PMID:1556102
| Citation |
Yu, Y, Russell, RN, Thorson, JS, Liu, LD and Liu, HW (1992) Mechanistic studies of the biosynthesis of 3,6-dideoxyhexoses in Yersinia pseudotuberculosis. Purification and stereochemical analysis of CDP-D-glucose oxidoreductase. J. Biol. Chem. 267:5868-75 |
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| Abstract |
An NAD(+)-dependent CDP-D-glucose oxidoreductase which catalyzes the first step of the biosynthesis of CDP-ascarylose (CDP-3,6-dideoxy-L-arabino-hexose), converting CDP-D-glucose to CDP-4-keto-6-deoxy-D-glucose, was isolated from Yersinia pseudotuberculosis. A protocol consisting of DEAE-cellulose, Matrex Blue-A, hydroxylapatite, DEAE-Sephadex, Sephadex G-100, and NAD(+)-agarose column chromatography was used to purify this enzyme 6000-fold to homogeneity. This enzyme consists of two identical subunits, each with a molecular weight of 42,500. Using CDP-D-glucose as the substrate, the Km and Vmax of this catalysis were determined to be 222 microM and 8.3 mumols mg-1 min-1, respectively. Unlike most other oxidoreductases of its class which have a tightly bound NAD+, this highly purified CDP-D-glucose oxidoreductase showed an absolute requirement of NAD+ for its activity. Using chemically synthesized (6S)- and (6R)-CDP-D-[4-2H,6-3H]glucose as substrates, a stereochemical analysis showed this enzymatic reaction involves an intramolecular hydrogen migration from C-4 to C-6, and the displacement of C-6 hydroxyl group by the C-4 hydrogen occurs with inversion. Thus, despite the low cofactor affinity, this enzyme undergoes a mechanism consistent with that followed by other members of its type. Such a mechanistic and stereochemical convergency found for all sugar oxidoreductases so far characterized suggests the presence of a common progenitor of this class of enzyme. |
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| Keywords |
Amino Acids/analysis; Carbohydrate Dehydrogenases/isolation & purification; Carbohydrate Dehydrogenases/metabolism; Chromatography, Affinity; Chromatography, Gel; Chromatography, Ion Exchange; Deoxy Sugars/biosynthesis; Deuterium; Glucose/analogs & derivatives; Glucose/chemical synthesis; Hexoses/biosynthesis; Isomerism; Kinetics; Molecular Weight; NAD/metabolism; Nucleoside Diphosphate Sugars/chemical synthesis; Tritium; Yersinia pseudotuberculosis/enzymology |
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