Mechanistic and Structural Insights into the specificity and Biological Functions of E. coli HAD superfamily phosphatase HAD4/YihX

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Mitochondria are dynamic organelles undergoing constant changes during the lifetime of a cell. Mitochondria contain multiple copies of circular mitochondrial DNA (mtDNA). To maintain a sustainable mtDNA copy number, a constant supply of dNTPs is essential because mtDNA replicates in a manner independent of the cell cycle. Any limitation of one or more dNTPs will stall mtDNA synthesis and result in mtDNA depletion, causing mitochondrial disease. Mitochondrial Thymidine Kinase 2 (TK2) catalyses transfer of a gamma-phosphate group from ATP to the 5'-hydroxyl group of thymidine, deoxycytidine, or deoxyuridine to form their 5'-mono-phosphates. Deficiency in TK2 activity due to genetic mutation causes devastating mitochondrial DNA depletion syndrome (MDS), affecting mainly liver and skeletal muscle. Per contra active TK2 in human tumour cells can reduce effectiveness of the anti-cancer drug gemcitabine by phosphorylating deoxycytidine which eventually leads to the generation of dCTP. TK2 is thus proven to be a drug target for development of both activators and inhibitors.There is a fundamental lack of structural information about TK2, which is a real barrier to a clear understanding of its biological function and targeted drug design. We will use a powerful set of chembiological techniques, namely NMR and x-ray crystallography, in combination with the utilisation of inorganic and synthetic bisubstrate analogues to perform functional and structural characterization of TK2, and thereby significantly advance knowledge of this drugable mitochondrial enzyme. The aim of this project is to deliver much needed protein structures to resolve just how TK2 catalyses the phosphorylation of thymidine, and to deliver the first structures of TK2 with proven inhibitors, thereby validating chemical inhibition as a viable cancer therapeutic strategy.This highly multidisciplinary project should achieve the following objectives:1. Molecular biology, for cloning and mutating the gene for TK2, and for producing and purifying human TK2. 2. Kinetic analyses, for measuring catalytic parameters and negative cooperativity of TK2.3. Single and multidimensional Nuclear Magnetic Resonance (NMR) for assigning key residues in structures and screening for bisubstrate ligand binding. 19F NMR for monitoring formation of metal fluoride transition state analogues of the kinase.4. X-ray crystallography for generating atomic-resolution protein structures with relevant substrates or inhibitors bound.The proposed research is to investigate a key enzyme whose dysfunctionality leads to multiple mitochondrial diseases affecting patients' wellbeing, while functional enzyme reduces the effectiveness of antimetabolite cancer chemotherapeutic drugs. The biochemical and structural outcomes from this project will lead to high impact publications. Based on these much-needed findings, new structure-based activators and inhibitors will be designed and synthesised in an independent research grant in collaboration with Dr. Youcef Mehellou's research group in Pharmacy and Pharmaceutical Sciences, Cardiff University.