The structure of Mycobacteria 2C-methyl-D-erythritol-2,4-cyclodiphosphatesynthase, an essential enzyme, provides a platform for drug discovery
The structure of Mycobacteria 2C-methyl-D-erythritol-2,4-cyclodiphosphatesynthase, an essential enzyme, provides a platform for drug discovery
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DOI：
10.1186/1472-6807-7-68
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发表时间：
2007-10-23
影响因子：
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通讯作者：
Hunter, William N.
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文献类型：
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作者：
Buetow, Lori;Brown, Amanda C.;Hunter, William N.
背景资料：结核病的流行、这种疾病需要长期和昂贵的治疗以及抗药性的增加表明迫切需要新的治疗方法。类异戊二烯前体生物合成的1-脱氧-D-木酮糖-5-磷酸途径是一个有吸引力的化疗靶点，因为它存在于许多病原体中，包括结核分枝杆菌，并且在人类中不存在。为了支持未来的药物开发，重要的是要评估在这一生物合成途径中的酶是必不可少的实际pathogenes.Results的特征：这一途径的第五个酶，由ispF编码，是2C-甲基-D-β-2，4-环二磷酸合酶（IspF）。采用两步重组策略构建了M.结核病，但只有野生型双交换菌株被分离。当在整合质粒上提供第二个功能性拷贝时，染色体拷贝可以被删除，证明ispF在测试条件下是必需基因，从而证实其作为药物靶标的潜力。我们尝试了M.结核杆菌酶（MtlspF），但未能获得晶体。相反，我们分析了直向同源物M。在2.2埃分辨率下，具有73%氨基酸序列同一性的耻垢病IspF（MslspF）。高水平的序列保守性在活性位点中及其周围特别明显。MslspF是在其中心具有疏水空腔的三聚体，其含有与含二磷酸盐的类异戊二烯一致的密度。由两个亚基产生的活性位点包含刚性CDP-Zn 2+结合口袋，其具有柔性环以定位底物的2C-甲基-D-Zn 2+部分。序列结构的比较表明，活性位点和与配体的相互作用是高度保守的。结论：我们的研究遗传验证MtlspF作为一个治疗靶点，并提供了一个模型系统的结构为基础的配体设计。
Background: The prevalence of tuberculosis, the prolonged and expensive treatment that this disease requires and an increase in drug resistance indicate an urgent need for new treatments. The 1-deoxy-D-xylulose5-phosphate pathway of isoprenoid precursor biosynthesis is an attractive chemotherapeutic target because it occurs in many pathogens, including Mycobacterium tuberculosis, and is absent from humans. To underpin future drug development it is important to assess which enzymes in this biosynthetic pathway are essential in the actual pathogens and to characterize them.Results: The fifth enzyme of this pathway, encoded by ispF, is 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (IspF). A two-step recombination strategy was used to construct ispF deletion mutants in M. tuberculosis but only wild-type double crossover strains were isolated. The chromosomal copy could be deleted when a second functional copy was provided on an integrating plasmid, demonstrating that ispF is an essential gene under the conditions tested thereby confirming its potential as a drug target. We attempted structure determination of the M. tuberculosis enzyme (MtlspF), but failed to obtain crystals. We instead analyzed the orthologue M. smegmatis IspF (MslspF), sharing 73% amino acid sequence identity, at 2.2 angstrom resolution. The high level of sequence conservation is particularly pronounced in and around the active site. MslspF is a trimer with a hydrophobic cavity at its center that contains density consistent with diphosphate-containing isoprenoids. The active site, created by two subunits, comprises a rigid CDP-Zn2+ binding pocket with a flexible loop to position the 2C-methyl-D-erythritol moiety of substrate. Sequence-structure comparisons indicate that the active site and interactions with ligands are highly conserved.Conclusion: Our study genetically validates MtlspF as a therapeutic target and provides a model system for structure-based ligand design.