Novel mutations in the pheA gene of Escherichia coli K-12 which result in highly feedback inhibition-resistant variants of chorismate mutase/prephenate dehydratase.
The bifunctional enzyme chorismate mutase/prephenate dehydratase (EC 5.4.99.5/4.2.1.51), which is encoded by the pheA gene of Escherichia coli K-12, is subject to strong feedback inhibition by L-phenylalanine. Inhibition of the prephenate dehydratase activity is almost complete at concentrations of L-phenylalanine greater than 1 mM. The pheA gene was cloned, and the promoter region was modified to enable constitutive expression of the gene on plasmid pJN302. As a preliminary to sequence analysis, a small DNA insertion at codon 338 of the pheA gene unexpectedly resulted in a partial loss of prephenate dehydratase feedback inhibition. Four other mutations in the pheA gene were identified following nitrous acid treatment of pJN302 and selection of E. coli transformants that were resistant to the toxic phenylalanine analog beta-2-thienylalanine. Each of the four mutations was located within codons 304 to 310 of the pheA gene and generated either a substitution or an in-frame deletion. The mutations led to activation of both enzymatic activities at low phenylalanine concentrations, and three of the resulting enzyme variants displayed almost complete resistance to feedback inhibition of prephenate dehydratase by phenylalanine concentrations up to 200 mM. In all four cases the mutations mapped in a region of the enzyme that has not been implicated previously in feedback inhibition sensitivity of the enzyme.
Nelms
J
J
Biosciences Laboratory, Nutrasweet Research & Development, Mt. Prospect, Illinois 60056.
Edwards
R M
RM
Warwick
J
J
Fotheringham
I
I
D000595:Amino Acid Sequence; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D005246:Feedback; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011302:Prephenate Dehydratase / Q000037:antagonists & inhibitors / Q000235:genetics*; D011401:Promoter Regions, Genetic
D004269:DNA, Bacterial; D011302:Prephenate Dehydratase