The enterobactin biosynthetic intermediate 2,3‐dihydroxybenzoic acid is a competitive inhibitor of the <i>Escherichia coli</i> isochorismatase <scp>EntB</scp>
Bibliographic record
Abstract
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 ( 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.
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.001 |
| Science and technology studies | 0.001 | 0.002 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".