

Citation	Sikora, AL, Wilson, DJ, Aldrich, CC and Blanchard, JS (2010) Kinetic and inhibition studies of dihydroxybenzoate-AMP ligase from Escherichia coli. Biochemistry 49:3648-57
Abstract	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.
Links	PubMed PMC2860672 Online version:10.1021/bi100350c
Keywords	Binding Sites; Catalysis; Catalytic Domain; Dimerization; Enterobactin/metabolism; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins/antagonists & inhibitors; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Kinetics; Ligases/antagonists & inhibitors; Ligases/genetics; Ligases/metabolism; Models, Molecular; Multienzyme Complexes/metabolism; Pantothenic Acid/analogs & derivatives; Pantothenic Acid/metabolism; Peptide Synthases/metabolism; Substrate Specificity
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
ECOLI:ENTE	Contributes to	GO:0016874: ligase activity	ECO:0000314:		F		Figure 5: Shows that entE is associated with some type of inhibition	complete

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