Structures of Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase provide new insights into catalysis
Structures of Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase provide new insights into catalysis
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DOI：
10.1074/jbc.m701935200
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发表时间：
2007-07-06
影响因子：
4.8
通讯作者：
Jones, T. Alwyn
中科院分区：
文献类型：
--
作者：
Henriksson, Lena M.;Unge, Torsten;Jones, T. Alwyn
二磷酸异戊烯基是各种类异戊二烯的前体，对所有生物体都是必不可少的。甲羟戊酸在人体中通过甲羟戊酸途径产生，但在植物、原生动物和许多细菌中通过另一种途径产生。1-脱氧-d -木醛糖-5-磷酸还原异构酶催化非甲羟酸途径的第二步，包括nadph依赖性重排和1-脱氧-d -木醛糖-5-磷酸还原成2- c -甲基-d -赤藓糖醇4-磷酸。利用不同的途径，结合已报道的酶的重要性，使还原异构酶成为药物设计的一个非常有希望的目标。在这里，我们展示了结核分枝杆菌1-脱氧-d -木糖5-磷酸还原异构酶的几个高分辨率结构，代表了野生型和突变型酶与Mn2+， NADPH和已知抑制剂fosmidomycin的各种复合物。不对称单元对应于生物同型二聚体。虽然晶体接触稳定了B分子的开放活性位点，但A分子显示出封闭的构象，根据结合的配体有一些差异。一项用磷霉素进行的抑制研究估计IC50值为80 nM。双突变酶（D151N/E222Q）失去了结合金属的能力，因此也失去了活性。我们的结构信息与分子动力学模拟和自由能计算相结合，为新抑制剂的设计提供了框架，并为反应机理提供了新的见解。fosmidomycin与金属离子结合的构象与先前发表的结构不同，表明在催化过程中不需要中间体的重排。
Isopentenyl diphosphate is the precursor of various isoprenoids that are essential to all living organisms. It is produced by the mevalonate pathway in humans but by an alternate route in plants, protozoa, and many bacteria. 1-Deoxy-D-xylulose-5-phosphate reductoisomerase catalyzes the second step of this non-mevalonate pathway, which involves an NADPH-dependent rearrangement and reduction of 1-deoxy-D-xylulose 5-phosphate to form 2-C-methyl-D-erythritol 4-phosphate. The use of different pathways, combined with the reported essentiality of the enzyme makes the reductoisomerase a highly promising target for drug design. Here we present several high resolution structures of the Mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate reductoisomerase, representing both wild type and mutant enzyme in various complexes with Mn2+, NADPH, and the known inhibitor fosmidomycin. The asymmetric unit corresponds to the biological homodimer. Although crystal contacts stabilize an open active site in the B molecule, the A molecule displays a closed conformation, with some differences depending on the ligands bound. An inhibition study with fosmidomycin resulted in an estimated IC50 value of 80 nM. The double mutant enzyme (D151N/E222Q) has lost its ability to bind the metal and, thereby, also its activity. Our structural information complemented with molecular dynamics simulations and free energy calculations provides the framework for the design of new inhibitors and gives new insights into the reaction mechanism. The conformation of fosmidomycin bound to the metal ion is different from that reported in a previously published structure and indicates that a rearrangement of the intermediate is not required during catalysis.