Cloning of m-fluorophenylalanine-resistant gene and mutational analysis of feedback-resistant prephenate dehydratase from Corynebacterium glutamicum.
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.
Chan
M S
MS
Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan.
Hsu
W H
WH
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology; D001483:Base Sequence; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D004252:DNA Mutational Analysis; D017931:DNA Primers; D004352:Drug Resistance, Microbial / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009604:Nitrosoguanidines; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011302:Prephenate Dehydratase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D009604:Nitrosoguanidines; D011994:Recombinant Proteins; C027218:3-fluorophenylalanine; D010649:Phenylalanine; D011302:Prephenate Dehydratase