A single Ser-180 mutation desensitizes feedback inhibition of the phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthetase in Escherichia coli.
The Escherichia coli phenylalanine-sensitive DAHP (3-deoxy-D-arabino-heptulosonate 7-phosphate) synthetase (aroG product) is one of the DAHP synthetase isozymes that catalyze the first committed step in the biosynthesis of aromatic amino acids and vitamins. Through target-directed mutagenesis of the cloned aroG on a plasmid vector, followed by screening of phenylalanine-resistant colonies, we isolated a clone (pG908) showing feedback-insensitive mutation of DAHP synthetase. The mutations were identified as a T-->A mutation at nucleotide 22, and a C-->T mutation at nucleotide 539, causing a Leu-8 to Ile-8 mutation and a Ser-180 to Phe-180 substitution, respectively. The resulting enzyme exhibited comparable enzymatic activity to the wild type, but the degree of feedback inhibition had declined from approximately 60% to less than 10% in the presence of 20 mM phenylalanine in the assay medium. Replacement of Ile-8 by Leu, and substitution of Phe-180 with Ser, Asn and Cys, using site-directed mutagenesis, demonstrated that Ser-180 is a critical residue in the feedback inhibition of AroG. The result suggests that the major role played by Ser-180 may not involve a simple charge-size effect in the Phe-binding site of the enzyme molecule, but rather may involve more complicated molecular interactions occurring in the feedback inhibition mechanisms.
Ger
Y M
YM
Division of Microbiology, Development Center for Biotechnology, Taipei, Taiwan.
Chen
S L
SL
Chiang
H J
HJ
Shiuan
D
D
D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D004791:Enzyme Inhibitors / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000494:pharmacology; D012694:Serine / Q000737:chemistry*
D004791:Enzyme Inhibitors; D012694:Serine; D010649:Phenylalanine; D010723:3-Deoxy-7-Phosphoheptulonate Synthase