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

在利兹大学和先正达PLC之间为期4年的BBSRC iCASE博士研究生项目中，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。该项目由利兹大学阿斯特伯里结构分子生物学中心的Alex Breeze教授和Frank Soglit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，以进一步阐明非甲羟戊酸途径TPP依赖性酶（包括1-脱氧-D-木酮糖-5-磷酸合酶（DXS））的机制生物学和抑制模式。结果将是对潜在的抑制剂设计策略和机制的进一步理解，这反过来可能导致针对该途径的改进除草剂的开发。尽管属于普遍理解的一类酶，但对DXS及其抑制作用的机理理解仍然相对有限，至少部分是因为结构确定，特别是底物-，催化中间体-，通过X射线晶体学分析产物结合型或通道结合型已被证明是相当具有挑战性的。学生将使用尖端的超高场甲基NMR和氢氘交换质谱（HDX-MS）来建立我们早期的工作。具体来说，我们将转移我们的甲基NMR方法的基础上，E。coliDXS系统与植物直向同源物的同源性，将成为除草剂开发的更相关的模式系统。除了NMR，我们将使用结构MS（HDX-MS），蛋白质的快速光化学氧化（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AlphaFold 2结构模型的分子建模将被用作纳入我们的实验衍生约束的基础。

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.