title: 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).
abstract: The Escherichia coli siderophore enterobactin is synthesized in response to iron starvation. 2,3-Dihydro-2,3-dihydroxybenzoate dehydrogenase (EntA) produces 2,3-dihydroxybenzoate (DHB), a biosynthetic intermediate. 2,3-Dihydroxybenzoate-AMP ligase (EntE) adenylates DHB, activating it for attachment to the NRPS substrate holo-EntB. Using analytical ultracentrifugation, we found that EntA undergoes concentration-dependent dimer-tetramer self-association (K(D) = 12.3 μM). We further found that EntA can form a specific complex with EntE. Pull-down assays revealed that recombinant EntA bait pulled down EntE from E. coli lysates, whereas recombinant EntE bait could pull down EntA. Addition of the SMCC cross-linker to a mixture of EntA and EntE resulted in a cross-linked product with a molecular mass of >250 kDa, suggesting a complex stoichiometry of one EntA tetramer and four EntE monomers. The effect of EntA on EntE activity was also examined. Addition of a 4-fold excess of EntA to an EntE assay mixture resulted in a 6-fold stimulation of EntE activity. EntA was also found to perturb the FRET signal between EntE donor residues and EntE-bound DHB. By following the EntA-dependent decrease in the magnitude of the EntE-DHB FRET signal, EntA-EntE binding behavior was found to be sigmoidal, suggesting the presence of both low- and high-affinity binding sites. The EntA-EntE interaction was also directly measured by isothermal titration calorimetry at 10 °C. The resulting binding isotherm fit well to a model describing two binding sites, supporting our AUC and fluorescence data. Taken together, our data show that tetrameric EntA optimally interacts with EntE, resulting in an enhancement of EntE activity.
authors.affiliation: Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke Street West, Montreal, Quebec, Canada.
authors.forename: Sofia
authors.initials: S
authors.lastname: Khalil
authors.forename: Peter D
authors.initials: PD
authors.lastname: Pawelek
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D004758:Enterobactin / Q000096:biosynthesis / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D048168:Escherichia coli K12 / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D062385:Hydroxybenzoates / Q000737:chemistry / Q000378:metabolism*; D008025:Ligases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D025941:Protein Interaction Mapping / Q000379:methods*; D017262:Siderophores / Q000096:biosynthesis / Q000235:genetics
chemical_list: D029968:Escherichia coli Proteins; D062385:Hydroxybenzoates; D017262:Siderophores; D004758:Enterobactin; C009135:2,3-dihydroxybenzoic acid; D044925:Oxidoreductases Acting on CH-CH Group Donors; C061899:entA protein, E coli; D008025:Ligases; C061746:2,3-dihydroxybenzoate-AMP ligase, E coli