A novel type of D-mannitol dehydrogenase from Acetobacter xylinum: Occurrence, purification, and basic properties

We purified a novel type of D-mannitol dehydrogenase, which contains a c-type cytochrome and an unknown chromophore in the soluble fraction of an acetic acid bacterium, Acetobacter xylinum KU-1, to homogeneity. The enzyme showed the maximum activity at pH 5 and 40 degrees C. It was stable up to 60 degrees C at pH 6, and was inhibited by Hg2+ and p-quinone (K-i = 0.18 mM). The molecular weight of the enzyme was about 140,000, and those of the subunits were 69,000, 51,000, and 20,000; the enzyme is hetero-trimeric and contained 8 g-atoms of Fe per mole. The alpha-helix was estimated to be about 52.9%. The enzyme catalyzed phenazine methosulfate dependent oxidation of D-mannitol with an apparent K-m of 98 mu M (for D-mannitol) and V-max of 213 mu mol/min/mg. The reduced form of the enzyme showed the absorption maxima at 386, 416, 480, 518, 550, and 586 nm, which are attributable to a c-type cytochrome in the enzyme.