Allosteric communication and subunit interaction specificity in glutamine amidotransferases

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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.