Feedback Inhibition of Chorismate Mutase/Prephenate Dehydrogenase (TyrA) of Escherichia coli : Generation and Characterization of Tyrosine-Insensitive Mutants

Abstract

ABSTRACT In order to get insights into the feedback regulation by tyrosine of the Escherichia coli chorismate mutase/prephenate dehydrogenase (CM/PDH), which is encoded by the tyrA gene, feedback-inhibition-resistant (fbr) mutants were generated by error-prone PCR. The tyrA fbr mutants were selected by virtue of their resistance toward m -fluoro- d , l -tyrosine, and seven representatives were characterized on the biochemical as well as on the molecular level. The PDH activities of the purified His 6 -tagged TyrA proteins exhibited up to 35% of the enzyme activity of TyrA WT , but tyrosine did not inhibit the mutant PDH activities. On the other hand, CM activities of the TyrA fbr mutants were similar to those of the TyrA WT protein. Analyses of the DNA sequences of the tyrA genes revealed that tyrA fbr contained amino acid substitutions either at Tyr263 or at residues 354 to 357, indicating that these two sites are involved in the feedback inhibition by tyrosine.