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The enterobactin biosynthetic intermediate 2,3‐dihydroxybenzoic acid is a competitive inhibitor of the Escherichia coli isochorismatase EntB

Xue Bin,P. Pawelek

Published 2025 in Protein Science

ABSTRACT

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 (KD = 0.54 μM). Furthermore, apo‐EntB was found to bind to 2,3‐DHB with moderate affinity (KD = 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 (Ki ~ 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.

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