title: Crystallization and Properties of NADPH-Dependent L-Sorbose Reductase from Gluconobacter melanogenus IFO 3294.
abstract: NADPH-Dependent L-sorbose reductase (SORD, synonimously NADP-dependent D-srobitol dehydrogenase) was purified and crystallized for the first time from the cytosolic fraction of Gluconobacter melanogenus IFO 3294. The enzyme catalyzed oxidoreduction between D-sorbitol and L-sorbose in the presence of NADP or NADPH. Affinity chromatography by a Blue-dextran Sepharose 4B column was effective for purifying the enzyme giving about 770-fold purification with an overall yield of more than 50%. The crystalline enzyme showed a single sedimentation peak in analytical ultracentrifugation, giving an apparent sedimentation constant of 3.8 s. Gel filtration on a Sephadex G-75 column gave the molecular mass of 60 kDa to the enzyme, which dissociated into 30 kDa subunit on SDS-PAGE, indicating that the enzyme is composed of 2 identical subunits. Reduction of L-sorbose to D-sorbitol predominated in the presence of NADPH with the optimum pH of 5.0-7.0. Oxidation of D-sorbitol to L-sorbose was observed in the presence of NADP at the optimum pH of 7.0-9.0. The relative rate of L-sorbose reduction was more than seven times higher to that of D-sorbitol oxidation. NAD and NADH were inert for both reactions. D-Fructose reduction in the presence of NADPH did not occur with SORD. Since the reaction rate in L-sorbose reduction highly predominated over D-sorbitol oxidation over a wide pH range, the enzyme could be available for direct enzymatic measurement of L-sorbose. Even in the presence of a large excess of D-glucose and other substances, oxidation of NADPH to NADP was highly specific and stoichiometric to the L-sorbose reduced. Judging from the enzymatic properties, SORD would contribute to the intracellular assimilation of L-sorbose incorporated from outside the cells where L-sorbose is accumulated in huge amounts in the culture medium.
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: Y
authors.initials: Y
authors.lastname: Ano
authors.affiliation: a Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University.
authors.forename: D
authors.initials: D
authors.lastname: Moonmangmee
authors.affiliation: b Department of Chemical and Biological Engineering, Ube National College of Technology.
authors.forename: E
authors.initials: E
authors.lastname: Shinagawa
authors.affiliation: a Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University.
authors.forename: H
authors.initials: H
authors.lastname: Toyama
authors.affiliation: c Department of Microbiology, Faculty of Science, Kasetsart University.
authors.forename: G
authors.initials: G
authors.lastname: Theeragool
authors.affiliation: c 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 melanogenus; L-sorbose reductase; NADP-dependent D-sorbitol dehydrogenase; acetic acid bacteria