Purification and characterization of D-glucosaminitol dehydrogenase from Agrobacterium radiobacter.
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 degrees C, the K(m) 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 sulfhydryl 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.
Iwamoto
R
R
Department of Chemistry, Faculty of Science, Nara Women's University, Japan.
Sakamoto
C
C
Tamura
K
K
Mikata
Y
Y
Tanaka
M
M
D000595:Amino Acid Sequence; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D000431:Ethanol / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006865:Hydrogenation; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012231:Rhizobium / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D000431:Ethanol; D002237:Carbohydrate Dehydrogenases; C120012:D-glucosaminitol dehydrogenase