Department of Chemistry, Rutgers University, Newark, NJ 07102.
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Proc Natl Acad Sci U S A. 2017 Aug 29;114(35):9355-9360. doi: 10.1073/pnas.1619981114. Epub 2017 Aug 14.
The enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) is a key enzyme in the methylerythritol 4-phosphate pathway and is a target for the development of antibiotics, herbicides, and antimalarial drugs. DXPS catalyzes the formation of 1-deoxy-d-xylulose 5-phosphate (DXP), a branch point metabolite in isoprenoid biosynthesis, and is also used in the biosynthesis of thiamin (vitamin B) and pyridoxal (vitamin B). Previously, we found that DXPS is unique among the superfamily of thiamin diphosphate (ThDP)-dependent enzymes in stabilizing the predecarboxylation intermediate, C2-alpha-lactyl-thiamin diphosphate (LThDP), which has subsequent decarboxylation that is triggered by d-glyceraldehyde 3-phosphate (GAP). Herein, we applied hydrogen-deuterium (H/D) exchange MS (HDX-MS) of full-length DXPS to provide a snapshot of the conformational dynamics of this enzyme, leading to the following conclusions. () The high sequence coverage of DXPS allowed us to monitor structural changes throughout the entire enzyme, including two segments (spanning residues 183-238 and 292-317) not observed by X-ray crystallography. () Three regions of DXPS (spanning residues 42-58, 183-199, and 278-298) near the active center displayed both EX1 (monomolecular) and EX2 (bimolecuar) H/D exchange (HDX) kinetic behavior in both ligand-free and ligand-bound states. All other peptides behaved according to the common EX2 kinetic mechanism. () The observation of conformational changes on DXPS provides support for the role of conformational dynamics in the DXPS mechanism: The closed conformation of DXPS is critical for stabilization of LThDP, whereas addition of GAP converts DXPS to the open conformation that coincides with decarboxylation of LThDP and DXP release.
1-脱氧-D-木酮糖 5-磷酸合酶（DXPS）是甲基赤藓醇 4-磷酸途径中的一种关键酶，也是抗生素、除草剂和抗疟药物开发的靶标。DXPS 催化 1-脱氧-D-木酮糖 5-磷酸（DXP）的形成，DXP 是异戊烯基生物合成的分支点代谢物，也用于硫胺素（维生素 B）和吡哆醛（维生素 B）的生物合成。以前，我们发现 DXPS 在硫胺素二磷酸（ThDP）依赖性酶的超家族中是独特的，它能稳定预脱羧中间产物 C2-α-乳酰硫胺素二磷酸（LThDP），随后由甘油醛 3-磷酸（GAP）触发脱羧。在此，我们应用全长 DXPS 的氢氘（H/D）交换 MS（HDX-MS）提供了该酶构象动力学的快照，得出以下结论。()DXPS 的高序列覆盖率使我们能够监测整个酶的结构变化，包括 X 射线晶体学未观察到的两个片段（跨越残基 183-238 和 292-317）。()DXPS 的三个区域（跨越残基 42-58、183-199 和 278-298）靠近活性中心，在配体结合和非配体结合状态下均显示出单体（EX1）和双体（EX2）H/D 交换（HDX）动力学行为。所有其他肽段都遵循常见的 EX2 动力学机制。()在 DXPS 上观察到的构象变化为 DXPS 机制中构象动力学的作用提供了支持：DXPS 的封闭构象对于 LThDP 的稳定至关重要，而 GAP 的加入将 DXPS 转化为开放构象，与 LThDP 和 DXP 的释放同时发生脱羧。
