Structural Analysis of Mechanism and Regulation of Glutamine Amidotransferases

描述(申请人提供)：谷氨酰胺氨基转移酶(GATS)负责谷氨酰胺的水解，为多种生物合成途径产生氨。到目前为止，有16个已知的GAT，所有的都是模块化的，并根据它们的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群：N-末端亲核GATS(NTN)、三联体GATS、酰胺酶GAT和氰化酶GAT。除了它们的谷氨酰胺酶结构域，Gats在它们的合成酶结构域中还有第二个活性部位。在这个活性部位，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。从谷氨酰胺酶区域到合成酶区域的催化过程受到底物结合的高度调控，而实现这一调控的机制依赖于GAT。鉴于它们在生物合成途径中的重要性，它们是潜在的抗生素和抗癌治疗的靶点。此外，它们还提供了一个模型来研究具有层级调控的多步催化反应。在这项研究中，我们感兴趣的是研究受体底物结合或非底物小分子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。尽管所有的Gat都具有水解谷氨酰胺的能力，但它们的合成酶结构域是不同的。这种差异导致了每个GAT对其底物结合的反应方式不同。为了揭示哪些残基对不同祖先群体的GATS是关键的，三种不同的GATT(来自嗜硬脂酸菌的吡哆醇5‘磷酸合成酶和来自Aolius的GatCAB和胞嘧啶三磷酸合成酶)将与它们的底物共结晶，以捕捉其机制的快照。在目标1中，一个失活的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亚单位的C-末端尾部排序。一个对建筑群的功能至关重要的区域。这将演示多子单元Gat如何在子单元之间通信。目标2将侧重于GatCAB内结合的两种二价金属的作用，以及这些金属如何被用来在空间上定位其所有底物。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以增强活性。沙打旺CTP合成酶与其他生物的同源物相比是独特的，因为它对GTP有很高的亲和力。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一个范例。 与公共健康相关：谷氨酰胺氨基转移酶(GATs)是一种大的、多样化的模块化蛋白，与许多重要的生物学途径有关。这个项目的目标是阐明底物结合或这个蛋白质复合体中的小分子结合所引起的构象变化。这将提供对具有特定调控水平的模块蛋白质背后的机制的洞察。

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.