Enzymatic Adenylation of 2,3-Dihydroxybenzoate Is Enhanced by a Protein−Protein Interaction between Escherichia coli 2,3-Dihydro-2,3-dihydroxybenzoate Dehydrogenase (EntA) and 2,3-Dihydroxybenzoate-AMP Ligase (EntE)

摘要

The Escherichia coli siderophore enterobactin is synthesized in response to iron starvation. 2,3-nDihydro-2,3-dihydroxybenzoate dehydrogenase (EntA) produces 2,3-dihydroxybenzoate (DHB), a biosyntheticnintermediate. 2,3-Dihydroxybenzoate-AMP ligase (EntE) adenylates DHB, activating it for attachment to thenNRPS substrate holo-EntB. Using analytical ultracentrifugation, we found that EntA undergoes concentration-ndependent dimer-tetramer self-association (KD = 12.3 μM). We further found that EntA can form a specificncomplex with EntE. Pull-down assays revealed that recombinant EntA bait pulled down EntE from E. colinlysates, whereas recombinant EntE bait could pull down EntA. Addition of the SMCC cross-linker to a mixturenof EntA and EntE resulted in a cross-linked product with a molecular mass of>250 kDa, suggesting a complexnstoichiometry of one EntA tetramer and four EntE monomers. The effect of EntA on EntE activity was alsonexamined. Addition of a 4-fold excess of EntA to an EntE assay mixture resulted in a 6-fold stimulation of EntEnactivity. EntA was also found to perturb the FRET signal between EntE donor residues and EntE-bound DHB.nBy following the EntA-dependent decrease in the magnitude of the EntE-DHB FRET signal, EntA-EntEnbinding behavior was found to be sigmoidal, suggesting the presence of both low- and high-affinity binding sites.nThe EntA-EntE interaction was also directly measured by isothermal titration calorimetry at 10 u0001C. Thenresulting binding isotherm fit well to amodel describing two binding sites, supporting our AUC and fluorescencendata. Taken together, our data show that tetrameric EntA optimally interacts with EntE, resulting in annenhancement of EntE activity.