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The Escherichia coli enterobactin biosynthetic protein EntB is a bifunctional enzyme that catalyzes hydrolysis of isochorismate via its N-terminal isochorismatase (IC) domain, and then transfers phosphopantetheinylated 2,3-DHB to EntF via the EntB C-terminal aryl carrier protein (ArCP) domain. Here we used a fluorescence anisotropy binding assay to investigate the ability of 2,3-DHB to bind to enzymes in the DHB synthetic arm of the pathway. We found that 2,3-DHB binds to EntE as a natural substrate with high affinity (K_D = 0.54 µM). Furthermore, apo-EntB was found to bind to 2,3-DHB with moderate affinity (K_D = 8.95 µM), despite the fact that this intermediate is neither a substrate nor a product of EntB. Molecular docking simulations predicted a top-ranked ensemble in which 2,3-DHB is bound at the isochorismatase active site of apo-EntB. Steady-state coupled enzymatic assays revealed that 2,3-DHB is a competitive inhibitor of apo-EntB isochorismatase activity (K_i ~ 200 µM), consistent with modeling predictions. Monitoring the EntC-EntB coupled reaction in real time via isothermal titration microcalorimetry confirmed that EntB was required to drive the EntC reaction toward isochorismate formation. Furthermore, addition of 2,3-DHB to the ITC-monitored reaction resulted in a suppression of integrated reaction heats, consistent with our observation that the molecule acts as a competitive inhibitor of EntB. Finally, we found that 2,3-DHB lowered the efficiency of EntC-EntB isochorismate channeling by approximately 70%, consistent with steric blockage of the isochorismatase active site by bound 2,3-DHB. Given its inhibitory properties, we hypothesize that 2,3-DHB plays a regulatory role in feedback inhibition in order to maintain iron homeostasis upon intracellular accumulation of sufficient ferric enterobactin.