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

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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.