Membrane-bound Sugar Alcohol Dehydrogenase in Acetic Acid Bacteria catalyzes L-Ribulose Formation and NAD-Dependent Ribitol Dehydrogenase is Independent of the Oxidative Fermentation

• Published in: Bioscience, biotechnology, and biochemistry Vol. 65; no. 1; pp. 115 - 125
• Main Authors: ADACHI, Osao, FUJII, Yoshikazu, ANO, Yoshitaka, MOONMANGMEE, Duangtip, TOYAMA, Hirohide, SHINAGAWA, Emiko, THEERAGOOL, Gunjana, LOTONG, Napha, MATSUSHITA, Kazunobu
• Format: Journal Article
• Language: English
• Published: Tokyo Japan Society for Bioscience, Biotechnology, and Agrochemistry 01.01.2001; Japan Society for Bioscience Biotechnology and Agrochemistry; Oxford University Press
• Subjects: acetic acid bacteria; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; Cell Membrane - chemistry; Cell Membrane - metabolism; Crystallization; Electrophoresis, Polyacrylamide Gel; Fermentation; Fundamental and applied biological sciences. Psychology; Gluconobacter - cytology; Gluconobacter - enzymology; Gluconobacter - metabolism; Gluconobacter suboxydans; Hydrogen-Ion Concentration; Mannitol - metabolism; Mission oriented research; NAD - metabolism; Oxidation-Reduction; oxidative fermentation; pentitol oxidation; Pentoses - metabolism; Physiology and metabolism; Ribitol - metabolism; ribitol dehydrogenase; ribulose; Sorbitol - metabolism; Substrate Specificity; sugar alcohol dehydrogenase; Sugar Alcohol Dehydrogenases - chemistry; Sugar Alcohol Dehydrogenases - isolation & purification; Sugar Alcohol Dehydrogenases - metabolism; Sugar Alcohols - metabolism; Xylitol - metabolism; Acetobacteraceae; Purification; Enzyme; Fermentation; Gluconobacter oxydans; Enzymatic activity; Ribitol 2-dehydrogenase; Substrate specificity; Bacteria; Carbohydrate; Oxidation; Oxidoreductases; Acetic acid bacteria; Physicochemical properties
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.65.115

To identify the enzyme responsible for pentitol oxidation by acetic acid bacteria, two different ribitol oxidizing enzymes, one in the cytosolic fraction of NAD(P)-dependent and the other in the membrane fraction of NAD(P)-independent enzymes, were examined with respect to oxidative fermentation. The cytoplasmic NAD-dependent ribitol dehydrogenase (EC 1.1.1.56) was crystallized from Gluconobacter suboxydans IFO 12528 and found to be an enzyme having 100kDa of molecular mass and 5 s as the sedimentation constant, composed of four identical subunits of 25 kDa. The enzyme catalyzed a shuttle reversible oxidoreduction between ribitol and D-ribulose in the presence of NAD and NADH, respectively. Xylitol and L-arabitol were well oxidized by the enzyme with reaction rates comparable to ribitol oxidation. D-Ribulose, L-ribulose, and L-xylulose were well reduced by the enzyme in the presence of NADH as cosubstrates. The optimum pH of pentitol oxidation was found at alkaline pH such as 9.5-10.5 and ketopentose reduction was found at pH 6.0. NAD-Dependent ribitol dehydrogenase seemed to be specific to oxidoreduction between pentitols and ketopentoses and D-sorbitol and D-mannitol were not oxidized by this enzyme. However, no D-ribulose accumulation was observed outside the cells during the growth of the organism on ribitol. L-Ribulose was accumulated in the culture medium instead, as the direct oxidation product catalyzed by a membrane-bound NAD(P)-independent ribitol dehydrogenase. Thus, the physiological role of NAD-dependent ribitol dehydrogenase was accounted to catalyze ribitol oxidation to D-ribulose in cytoplasm, taking D-ribulose to the pentose phosphate pathway after being phosphorylated. L-Ribulose outside the cells would be incorporated into the cytoplasm in several ways when need for carbon and energy sources made it necessary to use L-ribulose for their survival. From a series of simple experiments, membrane-bound sugar alcohol dehydrogenase was concluded to be the enzyme responsible for L-ribulose production in oxidative fermentation by acetic acid bacteria.