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 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.
Jossek
R
R
Institut für Biotechnologie 1, Forschungszentrum Jülich GambH, Germany.
Bongaerts
J
J
Sprenger
G A
GA
D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000483:Alleles; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D025461:Feedback, Physiological / Q000187:drug effects; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013403:Sugar Phosphates / Q000378:metabolism*; D013696:Temperature; D014443:Tyrosine / Q000378:metabolism / Q000494:pharmacology
D013403:Sugar Phosphates; C013469:3-deoxy-D-arabino-heptulosonate-7-phosphate; D014443:Tyrosine; D010723:3-Deoxy-7-Phosphoheptulonate Synthase