Allosteric communication and subunit interaction specificity in glutamine amidotransferases

谷氨酰胺氨基转移酶（GATases）是由谷氨酰胺酶和合成酶亚基组成的双酶复合物，它们共同催化氮进入各种生物分子。每一种谷氨酰胺酶的两个亚基在结构和功能上是偶联的：合成酶底物的结合变构刺激谷氨酰胺酶处谷氨酰胺的水解。生成的氨通过分子间通道扩散到合成酶，在那里它与“等待”的底物反应，生成每种GATase特有的产物。在项目的第一部分，我们将使用咪唑甘油磷酸合成酶（ImGP-S；谷氨酰胺酶亚基：HisH；合成酶亚基：HisF）来破译底物结合HisF活性位点刺激HisH活性位点谷氨酰胺水解的分子机制。基于过去资助期的结果，将通过多维核磁共振、x射线晶体学和非天然氨基酸的掺入，比较分析变构能力和变构能力不足的ImGP-S变体以及具有组成型谷氨酰胺酶活性的变体。重点将放在HisH活性位点的保守组氨酸残基和HisF的保守天冬氨酸残基上，这些残基位于HisH-HisF界面，似乎对刺激谷氨酰胺水解起重要作用。在项目的第二部分，我们将分析不同gatase中特定谷氨酰胺酶合酶相互作用形成的结构基础。为此，我们将利用合理的蛋白质设计，将一种可以与两种不同合酶相互作用的混杂型谷氨酰胺酶转化为一种只与一种合酶相互作用的特异性谷氨酰胺酶。最后，我们将使用基于计算机的阳性和阴性蛋白设计来完全改变谷氨酰胺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.