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（由申请人提供）：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。迄今为止，已知的 GAT 共有 16 种，所有这些 GAT 都是模块化的，并按其谷氨酰胺酶结构域进行分类。这些结构域来自四个不同的祖先组：N 端亲核体 GAT (Ntn)、三联体 GAT、酰胺酶 GAT 和腈水解酶 GAT。除了谷氨酰胺酶结构域外，GAT 的合酶结构域内还有第二个活性位点。在这个活性位点，谷氨酰胺酶结构域产生的氨与受体底物结合，合成氨基酸、嘌呤和嘧啶核苷酸、氨基糖和辅酶。从谷氨酰胺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目标。此外，他们提供了一个模型来研究具有层次调节的多步催化。在这项研究中，我们有兴趣研究由受体底物的结合或非底物小分子的结合引起的结构变化，以及哪些特定残基传播对该机制至关重要的构象变化。尽管所有 GAT 都具有水解谷氨酰胺的能力，但它们的合成酶结构域有所不同。这种变化导致每个 GAT 对底物结合的反应方式发生变化。为了揭示哪些残基对来自不同祖先群体的 GAT 至关重要，三种不同的 GAT（来自 G. starothemophilus 的吡哆醛 5' 磷酸合酶，以及来自 A. aeolius 的 GatCAB 和胞嘧啶三磷酸合成酶）将与其底物共结晶，以便捕获快照他们的机制。在目标 1 中，无活性的 PLP 合酶突变体将与其底物共结晶。预计这种失活突变将使 PdxS 亚基的 C 末端尾部排序。对于综合体功能至关重要的区域。这将演示多子单元 GAT 如何在子单元之间进行通信。目标 2 将重点关注 GatCAB 中结合的两种二价金属的作用，以及如何使用这些金属对所有底物进行空间定向。目标 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构象变化，从而增强活性。与来自其他生物体的同源物相比，来自风神杆菌的 CTP 合成酶是独特的，因为它对 GTP 的亲和力显着更高。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 (GAT) 是一种大型、多样化的模块蛋白，与许多重要的生物学途径相关。该项目的目标是阐明该蛋白质复合物家族中底物结合或小分子结合引起的构象变化。这将有助于深入了解具有特定调节水平的模块化蛋白质背后的机制。