Mechanistic studies of the biosynthesis of 3,6-dideoxyhexoses in Yersinia pseudotuberculosis. Purification and stereochemical analysis of CDP-D-glucose oxidoreductase

Y. Yu, R. N. Russell, J. S. Thorson, L. D. Liu, H. W. Liu

Research output: Contribution to journalArticlepeer-review

48 Scopus citations

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 K(m) and V(max) of this catalysis were determined to be 222 μM and 8.3 μmol 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.

Original languageEnglish
Pages (from-to)5868-5875
Number of pages8
JournalJournal of Biological Chemistry
Volume267
Issue number9
StatePublished - 1992

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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