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標題: Mutational analysis of feedback inhibition and catalytic sites of prephenate dehydratase from Corynebacterium glutamicum
作者: Hsu, S.K.
Lin, L.L.
Lo, H.H.
Hsu, W.H.
關鍵字: prephenate dehydratase;site-directed mutagenesis;feedback inhibition;catalytic activity;Corynebacterium glutamicum;escherichia-coli k-12;cation-pi interaction;chorismate mutase;brevibacterium-flavum;active-site;phea-gene;proteins;phenylalanine;mutagenesis;mechanism
Project: Archives of Microbiology
期刊/報告no:: Archives of Microbiology, Volume 181, Issue 3, Page(s) 237-244.
Prephenate dehydratase is a key regulatory enzyme in the phenylalanine-specific pathway of Corynebacterium glutamicum. PCR-based random mutagenesis and functional complementation were used to screen for m-fluorophenylalanine (mFP)-resistant mutants. Comparison of the amino acid sequence of the mutant prephenate dehydratases indicated that Ser-99 plays a role in the feedback regulation of the enzyme. When Ser-99 of the wild-type enzyme was replaced by Met, the specific activity of the mutant enzyme was 30% lower than that of the wild-type. The Ser99Met mutant was active in the presence of 50 muM phenylalanine, whereas the wild-type enzyme was not. The functional roles of the eight conserved residues of prephenate dehydratase were investigated by site-directed mutagenesis. Glu64Asp substitution reduced enzyme activity by 15%, with a 4.5- and 1.7-fold increase in K-m and k(cat) values, respectively. Replacement of Thr-183 by either Ala or Tyr resulted in a complete loss of enzyme activity. Substitution of Arg-184 with Leu resulted in a 50% decrease of enzyme activity. The specific activity for Phe185Tyr was more than 96% lower than that of the wild-type, and the K-m value was 26-fold higher. Alterations in the conserved Asp-76, Glu-89, His-115, and Arg-236 residues did not cause a significant change in the K-m and k(cat) values. These results indicated that Glu-64, Thr-183, Arg-184, and Phe-185 residues might be involved in substrate binding and/or catalytic activity.
ISSN: 0302-8933
DOI: 10.1007/s00203-004-0649-5
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