Khalil Sofia, Pawelek Peter D
Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke Street, West, Montreal, Quebec, Canada.
J Mol Biol. 2009 Oct 30;393(3):658-71. doi: 10.1016/j.jmb.2009.08.036. Epub 2009 Aug 20.
Siderophores are small-molecule iron chelators that many bacteria synthesize and secrete in order to survive in iron-depleted environments. Biosynthesis of enterobactin, the Escherichia coli catecholate siderophore, requires adenylation of 2,3-dihydroxybenzoic acid (2,3-DHB) by the cytoplasmic enzyme EntE. The DHB-AMP product is then transferred to the active site of holo-EntB subsequent to formation of an EntE-EntB complex. Here we investigate the binding of 2,3-DHB to EntE and how DHB binding affects EntE-EntB interaction. We overexpressed and purified recombinant forms of EntE and EntB with N-terminal hexahistidine tags (H6-EntE and H6-EntB). Isothermal titration calorimetry showed that 2,3-DHB binds to H6-EntE with a 1:1 stoichiometry and a K(d) of 7.4 microM. Fluorescence spectra revealed enhanced 2,3-DHB emission at 440 nm (lambda(ex)=280 nm) when bound to H6-EntE due to fluorescence resonance energy transfer (FRET) between EntE intrinsic fluorophore donors and bound 2,3-DHB acceptor. A FRET signal was not observed when H6-EntE was mixed with either 2,5-dihydroxybenzoic acid or 3,5-dihydroxybenzoic acid. The H6-EntE-2,3-DHB FRET signal was quenched by H6-EntB in a concentration-dependent manner. From these data, we were able to determine the EC(50) of EntE-EntB interaction to be approximately 1.5 microM. We also found by fluorescence and CD measurements that H6-EntB can bind 2,3-DHB, resulting in conformational changes in the protein. Additional alterations in H6-EntB near-UV and far-UV CD spectra were observed upon mixture with H6-EntE and 2,3-DHB, suggesting that further conformational rearrangements occur in EntB upon interaction with substrate-loaded EntE. We also found that H6-EntB as a bait protein pulled down a higher concentration of chromosomally expressed EntE in the presence of exogenous 2,3-DHB. Taken together, our results show that binding of 2,3-DHB to EntE and EntB primes these proteins for efficient complexation, thus facilitating direct channeling of the siderophore precursor 2,3-DHB-AMP.
铁载体是许多细菌合成并分泌的小分子铁螯合剂，以便在缺铁环境中生存。大肠杆菌儿茶酚型铁载体肠杆菌素的生物合成需要细胞质酶EntE将2,3 - 二羟基苯甲酸（2,3 - DHB）腺苷化。DHB - AMP产物在形成EntE - EntB复合物后被转移至全酶EntB的活性位点。在此，我们研究2,3 - DHB与EntE的结合以及DHB结合如何影响EntE - EntB相互作用。我们过表达并纯化了带有N端六组氨酸标签的EntE和EntB的重组形式（H6 - EntE和H6 - EntB）。等温滴定量热法表明，2,3 - DHB以1:1的化学计量比与H6 - EntE结合，解离常数（K(d)）为7.4微摩尔。荧光光谱显示，由于EntE内在荧光供体与结合的2,3 - DHB受体之间的荧光共振能量转移（FRET），当2,3 - DHB与H6 - EntE结合时，其在440纳米（激发波长（lambda(ex)）= 280纳米）处的发射增强。当H6 - EntE与2,5 - 二羟基苯甲酸或3,5 - 二羟基苯甲酸混合时，未观察到FRET信号。H6 - EntE - 2,3 - DHB的FRET信号被H6 - EntB以浓度依赖的方式淬灭。根据这些数据，我们能够确定EntE - EntB相互作用的半数有效浓度（EC(50)）约为1.5微摩尔。我们还通过荧光和圆二色性测量发现，H6 - EntB可以结合2,3 - DHB，导致蛋白质构象发生变化。在与H6 - EntE和2,3 - DHB混合后，观察到H6 - EntB近紫外和远紫外圆二色光谱有额外变化，这表明EntB与负载底物的EntE相互作用时会发生进一步的构象重排。我们还发现，作为诱饵蛋白的H6 - EntB在存在外源2,3 - DHB的情况下，能拉下更高浓度的染色体表达的EntE。综上所述，我们的结果表明，2,3 - DHB与EntE和EntB的结合使这些蛋白质为高效络合做好准备，从而促进铁载体前体2,3 - DHB - AMP的直接通道运输。
