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

线粒体是一种动态的细胞器，在细胞的一生中不断发生变化。线粒体含有多个拷贝的环状线粒体DNA（mtDNA）。为了维持可持续的mtDNA拷贝数，dNTP的持续供应是必不可少的，因为mtDNA以独立于细胞周期的方式复制。一个或多个dNTP的任何限制都会阻止mtDNA合成，导致mtDNA耗尽，引起线粒体疾病。线粒体胸苷激酶2（TK 2）催化γ-磷酸基团从ATP转移到胸苷、脱氧胞苷或脱氧尿苷的5 '-羟基，形成其5'-单磷酸。由于基因突变导致的TK 2活性缺乏导致破坏性的线粒体DNA耗竭综合征（MDS），主要影响肝脏和骨骼肌。人肿瘤细胞中的反式活性TK 2可以通过磷酸化脱氧胞苷而降低抗癌药物吉西他滨的有效性，这最终导致dCTP的产生。因此，TK 2被证明是开发激活剂和抑制剂的药物靶点。关于TK 2的结构信息从根本上缺乏，这是清楚了解其生物学功能和靶向药物设计的真实的障碍。我们将使用一套强大的化学生物学技术，即NMR和X射线晶体学，结合无机和合成的双底物类似物的利用来进行TK 2的功能和结构表征，从而显着推进这种可药用线粒体酶的知识。该项目的目标是提供急需的蛋白质结构，以解决TK 2如何催化胸苷磷酸化，并提供第一个结构的TK 2与已证实的抑制剂，从而验证化学抑制作为一个可行的癌症治疗策略。分子生物学，用于克隆和突变TK 2的基因，以及用于生产和纯化人TK 2。2.动力学分析，用于测量TK 2.3的催化参数和负协同性。单维和多维核磁共振（NMR）用于分配结构中的关键残基和筛选双底物配体结合。用于监测激酶的金属氟化物过渡态类似物的形成的19 F NMR。X射线晶体学用于生成原子分辨率的蛋白质结构与相关底物或抑制剂结合。拟议的研究是调查一种关键酶，其功能障碍导致影响患者健康的多种线粒体疾病，而功能酶降低抗代谢癌症化疗药物的有效性。该项目的生物化学和结构成果将导致高影响力的出版物。基于这些急需的发现，新的基于结构的激活剂和抑制剂将与卡迪夫大学的Youcef Mehellou博士药学和药物科学研究小组合作，在独立的研究资助下设计和合成。

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.