Ligand Binding Induces an Ammonia Channel in 2-Amino-2-desoxyisochorismate (ADIC) Synthase PhzE
Ligand Binding Induces an Ammonia Channel in 2-Amino-2-desoxyisochorismate (ADIC) Synthase PhzE
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DOI：
10.1074/jbc.m110.183418
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发表时间：
2011-05-20
影响因子：
4.8
通讯作者：
Blankenfeldt, Wulf
中科院分区：
文献类型：
--
作者：
Li, Qi-Ang;Mavrodi, Dmitri V.;Blankenfeldt, Wulf
PhzE在吩嗪生物合成的第一步利用分支酸和谷氨酰胺合成2-氨基-2-脱氧异分支酸（ADIC）。PhzE单体包含由45个残基连接体连接的分支酸转化甲基萘醌、铁载体、色氨酸生物合成（MST）和1型谷氨酰胺转移酶（GATase 1）结构域。我们在这里提出的晶体结构的PhzE从伯克霍尔德氏菌383在一个无配体的开放和配体结合的封闭构象在2.9和2.1埃的分辨率，分别。PhzE排列在一个相互缠绕的二聚体中，使得一条链的GATase 1结构域为另一条链的MST结构域提供NH3。通过小角X射线散射证实了这种四级结构。发现在封闭形式的MST活性中心中转化为苯甲酸盐和丙酮酸盐的胆酸的结合导致结构重排，在二聚体的两个MST/GATase 1功能单元中的每一个内建立长度约25埃的氨转运通道。通过产物的质谱分析确认了PhzE作为ADIC合酶的归属，该产物也通过在PhzD的失活突变体（催化吩嗪生物合成中的后续步骤的异分支酶）的晶体中捕获而在1.9埃分辨率下可视化。与一些相关的邻氨基苯甲酸酯酶不同，在PhzE中未观察到变构抑制。这可归因于蛋白质的色氨酸残基阻断了潜在的调节位点。GATase 1活性中心的额外电子密度被鉴定为锌，并且证明Zn 2+、Mn 2+和Ni 2+降低PhzE的活性。
PhzE utilizes chorismate and glutamine to synthesize 2-amino-2-desoxyisochorismate (ADIC) in the first step of phenazine biosynthesis. The PhzE monomer contains both a chorismate-converting menaquinone, siderophore, tryptophan biosynthesis (MST) and a type 1 glutamine amidotransferase (GATase1) domain connected by a 45-residue linker. We present here the crystal structure of PhzE from Burkholderia lata 383 in a ligand-free open and ligand-bound closed conformation at 2.9 and 2.1 angstrom resolution, respectively. PhzE arranges in an intertwined dimer such that the GATase1 domain of one chain provides NH3 to the MST domain of the other. This quaternary structure was confirmed by small angle x-ray scattering. Binding of chorismic acid, which was found converted to benzoate and pyruvate in the MST active centers of the closed form, leads to structural rear-rangements that establish an ammonia transport channel approximately 25 angstrom in length within each of the two MST/GATase1 functional units of the dimer. The assignment of PhzE as an ADIC synthase was confirmed by mass spectrometric analysis of the product, which was also visualized at 1.9 angstrom resolution by trapping in crystals of an inactive mutant of PhzD, an isochorismatase that catalyzes the subsequent step in phenazine biosynthesis. Unlike in some of the related anthranilate synthases, no allosteric inhibition was observed in PhzE. This can be attributed to a tryptophan residue of the protein blocking the potential regulatory site. Additional electron density in the GATase1 active center was identified as zinc, and it was demonstrated that Zn2+, Mn2+, and Ni2+ reduce the activity of PhzE.