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

Tyrosine feedback-inhibits the 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase isoenzyme AroF of Escherichia coli. Here we show that an Asn-8 to Lys-8 substitution in AroF leads to a tyrosine-insensitive DAHP synthase. This mutant enzyme exhibited similar activities (v=30–40 U mg−1) and substrate affinities (Kmerythrose-4-phosphate=0.5 mM, positive cooperativity with respect to phospho(enol)pyruvate) as the wild-type AroF, but showed decreased thermostability. An engineered AroF enzyme lacking the seven N-terminal residues also was tyrosine-resistant. These results strongly suggest that the N-terminus of AroF is involved in the molecular interactions occurring in the feedback-inhibition mechanism.