No abstract

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 PQQH 2 . 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 PQQH 2 . PQQ1 is a non-covalently bound prosthetic group of most quinoprotein dehydrogenases in Gram-negative bacteria, which are involved in the oxidation of alcohols or aldose sugars in their periplasm (1).Membrane-bound quinoprotein GDH of Escherichia coli catalyzes oxidation of the C-1 hydroxyl group of the pyranose form of D-glucose to D-glucono-␦-lactone, which is spontaneously converted to D-gluconate, and concomitantly transfers electrons to ubiquinol oxidase through ubiquinone in the respiratory chain (2, 3). Topological analysis revealed that the monomeric GDH possesses five trans-membrane segments at the N-terminal portion (residues 1-154), which ensure strong anchorage of the protein in the inner membrane (4). The remaining C-terminal portion (residues 155-796) occurs at the periplasmic side of the membrane. This portion is assumed to have a catalytic domain including PQQ (5, 6) and Ca 2ϩ or Mg 2ϩ binding sites (7,8). Moreover, GDH of E. coli occurs as an apoenzyme (7, 8), and the exogenous addition of PQQ with the divalent cation leads to formation of the active enzyme (9).Three-dimensional structures of MDHs from three different bacteria have been determined by x-ray crystallography (10 -12), which reveals that the ␣ subunit is a superbarrel made up of eight topologically identical four stranded anti-parallel ␤ sheets, being arranged with radial symmetry like the blades of a propeller. PQQ is tightly stacked within a chamber of the active site in the ␣ subunit, and Ca 2ϩ helps PQQ to be maintained in the correct configuration. Amino acid residues interacting with PQQ and Ca 2ϩ are dispersed in the whole ␣ subunit.Alignment of the PQQ-binding proteins or subunits among quinoprotein dehydrogenases reveals that the periplasmic domain of GDH in E. coli has 26% sequence similarity to the ␣ subunit of MDH...

The membrane-bound pyrroloquinoline quinone (PQQ)-containing quinoprotein glucose dehydrogenase (mGDH) in Escherichia coli functions by catalyzing glucose oxidation in the periplasm and by transferring electrons directly to ubiquinone (UQ) in the respiratory chain. To clarify the intramolecular electron transfer of mGDH, quantitation and identification of UQ were performed, indicating that purified mGDH contains a tightly bound UQ 8 in its molecule. A significant increase in the EPR signal was observed following glucose addition in mGDH reconstituted with PQQ and Mg 2؉ , suggesting that bound UQ 8 accepts a single electron from PQQH 2 to generate semiquinone radicals. No such increase in the EPR signal was observed in UQ 8 -free mGDH under the same conditions. Moreover, a UQ 2 reductase assay with a UQ-related inhibitor (C49) revealed different inhibition kinetics between the wildtype mGDH and UQ 8 -free mGDH. From these findings, we propose that the native mGDH bears two ubiquinone-binding sites, one (Q I ) for bound UQ 8 in its molecule and the other (Q II ) for UQ 8 in the ubiquinone pool, and that the bound UQ 8 in the Q I site acts as a single electron mediator in the intramolecular electron transfer in mGDH. Escherichia coli mGDH,1 which contains PQQ as a prosthetic group (1, 2), catalyzes a direct oxidation of D-glucose to Dgluconate in the periplasm and concomitantly transfers electrons to UQH 2 oxidase via UQ in the respiratory chain (3-6). mGDH is an 88-kDa monomeric protein with an N-terminal hydrophobic domain and a large C-terminal periplasmic domain (6). The former consists of five transmembrane segments, and the latter has a ␤-sheet propeller fold superbarrel structure that is a catalytic domain bearing the PQQ-binding (7) and Ca 2ϩ -or Mg 2ϩ -binding (8, 9) sites. A substantial amount of information on the domains, equivalent to the latter in PQQcontaining quinoproteins, has been accumulated from the modeled structures of mGDH (7) and membrane-bound ADH III (10) and from x-ray structures of MDH (11), ADH I (12), ADH IIB (13), and soluble glucose dehydrogenase (14), which have been further confirmed by mutagenic analysis on several of the amino acid residues surrounding PQQ (15)(16)(17)(18)(19).Our understanding of the interaction with UQ or its involvement in catalytic reactions in membrane-bound PQQ-containing dehydrogenases, however, is limited. 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). Among other primary dehydrogenases, both the FAD-containing succinate dehydrogenase and the subunit NuoM of NADH-UQ oxidoreductase in E. coli include at least one UQ-binding site (23,24).Most of the information on UQ-binding sites ...

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...

The Escherichia coli membrane-bound glucose dehydrogenase (mGDH) as the primary component of the respiratory chain possesses a tightly bound ubiquinone (UQ) flanking pyrroloquinoline quinone (PQQ) as a coenzyme. Several mutants for Asp-354, Asp-466, and Lys-493, located close to PQQ, that were constructed by site-specific mutagenesis were characterized by enzymatic, pulse radiolysis, and EPR analyses. These mutants retained almost no dehydrogenase activity or ability of PQQ reduction. CD and high pressure liquid chromatography analyses revealed that K493A, D466N, and D466E mutants showed no significant difference in molecular structure from that of the wild-type mGDH but showed remarkably reduced content of bound UQ. A radiolytically generated hydrated electron (e aq ؊ ) reacted with the bound UQ of the wild enzyme and K493R mutant to form a UQ neutral semiquinone with an absorption maximum at 420 nm. Subsequently, intramolecular electron transfer from the bound UQ semiquinone to PQQ occurred. In K493R, the rate of UQ to PQQ electron transfer is about 4-fold slower than that of the wild enzyme. With D354N and D466N mutants, on the other hand, transient species with an absorption maximum at 440 nm, a characteristic of the formation of a UQ anion radical, appeared in the reaction of e aq ؊ , although the subsequent intramolecular electron transfer was hardly affected. This indicates that D354N and D466N are prevented from protonation of the UQ semiquinone radical. Moreover, EPR spectra showed that mutations on Asp-466 or Lys-493 residues changed the semiquinone state of bound UQ. Taken together, we reported here for the first time the existence of a semiquinone radical of bound UQ in purified mGDH and the difference in protonation of ubisemiquinone radical because of mutations in two different amino acid residues, located around PQQ. Furthermore, based on the present results and the spatial arrangement around PQQ, Asp-466 and Lys-493 are suggested to interact both with the bound UQ and PQQ in mGDH.The Escherichia coli membrane-bound glucose dehydrogenase (mGDH) 2 belongs to the quinoprotein family with PQQ as a coenzyme (1, 2), and it catalyzes D-glucose oxidation to D-gluconate at the periplasmic side to transfer electrons to ubiquinol oxidase via UQ in the respiratory chain (3-5). Topological analysis revealed that mGDH consists of an N-terminal hydrophobic domain with five membrane-spanning segments and a large C-terminal domain residing in the periplasm, which contains PQQ and Ca 2ϩ -or Mg 2ϩ -binding sites in a superbarrel structure, conserved in quinoproteins (6 -8). Although its tertiary structure has not been resolved, the arrangement of amino acid residues around PQQ has been modeled on the basis of the crystal structure of the quinoprotein methanol dehydrogenase (6) as depicted in Fig. 1. The arrangement has been confirmed by results of several experiments with site-directed amino acid substitutions (9 -13). The orthoquinone portion of PQQ is a vital part for the catalytic reaction, to which Lys-493 hydr...

The hpt gene, which encodes hypoxanthine phosphoribosyltransferase, is located next to, but transcribed in the opposite direction to, the gcd gene, which codes for a membrane-bound glucose dehydrogenase, at 3.1 min on the Escherichia coli genome. In their promoter-operator region, putative regulatory elements for integration host factor (IHF) and for the complex comprising 3', 5'-cyclic AMP (cAMP) and its receptor protein (CRP) are present, and they overlap the promoters for hpt and gcd, respectively. The involvement of IHF and cAMP-CRP, as well as the corresponding putative cis-acting elements, in the expression of the two genes was investigated by using lacZ operon fusions. In an adenylate cyclase-deficient strain, addition of cAMP increased the expression of hpt and reduced the expression of gcd. In agreement with this observation, the introduction of mutations into the putative binding element for the cAMP-CRP complex enhanced the expression of gcd. In contrast, mutations introduced into the putative IHF-binding elements increased the level of hpt expression. Similar results were obtained with IHF-defective strains. Thus, the expression of the two genes is regulated in a mutually exclusive manner. Additional experiments with mutations at the -10 sequence of the gcd promoter suggest that the binding of RNA polymerase to the hpt promoter interferes with the interaction of RNA polymerase with the gcd promoter, and vice versa.