Existence of a novel prosthetic group, PQQ, in mebrane‐bound, electron transport chain‐linked, primary dehydrogenases of oxidative bacteria

Ameyama, Minoru; Matsushita, Kazunobu; Ohno, Yasue; Shinagawa, Emiko; Adachi, Osao

doi:10.1016/0014-5793(81)81114-3

Cited by 113 publications

(55 citation statements)

References 13 publications

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“…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)(4)(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).…”

mentioning

confidence: 99%

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

Mustafa

Ishikawa

Kobayashi

et al. 2008

Journal of Biological Chemistry

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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...

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confidence: 99%

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

Mustafa

Ishikawa

Kobayashi

et al. 2008

Journal of Biological Chemistry

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“…It has been established recently that certain oxidoreductases from a variety of sources contain pyrrolo-quinoline quinone (PQQ) as a prosthetic group (2,4,10,11). These quinoproteins include bacterial methanol (9) and glucose dehydrogenases (12), which possess noncovalently associated PQQ, and bacterial methylamine dehydrogenase, which contains a covalently bound form of PQQ (8,21).…”

mentioning

confidence: 99%

Purification and properties of methylamine dehydrogenase from Paracoccus denitrificans

Husain¹,

Davidson²

1987

J Bacteriol

110

106

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Methylamine dehydrogenase from Paracoccus denitrificans was purified to homogeneity in two steps from the periplasmic fraction of methylamine-grown cells. The enzyme exhibited a pI value of 4.3 and was composed of two 46,700-dalton subunits and two 15,500-dalton subunits. Each small subunit possessed a covalently bound pyrrolo-quinoline quinone prosthetic group. The amino acid compositions of the large and small subunits are very similar to those of other methylamine dehydrogenases which have been isolated from taxonomically different sources. It has been established recently that certain oxidoreductases from a variety of sources contain pyrrolo-quinoline quinone (PQQ) as a prosthetic group (2, 4, 10, 11). These quinoproteins include bacterial methanol (9) and glucose dehydrogenases (12), which possess noncovalently associated PQQ, and bacterial methylamine dehydrogenase, which contains a covalently bound form of PQQ (8,21). Mammalian plasma amine oxidase (26) and choline dehydrogenase (3) also possess covalently attached PQQ. When grown on methylamine as a sole source of carbon and energy, Paracoccus denitrificans synthesizes a methylamine dehydrogenase which functions in the periplasm of this gramnegative bacterium and donates electrons to a periplasmic type I blue copper protein, amicyanin (16). We have previously reported the partial purification of methylamine dehydrogenase from P. denitrificans (16) and have characterized the physical and redox properties of amicyanin (14,16,18,25) and periplasmic c-type cytochromes (14, 17) which accept electrons from methylamine dehydrogenase via amicyanin. This paper reports a relatively simple procedure by which methylamine dehydrogenase was purified to homogeneity from P. denitrificans and describes several of the physical and kinetic properties of this enzyme. MATERIALS AND METHODSBacterial strains and culture conditions. P. denitrificans (ATCC 13543) was grown aerobically at 30°C in the medium of Kornberg and Morris (23), supplemented with 0.05% NaHCO3, 0.01% yeast extract, and 0.5% methylamine, 0.5% methanol, or 0.3% succinate.Preparation of proteins. Methylamine dehydrogenase was purified from the periplasmic fraction of methylamine-grown cells, which was prepared by the method of Alefounder and Ferguson (1). Routinely, 15 to 20 g (wet weight) of cells was * Corresponding author. fractionated at a time, and four such preparations were pooled, concentrated by ultrafiltration over an Amicon YM5 membrane (Amicon Corp., Lexington, Mass.), dialyzed against 20 mM potassium phosphate (pH 7.2), and applied to a DEAE-cellulose column (3.5 by 30 cm) which had been equilibrated in the same buffer that was used for dialysis. This column was eluted with a linear gradient (2.0 liters) of 0 to 400 mM NaCl in the starting buffer. Fractions containing methylamine dehydrogenase were pooled, concentrated by ultrafiltration over an Amicon PM 30 membrane, dialyzed against 20 mM potassium phosphate (pH 7.2), and applied to a DEAE-Trisacryl (LKB Instruments, Inc., Rockville, Md.) column...

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“…19 ) On the other hand, D-glucose dehydrogenase of P. fluorescens could react directly with ubiquinone in spite of being a single polypeptide primary dehydrogenase. The dehydrogenase is not a flavoprotein unlike other dehydrogenases described above, but a quinoprotein 20 ) having a novel prosthetic group, pyrrolo-quinoline quinone. 2 1) Thus, it may be characteristic of the quinoprotein, unlike flavoprotein, to be capable of direct reduction of ubiquinone, and this may show that the quinoprotein has a peculiar function in the bacterial electron transport chain.…”

Section: Reactivity Of O-glucose Dehydrogenase With Long-chain Q Homomentioning

confidence: 94%

Membrane-bound, Electron Transport-linked,d-Glucose Dehydrogenase ofPseudomonas fluorescens. Interaction of the Purified

Matsushita

Ohno

Shinagawa

et al. 1982

Agricultural and Biological Chemistry

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Existence of a novel prosthetic group, PQQ, in mebrane‐bound, electron transport chain‐linked, primary dehydrogenases of oxidative bacteria

Cited by 113 publications

References 13 publications

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

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

Purification and properties of methylamine dehydrogenase from Paracoccus denitrificans

Membrane-bound, Electron Transport-linked,d-Glucose Dehydrogenase ofPseudomonas fluorescens. Interaction of the Purified

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