Biochemical characterization of the TyrR proteins of Escherichia coli and Haemophilus influenzae

The TyrR proteins of E. coli and H. influenzae were studied biochemically. The TyrR protein of E. coli , a multidomain transcription factor containing 513 amino acid residues, was found to have both autokinase activity and phosphatase activity. The phosphatase activity lies in the central domain of the TyrR protein of E. coli . Attempts to find the autokinase domain were not successful but a phosphoryl transfer mechanism was operative between the N terminal domain and the central domain. Both activities were ligand responsive and interconnected. Evidence is provided to show that both the autokinase activity and the phosphatase activity are related to the activation function of the TyrR protein of E. coli. The TyrR protein of H. influenzae is a protein of 318 amino acid residues that has a high degree of amino acid similarity to the central and C terminal domains of the TyrR protein of E. coli . During storage, the TyrR protein of H. influenzae gave rise to two functional domains, a 28-kDa ligand binding domain and an 8-kDa DNA binding domain. The 8-kDa domain and the 28-kDa domain remain associated and were functionally indistinguishable from the intact TyrR protein of H. influenzae. Both fragments can be generated separately and purified to homogeneity. Fragment complementation occurs when the purified 8-kDa domain and 28-kDa domain are mixed. A hydrophobic patch was found to be responsible for the reconstitution of these two domains. NMR structural studies revealed that the 8-kDa domain mainly consists of a helix-turn-helix DNA binding motif. Studies of the 28-kDa domain are ongoing