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

• DOI: 10.1111/j.1574-6968.2001.tb10795.x
• Publication Date: 2006-01-09T14:36:15Z
• Authors: Ralf Jossek; Johannes Bongaerts; Georg A. Sprenger
• Keywords: info:eu-repo/classification/ddc/570; AroF; Feedback, Physiological; 0301 basic medicine; Sequence Homology, Amino Acid; tyrosine resistance; Temperature; 3-deoxy-D-arabino-heptulosonate synthase; 3. Good health; Kinetics; 03 medical and health sciences; Amino Acid Substitution; Enzyme Stability; Mutation; Escherichia coli; Tyrosine; 3-Deoxy-7-Phosphoheptulonate Synthase; Sugar Phosphates; Amino Acid Sequence; Alleles; feedback-inhibition

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 (K(m)(erythrose-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.