Cloning of m-fluorophenylalanine-resistant gene and mutational analysis of feedback-resistant prephenate dehydratase from Corynebacterium glutamicum

Biochemical and Biophysical Research Communications
M S Chan, W H Hsu

Abstract

Corynebacterium glutamicum was mutated by nitrosoguanidine and five m-fluorophenylalanine (mFP)-resistant mutants were isolated. The mutants were resistant to phenylalanine-mediated feedback inhibition of the prephenate dehydratase activity. Cloning and characterization of the mFP-resistant gene revealed that mutant prephenate dehydratase, encoded by the phe A gene, confers the mFP-resistant phenotype upon C. glutamicum. To determine the amino acid residues to which variation may result in the feedback resistance of prephenate dehydratase, the phe A gene was modified by site-directed mutagenesis and the activities of mutant enzymes were assayed in the presence of phenylalanine. The data indicated that Arg-202 and Gly-224 located at the C-terminal region of prephenate dehydratase were important residues regarding the feedback resistance. Variations of these residues rendered the enzyme insensitive to phenylalanine inhibition. The results also suggested that Gly-224 may reside at the entrance of phenylalanine-binding pocket.

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