Structural Analysis of Mechanism and Regulation of Glutamine Amidotransferases

谷氨酰胺酰胺转移酶机制和调控的结构分析

• 批准号: 7910122
• 负责人: Amber Marie Smith
• 金额: $ 3.24万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-01 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7910122
• 关键词: Active Sites; Affinity; Amidohydrolases; Amino Acids; Amino Sugars; Ammonia; Antibiotics; Binding; C-terminal; CTP synthase; Catalysis; Coenzymes; Complex; Cytosine; Family; Glutaminase; Glutamine; Goals; Guanosine Triphosphate; Homologous Gene; Hydrolysis; Lead; Ligase; Metals; Modeling; Multienzyme Complexes; Mutation; N-terminal; Nitrilase; Organism; Pathway interactions; Proteins; Purines; Pyrimidine Nucleotides; Regulation; Role; System; Tail; Triad Acrylic Resin; Variant; amidase; cancer therapy; divalent metal; inorganic phosphate; insight; interest; mutant; protein complex; public health relevance; purine; small molecule; tripolyphosphate

DESCRIPTION (provided by applicant): The glutamine amidotranferases (GATs) are responsible for the hydrolysis of glutamine to produce ammonia for a diverse array of biosynthetic pathways. Thus far, there are 16 known GATs, all of which are modular and classified by their glutaminase domains. These domains are from four different ancestral groups: N-terminal nucleophile GATs (Ntn), Triad GATs, amidase GAT and Nitrilase GAT. In addition to their glutaminase domains, GATs have a second active site within their synthase domains. In this active site the ammonia produced from the glutaminase domain is combined with acceptor substrates for the synthesis of amino acids, purine and pyrimidine nucleotides, amino sugars and coenzymes. The progression of catalysis from the glutaminase domain to the synthase domain is highly regulated by substrate binding and the mechanism by which this regulation is achieved is GAT dependent. Given their importance in biosynthetic pathways, they are targets for potential antibiotic and anticancer therapies. Furthermore, they provide a model to study multi-step catalysis with a hierarchy of regulation. In this study we are interested in investigating the structural changes induced by either binding of the acceptor substrates or binding of a non-substrate small molecule and which specific residues propagate the conformational changes that are essential to the mechanism. Although all GATs share the ability to hydrolyze glutamine, their synthetase domains vary. This variation has lead to variations in how each GAT responds to the binding of their substrates. To expose which residues are critical to GATs from different ancestral groups three different GATs (pyridoxial 5'phosphate synthase from G. stearothemophilus, and GatCAB and cytosine triphosphate synthetase from A. aeolius) will be co-crystallized with their substrates in order to capture snapshots of their mechanisms. In aim 1 an inactive PLP synthase mutant will be co-crystallized with its substrates. It is anticipated that this inactivating mutation will order the C-terminal tail of the PdxS subunit. A region that is essential to the complex's function. This will demonstrate how multi-subunit GATs communicate between subunits. Aim 2 will focus on the role of the two divalent metals bound within GatCAB and how these metals are used to spatially orient all its substrates. Aim 3 will identify why CTP synthetase uses a small molecule, GTP, instead of its substrates to induce conformational changes that enhance activity. CTP synthetase from A. aeolius is unique compared with its homologs from other organisms due to its significantly higher affinity for GTP. Understanding the mechanism behind these complex enzymes will provide a paradigm for structural studies for other multi-modular systems. PUBLIC HEALTH RELEVANCE: Glutamine amidotransferases (GATs) are large, diverse modular proteins associated with many biologically important pathways. The goal of this project is to elucidate the conformational changes induced by substrate binding or the binding of a small molecule within this family of protein complexes. This will provide insight into the mechanisms behind modular proteins with specific levels of regulation.

描述（由申请方提供）：谷氨酰胺酰胺转移酶（GAT）负责水解谷氨酰胺，以产生用于多种生物合成途径的氨。到目前为止，有16个已知的GAT，所有这些都是模块化的，并根据其转氨酶结构域进行分类。这些结构域来自四个不同的祖先组：N-末端亲核GAT（Ntn）、三联体GAT、酰胺酶GAT和腈水解酶GAT。除了它们的转氨酶结构域之外，GAT在它们的合酶结构域内具有第二活性位点。在该活性位点中，由丙氨酸氨基酶结构域产生的氨与受体底物结合，用于合成氨基酸、嘌呤和嘧啶核苷酸、氨基糖和辅酶。从转氨酶结构域到合酶结构域的催化进程受到底物结合的高度调节，并且实现这种调节的机制是GAT依赖性的。鉴于它们在生物合成途径中的重要性，它们是潜在抗生素和抗癌疗法的靶点。此外，他们提供了一个模型来研究多步催化与层次的监管。在这项研究中，我们感兴趣的是调查的受体底物的结合或非底物小分子的结合诱导的结构变化和特定的残基传播的构象变化是必不可少的机制。虽然所有的GAT都具有水解谷氨酰胺的能力，但它们的合成酶结构域各不相同。这种变化导致每个GAT如何响应其底物的结合的变化。为了揭示哪些残基对来自不同祖先群体的GAT是关键的，三种不同的GAT（来自G. stearothemophilus的GatCAB和胞嘧啶三磷酸合成酶。aeolius）将与它们的基底共结晶，以便捕获它们的机制的快照。在目的1中，失活的PLP合酶突变体将与其底物共结晶。预期该失活突变将使PdxS亚基的C末端尾部有序化。对复合体功能至关重要的区域。这将演示多亚基GAT如何在亚基之间通信。目标2将集中在GatCAB内结合的两种二价金属的作用，以及这些金属如何用于空间定向其所有基板。目的3将确定为什么CTP合成酶使用小分子GTP而不是其底物来诱导增强活性的构象变化。CTP合成酶。aeolius与其他生物的同源物相比是独特的，因为它对GTP的亲和力显著更高。了解这些复杂酶背后的机制将为其他多模块系统的结构研究提供范例。 公共卫生相关性：谷氨酰胺酰胺转移酶（GATs）是与许多生物学重要途径相关的大的、多样的模块蛋白。本项目的目标是阐明底物结合或小分子结合引起的构象变化，在这个家庭的蛋白质复合物。这将提供深入了解具有特定调节水平的模块化蛋白质背后的机制。