title: Mechanistic studies of the biosynthesis of 3,6-dideoxyhexoses in Yersinia pseudotuberculosis. Purification and stereochemical analysis of CDP-D-glucose oxidoreductase.
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.
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis 55455.
authors.forename: Y
authors.initials: Y
authors.lastname: Yu
authors.forename: R N
authors.initials: RN
authors.lastname: Russell
authors.forename: J S
authors.initials: JS
authors.lastname: Thorson
authors.forename: L D
authors.initials: LD
authors.lastname: Liu
authors.forename: H W
authors.initials: HW
authors.lastname: Liu
mesh_terms: D000596:Amino Acids / Q000032:analysis; D002237:Carbohydrate Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003837:Deoxy Sugars / Q000096:biosynthesis*; D003903:Deuterium; D005947:Glucose / Q000031:analogs & derivatives / Q000138:chemical synthesis; D006601:Hexoses / Q000096:biosynthesis*; D007536:Isomerism; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009702:Nucleoside Diphosphate Sugars / Q000138:chemical synthesis; D014316:Tritium; D015011:Yersinia pseudotuberculosis / Q000201:enzymology*
chemical_list: D000596:Amino Acids; D003837:Deoxy Sugars; D006601:Hexoses; D009702:Nucleoside Diphosphate Sugars; C073881:cytidine diphosphate-glucose; D009243:NAD; D014316:Tritium; D003903:Deuterium; D002237:Carbohydrate Dehydrogenases; C073880:CDP-glucose oxidoreductase; D005947:Glucose