Improving the catalytic activity of a thermostable anthranilate phosphoribosyl transferase by directed evolution

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Enzymes from hyperthermophilic microorganisms are extremely thermostable and catalytically highly active at temperatures close to the boiling point of water. However, most hyperthermophilic enzymes are only marginally active at the optimum growth temperatures of mesophiles. It will be attempted to increase the low activity at 37°C of anthranilate phosphoribosyltransferase from the hyperthermophilic archaeon Sulfolobus solfataricus (sTrpD) by directed evolution. To this end, cells of an auxotrophic Escherichia coli strain lacking a functional trpD gene will be transformed with a strpD gene library that has been generated by random mutagenesis, plated on minimal medium without tryptophan and incubated at 37°C. sTrpD variants with improved catalytic activities compared to the wild-type enzyme will be identified by a faster growth of the transformed cells. The characterisation of the purified variants by multiparameter fluorescence detection (MFD) of single molecules, steady-state enzyme kinetics, stopped-flow techniques, chemical and thermal unfolding experiments, and limited proteolysis will show to what extent and by what mechanisms catalytic activity and conformational stability are coupled. Moreover, MFD will quantitatively describe the extent and the dynamics of the structural changes during catalysis as well as their relation to enzymatic activity. The obtained results will be analysed on the basis of the known X-ray structure of sTrpD.