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KMID : 0380219990320040358
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Journal of Biochemistry and Molecular Biology
1999 Volume.32 No. 4 p.358 ~ p.362
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Mechanism Study of dTDP-D-Glucose 4,6-Dehydratase: General Base in Active Site Domain
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Sohng Jae-Kyung
Noh Hyung-Rae
Yoo Jin-Cheol
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Abstract
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dTDP-D-glucose 4,6-dehydratase as an oxidoreductase catalyzes the conversion of dTDP-D-glucose to dTDP-4-keto-6-deoxy-D-glucose, which is essential for the formation of 6-deoxysugars. dTDP-D-glucose 4,6-dehydratase shows remarkable sterochemical convergence in which displacement of the C-6 hydroxyl group by a C-4 hydrogen proceeds intramolecularly with inversion of configuration. The reaction mechanism is known to be oxidation, dehydration, and reduction by bases mediating proton transfer and NAD+ cofactor. In this study, the bases in the active site domain are proposed to be His-79 and His-300 from a comparison of the peptides of the dehydratase and UDP-D-glucose epimerase. His-79 and His-300 were mutated to prepare the mutants H79L (mutation of histidine to leucine at the 79th amino acid) and H300A (mutation of histidine to alanine at the 300th amino acid) by site-directed mutagenesis. The H79L protein was inactive, showing that His-79 participates in the reaction mechanism.
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KEYWORD
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dTDP-D-glucose 4,6-dehydratase, Mechanism, Site-directed mutagenesis, UDP-D-glucose epimerase
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