title: Crystallization and Properties of NAD-Dependent D-Sorbitol Dehydrogenase from Gluconobacter suboxydans IFO 3257.
abstract: NAD-dependent D-sorbitol dehydrogenase (EC 1.1.1.14) was crystallized from the cytosolic fraction of Gluconobacter suboxydans IFO 3257. This is the first example of the enzyme crystallized from acetic acid bacteria. The enzyme catalyzed oxidoreduction between D-sorbitol and D-fructose in the presence of NAD or NADH. The crystalline enzyme showed a single sedimentation peak in analytical ultracentrifugation, giving an apparent sedimentation constant of 5.1s. Gel filtration on a Sephadex G-200 column gave the molecular mass of 98 kDa for the enzyme, which dissociated into 26-kDa subunits on SDS-PAGE, indicating that the enzyme is composed of four identical subunits. Oxidation of D-sorbitol to D-fructose and xylitol to D-xylulose predominated in the presence of NAD at the optimum pH of 9.5-10.0. Reductions of D-fructose to D-sorbitol and D-xylulose to xylitol were also observed in the presence of NADH with the optimum pH around 6.0. The relative rate of D-fructose reduction was about one-fourth of that of D-sorbitol oxidation. NADP and NADPH were inert for the both reactions. Since the reation rate in D-sorbitol oxidation predominated over D-fructose reduction at some alkaline pH, the enzyme could be available for direct enzymatic measurement of D-sorbitol. Even in the presence of a large excess of D-glucose and other substances, reduction of NAD to NADH was highly specific and stoichiometric to the D-sorbitol oxidized.
authors.affiliation: a Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University.
authors.forename: O
authors.initials: O
authors.lastname: Adachi
authors.affiliation: a Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University.
authors.forename: H
authors.initials: H
authors.lastname: Toyama
authors.affiliation: b Department of Microbiology, Faculty of Science, Kasetsart University.
authors.forename: G
authors.initials: G
authors.lastname: Theeragool
authors.affiliation: b Department of Microbiology, Faculty of Science, Kasetsart University.
authors.forename: N
authors.initials: N
authors.lastname: Lotong
authors.affiliation: a Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University.
authors.forename: K
authors.initials: K
authors.lastname: Matsushita
keywords: Gluconobacter suboxydans; NAD-dependent D-sorbitol dehydrogenase; acetic acid bacteria; fructose reductase; sorbitol dehydrogenase