Structure of Active IspH Enzyme from Escherichia coli Provides Mechanistic Insights into Substrate Reduction
Structure of Active IspH Enzyme from Escherichia coli Provides Mechanistic Insights into Substrate Reduction
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DOI：
10.1002/anie.200900548
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发表时间：
2009-01-01
影响因子：
16.6
通讯作者：
Groll, Michael
中科院分区：
文献类型：
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作者：
Graewert, Tobias;Rohdich, Felix;Groll, Michael
真核生物和大多数原核生物需要异戊烯基二磷酸（IPP）和二甲基烯丙基二磷酸（DMAPP）作为萜烯的生物合成前体。虽然动物通过甲羟戊酸途径产生这些必需的代谢物，[1]许多人类病原体，包括恶性疟原虫和结核分枝杆菌，已知使用最近确定的非甲羟戊酸途径，这是药物开发的潜在目标。[2-4]该途径的最后一步由IspH蛋白催化，其通过1-羟基-2-甲基-2-（E）-丁烯基-4-二磷酸（HMBPP，图1a）的还原脱水产生IPP和DMAPP的混合物。[5-11]最近，Rekittke等人描述了来自超嗜热真细菌Aquifex aeolicus的IspH蛋白在其开放状态下的第一个X射线结构。[12]在本文中，我们报告了来自大肠杆菌的IspH蛋白的晶体结构[11]，其处于闭合构象，这是详细讨论催化途径的基础。重组大肠coli IspH蛋白（包含N-末端His 6融合标签）在厌氧条件下纯化并结晶。用单波长反常衍射方法测定了其结构，分辨率为1.8。每蛋白质单位的三个铁位点被定位在异常差异Patterson图中，并用于定相。连续几轮的模型构建和优化为整个IspH分子提供了明确定义的电子密度，除了N-末端His 6标签和5个C-末端氨基酸残基（Rfree= 23.8%，SupportingInformation，表S2）。不对称单元中两个蛋白质分子的Cα位置之间的均方根（rms）偏差小于0.3。单体蛋白的折叠模式涉及三个结构相似的结构域，D1至D3，它们通过假C3对称性相关，但缺乏可检测的序列相似性（图1b和支持信息，图S1）。相对于域D1，域D2和D3看起来分别旋转了大约100 °和140 °的角度。每个结构域以一个保守的半胱氨酸残基开始，该半胱氨酸残基突出到蛋白质中心的空腔中，在那里它与[3Fe-4S]簇的一个相应的铁原子配位。该集群似乎是倾斜相对于pseudotrigonal轴的apoprotein约208。三角对称的[3Fe-4S]簇位于中心腔的疏水口袋中，其由位于D1（G14和V15）、D2（P97和V99）、D3（A199）以及C-末端（F302和P305）上的残基形成，其稳定了各个结构域的排列。此外，D2中C96的亚甲基部分向内翻转，产生原子Fe 2的额外疏水屏蔽（参见图2）。位于中心腔内部的残留电子密度被确定为无机二磷酸盐（PPi;参见支持性
Eukaryotes and most prokaryotes require isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) as biosynthetic precursors of terpenes. Whereas animals generate these essential metabolites via the mevalonate pathway,[1] many human pathogens including Plasmodium falciparum and Mycobacterium tuberculosis are known to use the more recently identified non-mevalonate pathway, which is a potential target for drug development.[2–4] The final step of this pathway is catalyzed by IspH protein, which generates a mixture of IPP and DMAPP by reductive dehydration of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate(HMBPP, Figure 1 a).[5–11] Recently, Rekittke et al. described the first X-ray structure of IspH protein from the hyperthermophilic eubacterium Aquifex aeolicus in its open state.[12] Herein, we report the crystal structure of the IspH protein from Escherichia coli [11] in its closed conformation, which serves as basis for a detailed discussion of the catalytic pathway. Recombinant E. coli IspH protein (comprising an N-terminal His6 fusion tag) was purified and crystallized under anaerobic conditions. Its structure was determined to a resolution of 1.8 by single-wavelength anomalous diffraction methods. Three iron sites per protein unit were localized in the anomalous difference Patterson map and were used for phasing. Successive rounds of model building and refinement afforded a well-defined electron density for the entire IspH molecule except for the N-terminal His6 tag and five C-terminal amino acid residues (Rfree= 23.8%, SupportingInformation, Table S2). The root mean square (rms) deviation between the Cα positions of the two protein molecules in the asymmetric unit is less than 0.3. The folding pattern of the monomeric protein involves three structurally similar domains, D1 to D3, which are related by pseudo-C3 symmetry but are devoid of detectable sequence similarity (Figure 1b and Supporting Information, FigureS1). Relative to domain D1, domains D2 and D3 appear rotated by angles of approximately 1008 and 1408, respectively. Each domain starts with a conserved cysteine residue that protrudes into a cavity at the center of the protein where it coordinates one respective iron atom of a [3Fe-4S] cluster. The cluster appears to be tilted relative to the pseudotrigonal axis of the apoprotein by about 208. The trigonal symmetric [3Fe-4S] cluster is located in a hydrophobic pocket of the central cavity, which is formed by residues located on D1 (G14 and V15), D2 (P97 and V99), D3 (A199) as well as the C-terminus (F302 and P305), which stabilizes the arrangement of the individual domains. Furthermore, the methylene moiety of C96 in D2 is turned inward generating additional hydrophobic shielding of atom Fe2 (see Figure 2). Residual electron density located inside the central cavity was identified as inorganic diphosphate (PPi; see Supporting