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Membrane-bound quinoprotein glucose dehydrogenase (GDH) in Escherichia colidonates electrons directly to ubiquinone during the oxidation of d-glucose as a substrate, and these electrons are subsequently transferred to ubiquinol oxidase in the respiratory chain. To determine whether the specific ubiquinone-reacting site of GDH resides in the N-terminal transmembrane domain or in the large C-terminal periplasmic catalytic domain (cGDH), we constructed a fusion protein between the signal sequence of β-lactamase and cGDH. This truncated GDH was found to complement a GDH gene-disrupted strain in vivo. The signal sequence of the fused protein was shown to be cleaved off, and the remaining cGDH was shown to be recovered in the membrane fraction, suggesting that cGDH has a membrane-interacting site that is responsible for binding to membrane, like peripheral proteins. Kinetic analysis and reconstitution experiments revealed that cGDH has ubiquinone reductase activity nearly equivalent to that of the wild-type GDH. Thus, it is likely that the C-terminal periplasmic domain of GDH possesses a ubiquinone-reacting site and transfers electrons directly to ubiquinone.

Several mutants of quinoprotein glucose dehydrogenase (GDH) in Escherichia coli, located around its cofactor pyrroloquinoline quinone (PQQ), were constructed by site-specific mutagenesis and characterized by enzymatic and kinetic analyses. Of these, critical mutants were further characterized after purification or by different amino acid substitutions. H262A mutant showed reduced affinities both for glucose and PQQ without significant effect on glucose oxidase activity, indicating that His-262 occurs very close to PQQ and glucose, but is not the electron acceptor from PQQH2. W404A and W404F showed pronounced reductions of affinity for PQQ, and the latter rather than the former had equivalent glucose oxidase activity to the wild type, suggesting that Trp-404 may be a support for PQQ and important for the positioning of PQQ. D466N, D466E, and K493A showed very low glucose oxidase activities without influence on the affinity for PQQ. Judging from the enzyme activities of D466E and K493A, as well as their absorption spectra of PQQ during glucose oxidation, we conclude that Asp-466 initiates glucose oxidation reaction by abstraction of a proton from glucose and Lys-493 is involved in electron transfer from PQQH2.

Several mutants of quinoprotein glucose dehydrogenase (GDH) in Escherichia coliwere obtained and characterized. Of these, significant mutants were further characterized by kinetic analysis after purification or by site-directed mutagenesis to introduce different amino acid substitutions. H775R and H775A showed a pronounced reduction of affinity for a prosthetic group, pyrroloquinoline quinone (PQQ), suggesting that His-775 may directly interact with PQQ. D730N and D730A showed low glucose oxidase activity without influence on the affinity for PQQ, Mg2+, or substrate, but D730R showed reduced affinity for PQQ. The spectrum of tryptophan fluorescence revealed that the local structure surrounding PQQ was not changed by D730N mutation. Based on these data, we assume that Asp-730 may occur close to PQQ and function as a proton (and also electron) donor to PQQ or acceptor from PQQH2. Substitutions of Gly-689, that are located at the end of a unique segment of GDH among homologous quinoprotein dehydrogenases, directed reduction of the affinity for PQQ or GDH activity. Therefore, the unique segment and Asp-730 may play a specific role for GDH, which might be related to the intramolecular electron transfer from PQQ to ubiquinone.