Mechanistic understanding and inhibitor design strategies for non-mevalonate pathway TPP-dependent enzymes

在英国利兹大学和先正达公司为期4年的BBSRC iCASE博士学位课程中，学生将研究对农业科学产业重要的植物酶的结构、功能和机制。该项目由利兹大学的Astbury结构分子生物学中心共同监督，由Alex Breeze教授和Frank Sobott教授共同监督，建立在先正达和利兹大学之前的合作基础上，进一步阐明非甲羟戊酸途径tpp依赖酶的机制生物学和抑制模式，包括1-脱氧-d -木酮糖-5-磷酸合成酶（DXS）。结果将是对潜在抑制剂设计策略和机制的更好理解，这反过来可能导致针对这一途径的改进除草剂的开发。尽管DXS是一种被广泛理解的酶，但对DXS及其抑制作用的机制理解仍然相对有限，至少部分原因是通过x射线晶体学确定结构，特别是底物，催化中间体，产物或抑制剂结合形式已被证明是相当具有挑战性的。该学生将使用尖端的超高场甲基核磁共振和氢-氘交换质谱（HDX-MS）来建立我们早期的工作。具体来说，我们将把基于大肠杆菌DXS系统的甲基核磁共振方法转移到来自植物物种的同源物上，这些同源物将成为除草剂抑制剂开发更相关的模型系统。除了核磁共振，我们将使用结构质谱（HDX-MS）、快速光化学氧化蛋白(FPOP)-MS和离子迁移谱(IMS)-MS来绘制底物、催化/过渡态中间体和抑制剂的结合位点和相互作用模式。基于现有PDB结构和AlphaFold2结构模型的分子建模将被用作结合我们实验推导的约束的基础。

In this 4-year BBSRC iCASE PhD studentship between the University of Leeds and Syngenta PLC, the student will investigate the structure, function and mechanism of plant enzymes important to the agriscience industry. The project, which is co-supervised in the Astbury Centre for Structural Molecular Biology, University of Leeds by Profs Alex Breeze and Frank Sobott, builds on previous collaboration between Syngenta and the University of Leeds to further elucidate the mechanistic biology and modes of inhibition ofnon-mevalonate pathway TPP-dependent enzymes including 1-deoxy-D-xylulose-5-phosphate synthase (DXS). The outcome will be an enhanced understanding of potential inhibitor design strategies and mechanisms, that in turn may lead to the development of improved herbicidal agents targeting this pathway.Despite belonging to a generally well-understood class of enzymes, mechanistic understanding of DXS and its inhibition is still relatively limited, at least in part because structure determination, particularly of substrate-, catalytic intermediate-, product- or inhibitor-bound forms by X-ray crystallography has proved to be quite challenging. The student will use cutting-edge ultra-high field methyl NMR and hydrogen-deuteriumexchange mass spectrometry (HDX-MS) to build on our earlier work. Specifically, we willtransfer our methyl NMR approach based on the E. coli DXS system to orthologue(s) fromplant species that will be more relevant model systems for herbicidal inhibitordevelopment. In addition to NMR, we will use structural MS (HDX-MS, fast photochemicaloxidation of proteins (FPOP)-MS and ion-mobility spectrometry- (IMS)-MS to map the binding sites and modes of interaction of substrates, catalytic / transition state intermediates and inhibitors. Molecular modelling based on existing PDB structures and AlphaFold2 structural models will be used as a basis for incorporating our experimentally-derived restraints.