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

线粒体是在细胞的生命周期中不断变化的动态细胞器。线粒体含有多个拷贝的环状线粒体DNA(MtDNA)。为了保持可持续的线粒体DNA拷贝数，dNTPs的持续供应是必不可少的，因为mtDNA以一种独立于细胞周期的方式进行复制。任何一个或多个dNTPs的限制都会阻碍mtDNA的合成，导致mtDNA枯竭，导致线粒体疾病。线粒体胸苷激酶2(TK2)催化γ-磷酸基团从三磷酸腺苷转移到胸腺嘧啶核苷、脱氧胞苷或脱氧尿苷的5‘-羟基，形成它们的5’-单磷酸。由于基因突变导致的TK2活性缺陷会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。相反，人类肿瘤细胞中的TK2活性可以通过磷酸化脱氧胞苷而降低抗癌药物吉西他滨的疗效，最终导致dCTP的产生。因此，TK2被证明是开发激活剂和抑制剂的药物靶点。关于TK2的结构信息基本上是缺乏的，这是清楚了解其生物学功能和靶向药物设计的真正障碍。我们将使用一套强大的化学生物学技术，即核磁共振和X射线结晶学，结合使用无机和合成双底物类似物来进行TK2的功能和结构表征，从而大大促进对这种可药物的线粒体酶的了解。这个项目的目的是提供急需的蛋白质结构，以解决TK2如何催化胸苷磷酸化的问题，并提供TK2的第一个结构和经证实的抑制剂，从而验证化学抑制作为一种可行的癌症治疗策略。这一高度多学科的项目应实现以下目标：1.分子生物学，克隆和突变TK2的基因，生产和纯化人TK2。2.动力学分析，测定TK2.3的催化参数和负协同性。一维和多维核磁共振用于结构中关键残基的分配和双底物配体结合的筛选。~(19)F-核磁共振用于监测金属氟化物过渡态类似物的形成。X射线结晶学用于产生具有相关底物或抑制物结合的原子分辨蛋白质结构。拟议的研究旨在研究一种关键酶，其功能障碍会导致多种线粒体疾病影响患者的健康，而功能酶则会降低抗代谢癌症化疗药物的有效性。该项目的生化和结构成果将导致影响很大的出版物。根据这些亟需的发现，新的基于结构的激活剂和抑制剂将在一项独立的研究拨款中设计和合成，该基金将与加的夫大学尤塞夫·梅赫鲁博士的药学和药学研究小组合作。

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.