Membrane Bound DGlucose Dehydrogenase mGDH

D-Glucose ^ D-glucono-S -lactone

The quinoprotein D-glucose dehydrogenase (EC 1.1.99.17) (GDH) occurs on the outer surface of the cytoplasmic membrane of oxidative bacteria such as Pseudomonas and Gluconobacter strains and catalyzes direct oxidation of D-glucose to D-gluconate via D-glucono-S-lactone (membrane-bound D-glucose dehydrogenase, m-GDH). It is known as an alternative pathway to the phosphotransferase system of bacteria to catalyze D-glucose assimilation. m-GDH is found in a variety of bacteria including Gram-negative facultative anaerobes such as enteric bacteria and Zymomonas, as well as aerobic bacteria such as pseudomonads and acetic acid bacteria.

GDH was originatally investigated in Acinetobacter calcoaceticus in the early 1960s by Hauge [32-35] and subsequently in the late 1970s by Duine et al. [36], who showed the enzyme to be a quinoprotein. A. calcoaceticus contains a soluble form of GDH (s-GDH) in addition to a membrane-bound form (m-GDH). Thus, they had been believed for many years to be the same enzyme or interconvertible forms. However, recent evidence has shown that this soluble enzyme is not a typical m-GDH. The membranes from several strains such as Escherichia coli, Klebsiella aerogenes, Pseudomonas aeruginosa, Gluconobacter suboxydans, Aceto-bacter aceti, and A. calcoaceticus contain antigens cross-reactive with an antibody of m-GDH purified from P. fluorescens [37], while s-GDH purified from the soluble fraction of A. calcoaceticus does not cross-react with the antibody [38]. Subsequently, s-GDH and m-GDH were purified separately from A. calcoaceticus and shown to be distinctive in all aspects, including optimum pH, kinetics, substrate specificity, ubiquinone reactivity, molecular size, and immunoreactivity [39]. As described below, s-GDH of A. calcoaceticus is a monomer consisting of a single polypeptide of 48-55 kDa containing one molecule of PQQ.

When m-GDH was solubilized and purified from Gluconobacter suboxydans IFO 12528, other species of membrane-bound dehydrogenases were eliminated by treating the enzyme solution at pH 2.5 in the initial stage of enzyme purification. The purified m-GDH was homogeneous in analytical ultracentrifugation (4.2 S) and sucrose density gradient centrifugation [40]. m-GDH from acetic acid bacteria was highly hydrophobic and 87 kDa of its molecular mass has been determined by SDS-PAGE in the presence of urea. The existence of PQQ as the primary coenzyme has been confirmed with the purified enzyme. The optimum pH of D-glucose oxidation is found to be pH 3.0 with potassium ferricyanide and pH 6.0 with PMS-DCIP. The substrate specificity of the enzyme seems to be restricted to D-glucose, and other sugars are not oxidized except for maltose, which is oxidized at a low rate. Due to the hydrophobicity, the enzyme is regarded as a typical integral membrane protein in acetic acid bacteria.

Since E. coli does not have the PQQ gene, the bacterium produces m-GDH in the apo-form. The apoenzyme was converted to active holoenzyme by the addition of PQQ and Mg2+ [41]. In contrast to the m-GDH found in acetic acid bacteria [40], PMS-DCIP is a convenient electron acceptor for measuring D-glucose oxidation by this enzyme. The enzyme is a typical membrane-bound enzyme highly embedded in the cytoplasmic membrane, with five membrane-spanning domains [42. 43]. The active site of m-GDH faces the periplasmic space. m-GDH was solu-bilized from the cytoplasmic membrane with the aid of detergent and further purified to a homogeneous state. The substrate specificity of E. coli m-GDH looks similar to that of A. calcoaceticus [39].

D-Gluconate is an important substance for the food industry as a food additive. Ca-D-gluconate is widely used as a metal polish in the lens industry. In aerobic Gram-negative bacteria including acetic acid bacteria, D-gluconate is produced from D-glucose by the action of m-GDH. It is distinct from a fungal D-glucose oxidase producing D-gluconate that the bacterial enzyme does not link to molecular oxygen directly while the fungal enzyme utilizes molecular oxygen-producing hydrogen peroxide. m-GDH has been shown to donate electrons to ubiquinone, embedded in the membrane phospholipids, and then to the terminal oxidase [1]. Unlike the previous assumption, however, m-GDH does not seem to have a ubiquinone reacting site in the deeply embedded region of the membrane. Despite having a transmembrane domain, m-GDH has been shown to react with ubiquinone near the surface of the membrane [44]. This notion has been strengthened by the recent finding that the C-terminal half of E. coli m-GDH, the N-terminal transmembrane domain being deleted, maintains ubiquinone reductase activity [45].

To clarify the intermolecular electron transfer of m-GDH, quantitation and identification of ubiquinone have been done, indicating that E. coli m-GDH contains a tightly bound ubiquinone-8 (UQ8) in its molecule. A significant increase in the EPR signal was observed following D-glucose addition in m-GDH reconstituted with PQQ and Mg2+ after the addition of D-glucose, suggesting that bound UQ8 accepts a single electron from PQQH2 to generate semiquinone radicals. No such increase in the EPR signal was observed in UQ8-free m-GDH under the same conditions. Moreover, a UQ2 reductase assay with a ubiquinone-related inhibitor (C49) revealed different inhibition kinetics between the wild-type m-GDH and UQ8-free m-GDH. It is proposed that the native m-GDH bears two ubiquinone-binding sites, one (Qj) for bound UQ8 in its molecule and the other (Qn) for UQ8 in the ubiquinone pool, and the bound UQ8 in the QI site acts as a single electron mediator in the intramolecular electron transfer in m-GDH [46]. It should be noted that there is no specific D-xylose dehydrogenase other than m-GDH. D-Xylose oxidation is done by m-GDH.

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