Allosteric communication and subunit interaction specificity in glutamine amidotransferases

谷氨酰胺酰胺转移酶（GATases）是由谷氨酰胺酶和合酶亚基组成的双酶复合物，它们一起催化氮掺入各种生物分子中。每个GAT酶的两个亚基在结构和功能上是偶联的：合酶底物的结合变构刺激谷氨酰胺在谷氨酰胺酶的水解。产生的氨通过分子间通道扩散到合酶，在那里它与“等待”底物反应，生成对每个GAT酶特异的产物。在该项目的第一部分中，我们将使用咪唑甘油磷酸合酶（ImGP-S;丙氨酸氨基酶亚基：HisH;合酶亚基：HisF）来破译在HisH的活性位点通过底物结合到HisF的活性位点来刺激谷氨酰胺水解的分子机制。基于过去资助期的结果，将通过多维NMR、X射线晶体学和掺入非天然氨基酸来比较分析具有变构能力和无变构能力的ImGP-S变体以及显示组成型丙氨酸氨基转移酶活性的变体。一个特别的重点将放在一个保守的组氨酸残基的活性位点的HisH和一个保守的天冬氨酸残基的HisF，这是位于HisH-HisF接口，似乎发挥了至关重要的作用，刺激谷氨酰胺水解。在该项目的第二部分，我们将分析不同GATases内形成特异性精氨酸酶合酶相互作用的结构基础。为此，我们将使用合理的蛋白质设计，将一种可以与两种不同的脱氢酶相互作用的混杂的脱氢酶转化为一种只能与单一合酶相互作用的特异性脱氢酶。最后，我们将使用基于计算机的阳性和阴性蛋白质设计来完全改变转氨酶的相互作用特异性。

Glutamine amidotransferases (GATases) are bi-enzyme complexes consisting of a glutaminase and a synthase subunit that together catalyze the incorporation of nitrogen into various biomolecules. The two subunits of each GATase are structurally and functionally coupled: Binding of the synthase substrate allosterically stimulates the hydrolysis of glutamine at the glutaminase. The generated ammonia diffuses through an intermolecular channel to the synthase where it reacts with the “waiting” substrate to the products that are specific for each GATase. In the first part of the project we will use imidazole glycerol phosphate synthase (ImGP-S; glutaminase subunit: HisH; synthase subunit: HisF) to decipher the molecular mechanisms underlying the stimulation of glutamine hydrolysis at the active site of HisH by substrate binding to the active site of HisF. Based on the results of the past funding period, allosterically competent and allosterically incompetent ImGP-S variants as well as variants showing constitutive glutaminase activity will be comparatively analyzed by multi-dimensional NMR, X-ray crystallography, and the incorporation of a non-natural amino acid. A special emphasis will be put on a conserved histidine residue at the active site of HisH and a conserved aspartate residue of HisF, which is located at the HisH-HisF interface and seems to play an essential role for the stimulation of glutamine hydrolysis. In the second part of the project, we will analyze the structural basis underlying the formation of specific glutaminase-synthase interactions within different GATases. For this purpose, we will use rational protein design to convert a promiscuous glutaminase, which can interact with two different synthases, into a specific glutaminase, which can interact with only a single synthase. Finally, we will use computer-based positive and negative protein design to fully change the interaction specificity of a glutaminase.