Mutational analysis of feedback inhibition and catalytic sites of prephenate dehydratase from<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis> |
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Authors: | Shih-Kuang?Hsu Long-Liu?Lin Hsueh-Hsia?Lo Email author" target="_blank">Wen-Hwei?HsuEmail author |
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Institution: | (1) Department of Dental Technology, Chungtai Institute of Health Sciences and Technology, 406 Taichung, Taiwan;(2) Department of Medical Technology, Chungtai Institute of Health Sciences and Technology, 406 Taichung, Taiwan;(3) Department of Applied Chemistry, National Chiayi University, 60083 Chiayi , Taiwan;(4) Institute of Molecular Biology, National Chung Hsing University, 402 Taichung, Taiwan |
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Abstract: | 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 M 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. |
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Keywords: | Prephenate dehydratase Site-directed mutagenesis Feedback inhibition Catalytic activity Corynebacterium glutamicum |
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