Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke Street West, Montreal, Quebec, Canada.
Biochemistry. 2011 Feb 1;50(4):533-45. doi: 10.1021/bi101558v. Epub 2010 Dec 31.
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.
大肠杆菌铁载体依铁菌素是在缺铁的情况下合成的。2,3-二氢-2,3-二羟基苯甲酸脱氢酶（EntA）产生 2,3-二羟基苯甲酸（DHB），这是一种生物合成中间体。2,3-二羟基苯甲酸-AMP 连接酶（EntE）将 DHB 腺苷酸化，使其附着在 NRPS 底物全同 EntB 上。通过分析超速离心，我们发现 EntA 会发生浓度依赖性的二聚体-四聚体自组装（K(D) = 12.3 μM）。我们还发现 EntA 可以与 EntE 形成特定的复合物。下拉实验表明，重组 EntA 诱饵从大肠杆菌裂解物中拉下 EntE，而重组 EntE 诱饵可以拉下 EntA。将 SMCC 交联剂添加到 EntA 和 EntE 的混合物中，会产生一个分子量大于 250 kDa 的交联产物，这表明 EntA 四聚体和 EntE 单体的复合物计量比为 1:4。我们还研究了 EntA 对 EntE 活性的影响。在 EntE 测定混合物中加入 4 倍过量的 EntA，EntE 的活性会增加 6 倍。还发现 EntA 会干扰 EntE 供体残基与 EntE 结合的 DHB 之间的 FRET 信号。通过跟踪 EntA 依赖性 EntE-DHB FRET 信号强度的降低，发现 EntA-EntE 结合行为呈 S 型，这表明存在低亲和性和高亲和性结合位点。EntA-EntE 相互作用也通过在 10°C 下进行等温滴定量热法直接测量。得到的结合等温线很好地符合描述两个结合位点的模型，支持我们的 AUC 和荧光数据。总的来说，我们的数据表明，四聚体 EntA 与 EntE 最佳相互作用，从而增强 EntE 的活性。
