Keto Dgluconate Dehydrogenase Membrane Bound

2-Keto-D-gluconate ^ 2,5-diketo-D-gluconate

The enzyme catalyzing 2-ketogluconate oxidation, yielding 2,5-diketo-D-gluconate, has been characterized as a flavohemoprotein with three different subunits [99]. From the membrane fraction of Gluconobacter melanogenus IFO 3293, 2-keto-D-gluconate dehydrogenase (KGDH) is solubilized and purified with high yield to a homogeneous state examined by the criteria of electrophoresis and analytical ultracentrifugation. The purified KGDH is homogeneous in analytical ultracentrifugation, with a sedimentation coefficient of 5.9 S, and also in native-PAGE, with a single protein band. After solubilization of the enzyme performed with 2% Na-cholate and 0.2 mol L-1 KCl, two-step column chromatography gave a purified enzyme. The molecular mass was measured to be 133 kDa and SDS-PAGE shows the presence of three different subunits of 61 kDa (flavoprotein), 47 kDa (cytochrome c), and 25 kDa. The flavoprotein contained a covalently bound FAD [93].

Purified KGDH has a characteristic deep rose-red color due to the cytochome component. The typical reduced cytochrome c type absorption spectrum has maxima at 554, 523, and 417 nm. The cytochrome component of the enzyme is reduced on addition of either 2-keto-D-gluconate or sodium dithionite. A

successful purification of KGDH came from the finding of fundamental differences in hydrophobicity between D-gluconate dehydrogenase (GADH) (see Section 1.4.2) and KGDH. KGDH was not solubilized in the absence of a chaotropic agent, KCl. With Brij 35 and Tween 80, even in the presence of KCl, KGDH was scarcely solubilized, but GADH was solubilized with a final recovery of 100%. Thus, the membrane fraction of G. melanogenus IFO 3293 was treated first with 2% Brij 35 and 0.2 mol L-1 KCl to remove GADH, and after centrifugation the precipitate containing KGDH was collected. The resulting membrane precipitates were suspended in 0.01 mol L-1 Tris-HCl, pH 8.0, and treated with Triton X-100, Na-cholate, or Na-deoxycholate for 3 h at 5 °C. KGDH was solubilized in the presence of 0.3 mol L-1 KCl together with 2% Triton X-100. Alternatively, solubilization of KGDH was carried out overnight with 2% Na-cholate and 0.2 mol L-1 KCl, which was favorable to the following purification procedure involving ammonium sulfate fractionation.

The enzyme activity of KGDH is most active at pH 4.0, and 2-keto-D-gluconate is the only substrate oxidized by the enzyme. Similar compounds such as 5-keto-D-gluconate, 2-keto-D-galactonate, and 2-keto-D-gulonate are inert. KGDH catalyzes 2-keto-D-gluconate oxidation to 2,5-diketo-D-gluconate, which is an important step of bioconversion in a novel pathway to L-ascorbate developed by Sonoyama et al. [100]. D-Glucose was first converted to Ca-2,5-diketo-D-gluconate by a mutant strain of Erwinia sp. with 94.5% yield after 26 h cultivation. Then, a mutant strain of Coprynebacterium sp. reduced 2,5-diketo-D-gluconate stereospe-cifically with 2,5-diketo-D-gluconate reductase to Ca-2-keto-L-gulonate with a yield of 84.5%. The occurrence of KGDH is known in strains of Erwinia, Pantoea, and Pseudomonas, as well as Gluconobacter. Ameyama and Kondo noted the importance of 2,5-diketo-D-gluconate as the precursor of D-lyxuronic acid [101].

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