Purification and Characterization of D-Glucosaminitol Dehydrogenase from Agrobacterium radiobacter.

DOI Web Site Web Site PubMed 23 References
  • IWAMOTO Ryoko
    Department of Chemistry, Faculty of Science, Nara Women’s University
  • SAKAMOTO Chieko
    Department of Chemistry, Faculty of Science, Nara Women’s University
  • TAMURA Keiko
    Department of Chemistry, Faculty of Science, Nara Women’s University
  • MIKATA Yuji
    Department of Chemistry, Faculty of Science, Nara Women’s University
  • TANAKA Mieko
    Department of Chemistry, Faculty of Science, Nara Women’s University

Bibliographic Information

Other Title
  • Purification and Characterization of D-Glucosaminitol Dehydrogenase from Agrobacterium radiobactor
  • Purification and Characterization of<scp>D</scp>-Glucosaminitol Dehydrogenase from<i>Agrobacterium radiobacter</i>

Search this article

Abstract

  D-Glucosaminitol dehydrogenase, which catalyzes the conversion of D-glucosaminitol to 3-keto-D-glucosaminitol, was purified to apparent homogeneity from extracts of Agrobacterium radiobacter. This organism has constitutively depressed levels of the enzyme but expression of the enzyme is induced by addition of D-glucosamine to the medium. Purification included ammonium sulfate fractionation and chromatography on columns of DEAE-Sephacel, Octyl-Sepharose CL-4B, and Cellulofine. The purified enzyme migrated as a single band, coinciding with dehydrogenase activities specific for D-glucosaminitol and ethanol, when electrophoresed on a 7.5% polyacrylamide gel at pH 8.0. Electrophoresis on a 12.5% PAGE in the presence of 1% SDS also yielded a single band. The enzyme had an apparent molecular mass of 79 kDa, as measured by the pattern of elution from a column of Cellulofine. The results indicated that the enzyme was a dimer of identical (or nearly identical) subunits of 39.5 kDa. D-Glucosaminitol dehydrogenase required NAD+ as a cofactor and used ethanol as the preferred substrate, as well as aliphatic alcohols with 2 to 4 carbon atoms, D-glucosaminitol, D-glucosaminate, DL-allothreonine, glycerol, and erythritol as additional substrates. In 50 mM Tris-HCl buffer (pH 9.0) at 25°C, the Km for D-glucosaminitol, ethanol, and NAD+ were 2.2, 2.0, and 0.08 mM, respectively. The enzyme had a pH optimum of 10 for D-glucosaminitol and 8.5 for ethanol. The enzyme lost substantial activity when treated with pyrazole, with certain reagents that react with sul- fhydryl groups and with Zn2+ ion. The various results together suggest that the enzyme exploits different amino acid residues for the dehydrogenation of ethanol and of D-glucosaminitol.<br>

Journal

References(23)*help

See more

Keywords

Details 詳細情報について

Report a problem

Back to top