C-terminal Periplasmic Domain of Escherichia coli Quinoprotein Glucose Dehydrogenase Transfers Electrons to Ubiquinone

Abstract

Membrane-bound quinoprotein glucose dehydrogenase (GDH) in Escherichia coli donates 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 ␤-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 membraneinteracting 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 ubiquinonereacting site and transfers electrons directly to ubiquinone.Membrane-bound GDH 1 in Escherichia coli is a PQQ-containing quinoprotein that catalyzes a direct oxidation of Dglucose to D-gluconate in the periplasm and concomitantly transfers electrons to ubiquinol oxidase through ubiquinone in the respiratory chain (1-3). GDH is an 88-kDa monomeric protein with five transmembrane segments at the N-terminal portion (residues 1-140), which ensure a strong anchorage of the protein to the inner membrane (4, 5). The remaining large C-terminal portion (residues 141-796) has a catalytic domain including PQQ-(6, 7) and Ca 2ϩ or Mg 2ϩ -binding sites (8, 9) that is located in the periplasmic side. A model structure of GDH based on the x-ray crystallographic structure of the ␣-subunit of MDH in Methylobacterium extorquens has been proposed (10), and the putative structure of the PQQ-binding catalytic site has been further confirmed and characterized by mutagenic analysis of several amino acid residues around PQQ (11-15).The ubiquinone-reacting site in GDH has also been analyzed. Friedrich et al. (16) proposed that the ubiquinone-reacting site may be located at the N-terminal transmembrane domain of Acinetobacter calcoaceticus GDH in which Arg-91 and Asp-93 may be involved in interaction with ubiquinone. The topological model of the N-terminal transmembrane domain of E. coli GDH has shown that the corresponding amino acid residues, Arg-93 and Asp-95, are located near the membrane surface of the periplasmic side (5). Furthermore, using depth-dependent fluorescent ubiquinone analogues, Miyoshi et al. (17) demonstrated that the ubiquinone-reduction site of GDH is located close to the membrane surface rather than in the hydrophobic interior. X-ray crystallographic structures of cytochrome bo in E. coli (18,19) and cytochrome bc 1 complex (Q o and Q i centers) in bovine heart mitochondria have recently been determined (20, 21), and it has b...

Occurrence of a Bound Ubiquinone and Its Function in Escherichia coli Membrane-bound Quinoprotein Glucose Dehydrogenase

“…Considering the molecular similarity, C49 may also occupy the bound UQ 8 site, and the binding may cause a structural change in the bulk UQ site in ⌬UbiA mGDH, which results in a reduction of the V max value of UQ 2 reductase activity. On the basis of the findings in this and previous studies (20,21), we propose the following mechanism of intramolecular electron transfer in mGDH. There are two UQ-binding sites, one for bound UQ 8 and another for the bulk UQ pool (designated as Q I and Q II , respectively).…”

“…The UQ reduction site (interacting with bulk UQ) in mGDH has been shown to be located near the membrane surface (20), which idea was strengthened from the findings that its C-terminal periplasmic domain, interacting peripherally with the membrane, possesses the UQ reduction site (21). ADH III in Gluconobacter suboxydans has been postulated to have two discrete sites for UQH 2 oxidation and UQ reduction in its subunit II (22).…”

“…In UQ 2 reductase assay, the K m and V max values and the K i value for C49 were estimated on the basis of the Lineweaver-Burk plots. Absolute absorption spectra of apoform and holoform of the wild-type mGDH and ⌬UbiA mGDH were taken before and after the addition of glucose as described (21).…”

Main Polyol Dehydrogenase ofGluconobacter suboxydansIFO 3255, Membrane-boundD-Sorbitol Dehydrogenase, That Needs Product of Upstream Gene,sldB, for Activity

“…According to Elias et al,24) N-terminal-truncated Dglucose dehydrogenase, cGDH, the large C-terminal periplasmic catalytic domain, binds to membrane and maintains ubiquinone reductase activity as wildtype D-glucose dehydrogenase does.…”

Amino Acid Residues Interacting with Both the Bound Quinone and Coenzyme, Pyrroloquinoline Quinone, in Escherichia coli Membrane-bound Glucose Dehydrogenase

“…PMS reductase activity was measured spectrophotometrically (U-2000A, Hitachi) with PMS and 2,4-dichlorophenol indophenol as an electron mediator and acceptor, respectively, as described previously (3). UQ 2 reductase activity was also measured spectrophotometrically in the presence of 0.0025% Tween 20 (11,42). The contents of mGDH protein in membrane fractions were compared by Western blot analysis using an antibody against E. coli mGDH (36).…”