Soluble and Membrane-bound Quinoprotein<scp>D</scp>-Glucose Dehydrogenases of the<i>Acinetobacter calcoaceticus</i>: The Binding Process of PQQ to the Apoenzymes

Matsushita, Kazunobu; Toyama, Hirohide; Ameyama, Minoru; Adachi, Osao; Dewanti, Aster; Duine, Johannis A.

doi:10.1271/bbb.59.1548

Cited by 43 publications

(42 citation statements)

References 18 publications

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“…PQQ reduction abilities during glucose oxidation of mutant GDHs were compared with that of the wild type by their spectral changes in between 300 and 350 nm. Similar changes of absolute spectra were reported with membranebound apo-GDH from Acinetobacter calcoaceticus after adding PQQ and PQQ ϩ substrate (37). stricted distance between Asp-466 and the C-1 hydroxyl group of glucose.…”

Section: Table II Pms Reductase and Glucose Oxidase Activities Of Mutsupporting

confidence: 80%

Functions of Amino Acid Residues in the Active Site ofEscherichia coli Pyrroloquinoline Quinone-Containing Quinoprotein Glucose Dehydrogenase

Elias

Tanaka

Izu

et al. 2000

Journal of Biological Chemistry

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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...

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Section: Table II Pms Reductase and Glucose Oxidase Activities Of Mutsupporting

confidence: 80%

Functions of Amino Acid Residues in the Active Site ofEscherichia coli Pyrroloquinoline Quinone-Containing Quinoprotein Glucose Dehydrogenase

Elias

Tanaka

Izu

et al. 2000

Journal of Biological Chemistry

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“…A. calcoaceticus AC3 (PQQstrain) was the same strain used in the previous study on quinoprotein glucose dehydrogenase, which cannot produce PQQ. 19) A. calcoaceticus SA1 and other Acinetobacter strains were isolated from urine, soil, fruits, and ‰owers in Thailand by enrichment culture on quinate. The culture medium consisted of 2 g of sodium quinate, 1 g of yeast extract, and 1 g of Polypepton in 1 liter of tap water.…”

Section: Methodsmentioning

confidence: 99%

“…The apparent binding constant was comparable to that of the membrane-bound apo-GDH. 19) Unlike other membrane-bound polyol dehydrogenases in acetic acid bacteria, most of which are readily inhibited by chelation of Ca 2＋ with EDTA, allowing release of PQQ, holo-QDH in the membrane fraction of Gluconobacter and Acinetobacter strains was quite resistant to EDTA To both QDH solutions, ammonium sulfate (AmSO 4 ) was added to 2.5 M and the pH was adjusted to 7.0 with ammonia water. The enzyme solution was adsorbed onto two columns of butyl-Toyopearl (1.5×10 cm) which had been equilibrated with the same buŠer containing 2.5 M AmSO 4 .…”

Section: Titration Of Apo-qdh With Pqqmentioning

confidence: 99%

Purification and Characterization of Membrane-bound Quinoprotein Quinate Dehydrogenase

Adachi

Yoshihara

Tanasupawat

et al. 2003

Bioscience, Biotechnology, and Biochemistry

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“…However, in contrast with the latter enzymes, mGDH prefers Mg2+ instead of Ca2+ in the reconstitution of apoenzyme with PQQ to active holoenzyme [4]. sGDH has so far been found only in A. calcoaceticus strains [5]. Although the enzyme is an extremely efficient catalyst for in vitro glucose oxidation, making it an attractive candidate for analytical applications [6], such a role is absent in whole cells, implying that the physiological function of sGDH is unknown [7].…”

mentioning

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Ca²⁺ and its Substitutes have Two Different Binding Sites and Roles in Soluble, Quinoprotein (Pyrroloquinoline‐Quinone‐Containing) Glucose Dehydrogenase

Olsthoorn

Otsuki

Duine

1997

European Journal of Biochemistry

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To investigate the mode of binding and the role of Ca2+ in soluble, pyrroloquinoline-quinone (PQQ)-containing glucose dehydrogenase of the bacterium Acinetobacter calcoaceticus (sGDH), the following enzyme species were prepared and their interconversions studied : monomeric apoenzyme (M) ; monomer with one firmly bound Ca2+ ion (M*); dimer consisting of 2 M* (D); dimer consisting of 2 M and 2 PQQ (Holo-Y); dimer consisting of D with 2 PQQ (Holo-X); fully reconstituted enzyme consisting of Holo-X with two extra Ca2+ ions (Holo) or substitutes for Ca2+ (hybrid Holo-enzymes). D and Holo are very stable enzyme species regarding monomerization and inactivation by chelator, respectively, the bound CaZi being locked up in such a way that it is not accessible to chelator. D can be converted into M* by heat treatment and the tightly bound Ca'+ can be removed from M* with chelator, transforming it into M. Reassociation of M* to D occurs spontaneously at 20°C; reassociation of M to D occurs by adding a stoichiometric amount of Ca2+. Synergistic effects were exerted by bound Ca2+ and PQQ, each increasing the affinity of the protein for the other component. Dimerization of M to D occurred with Ca2', Cd2+, Mn2+, and Sr2+ (in decreasing order of effectiveness), but not with Mg", Ba2+, Cozi, Ni", Znz+, or monovalent cations. Conversion of inactive Holo-X into active Holo, was achieved with Ca2+ or metal ions effective in dimerization. Although it is likely that activation of Holo-X involves binding of metal ion to PQQ, the spectral and enzymatic activity differences between normal Holo-and hybrid Holo-enzymes are relatively small. Titration experiments revealed that the two Caz+ ions required for activation of Holo-X are even more firmly bound than the two required for dimerization of M and anchoring of PQQ. Although the two binding sites related with the dual function of Ca2+ show similar metal ion specificity, they are not identical. The presence of two different sites in sGDH appears to be unique because in other PQQ-containing dehydrogenases, the PQQ-containing subunit has only one site. Given the broad spectrum of bivalent metal ions effective in reconstituting quinoprotein dehydrogenase apoenzymes to active holoenzymes, but the limited spectrum for an individual enzyme, the specificity is not so much determined by PQQ but by the variable metal-ion-binding sites.Keywords: quinoprotein ; pyrroloquinoline quinone ; glucose dehydrogenase; reconstitution; calcium.The bacterium Acinetobacter calcoaceticus produces two quite different PQQ-containing (quinoprotein) glucose dehydrogenases, the membrane-bound enzyme (mGDH) and the soluble enzyme (sGDH). mGDH occurs in many gram-negative bacteria [l] and glucose oxidation via this enzyme provides the organism with useful energy [2]. The deduced amino acid sequence of this Correspondence to J. A. Duine,

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Soluble and Membrane-bound QuinoproteinD-Glucose Dehydrogenases of theAcinetobacter calcoaceticus: The Binding Process of PQQ to the Apoenzymes

Cited by 43 publications

References 18 publications

Functions of Amino Acid Residues in the Active Site ofEscherichia coli Pyrroloquinoline Quinone-Containing Quinoprotein Glucose Dehydrogenase

Functions of Amino Acid Residues in the Active Site ofEscherichia coli Pyrroloquinoline Quinone-Containing Quinoprotein Glucose Dehydrogenase

Purification and Characterization of Membrane-bound Quinoprotein Quinate Dehydrogenase

Ca²⁺ and its Substitutes have Two Different Binding Sites and Roles in Soluble, Quinoprotein (Pyrroloquinoline‐Quinone‐Containing) Glucose Dehydrogenase

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Soluble and Membrane-bound QuinoproteinD-Glucose Dehydrogenases of theAcinetobacter calcoaceticus: The Binding Process of PQQ to the Apoenzymes

Cited by 43 publications

References 18 publications

Functions of Amino Acid Residues in the Active Site ofEscherichia coli Pyrroloquinoline Quinone-Containing Quinoprotein Glucose Dehydrogenase

Functions of Amino Acid Residues in the Active Site ofEscherichia coli Pyrroloquinoline Quinone-Containing Quinoprotein Glucose Dehydrogenase

Purification and Characterization of Membrane-bound Quinoprotein Quinate Dehydrogenase

Ca2+ and its Substitutes have Two Different Binding Sites and Roles in Soluble, Quinoprotein (Pyrroloquinoline‐Quinone‐Containing) Glucose Dehydrogenase

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Ca²⁺ and its Substitutes have Two Different Binding Sites and Roles in Soluble, Quinoprotein (Pyrroloquinoline‐Quinone‐Containing) Glucose Dehydrogenase