Characterization of a new feedback-resistant 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase AroF of Escherichia coli

Tyrosine feedback-inhibits the 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase isoenzyme AroF of Escherichia coli. Here we show that an As n-8 to Lys-8 substitution in AroF leads to a tyro sine-insensitive DAHP syn thase. This mutant enzyme exhibited similar activities (v = 30-40 U mg(-1)) and substrate affinities (K-m(erythrose-4-phosphate) = 0.5 mM, positive co operativity with respect to phospho(enol)pyruvate) as the wild-type AroF, b ut showed decreased thermostability. An engineered AroF enzyme lacking the seven Ni-terminal residues also was tyrosine-resistant. These results stron gly suggest that the N-terminus of AroF is involved in the molecular intera ctions occurring in the feedback-inhibition mechanism. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. A ll rights reserved.