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PubMed1999

Role of calcium in the reaction between pyrroloquinoline quinone and pyridine nucleotides monomers and dimers.

Redox reactions were carried out in aerobiosis and anaerobiosis between NAD(P) dimers or NAD(P)H and pyrroloquinoline quinone (PQQ) in different buffers. The buffer system and pH significantly affected the oxidation rates of nucleotides and the ESR signal intensity of the PQQ(*) radical formed in anaerobiosis by comproportion between the quinone and quinol forms. The relative reactivity of the four nucleotides toward PQQ was affected by pH and buffer nature. PQQ, which behaves as an electron shuttle from nucleotides to oxygen, was first converted to PQQH(2) and then rapidly reoxidized by oxygen, with formation of hydrogen peroxide. Both NAD(P) dimers and NAD(P)H consumed 1 mol of oxygen per mole of reacted molecule of pyridine nucleotide, yielding 1 or 2 mol of NAD(P)(+) from NAD(P)H or from NAD(P) dimers, respectively. Chelating agents such as EDTA and phytate strongly decreased the reaction rate and the PQQ(*) radical signal intensity. Kinetics carried out in the presence of metal ions showed instead an increased reaction rate in the order Ca(2+) >> Mg(2+) > Na(+) >> K(+). Spectrofluorimetric measurements of PQQ with increasing concentrations of Ca(2+) showed a fluorescence quenching and shift of the maximum emission toward lower wavelengths, while other metal ions showed minor effects, if any. Therefore, it is demonstrated that Ca(2+) binds to PQQ, probably forming a complex which is more reactive with both one-electron (NAD(P) dimers) or two-electron donors (NAD(P)H) in nonenzymic reactions. It is important to recall that Ca(2+) was already found to play active role in PQQ-containing enzymes.

Calcium↗
PubMed1993

Failure to verify high levels of pyrroloquinoline quinone in eggs and skim milk.

The levels of pyrroloquinoline quinone (PQQ) in eggs, skim milk and milk was re-examined by the use of gas chromatography/mass spectrometry. PQQ was extracted from the samples, after addition of [U-13C]PQQ as internal standard, with n-butanol and Sep-Pak C18 cartridges. After derivatization of PQQ with phenyltrimethylammonium hydroxide, molecular peaks at m/z 448 and 462 were used for detection of PQQ and [U-13C]PQQ, respectively, by selected ion monitoring. PQQ could be detected in eggs, skim milk and milk, but their levels were very low (e.g., egg yolk: 7.0 ng/ml; skim milk: 2.5 ng/g dry weight). Our data for eggs and skim milk are far below those measured by the redox cycling method of Gallop's group.

Animals↗
PubMed1993

Pyrroloquinoline quinone acts with flavin reductase to reduce ferryl myoglobin in vitro and protects isolated heart from re-oxygenation injury.

Pyrroloquinoline quinone has been isolated from bacteria and recently has been detected in mammalian tissues and fluids. We report in vitro studies which show that pyrroloquinoline quinone serves as a high-affinity substrate for an erythrocyte "flavin reductase" and that the pyrroloquinoline quinol generated by this catalysis reacts rapidly with ferryl myoglobin radical. Western blot analysis of rat and rabbit heart homogenates detects a cross-reactive protein which has a molecular weight identical to the erythrocyte reductase from the same species. Low concentrations of pyrroloquinoline quinone protect isolated rabbit heart from re-oxygenation injury, serving as an effective tissue-protective agent in this model for cellular oxidative damage. We propose that this tissue protection is due to a pyrroloquinoline quinol-mediated reduction of reactive oxygen species.

Animals↗
PubMed1995

Elucidation of the region responsible for EDTA tolerance in PQQ glucose dehydrogenases by constructing Escherichia coli and Acinetobacter calcoaceticus chimeric enzymes.

We constructed various chimeric PQQ glucose dehydrogenases (PQQGDHs) from an EDTA-sensitive PQQGDH from Escherichia coli and an EDTA-tolerant PQQGDH from Acinetobacter calcoaceticus by homologous recombination of their structural genes. The EDTA tolerance of the resulting chimeric enzymes was investigated. Our results demonstrated that EDTA tolerance of PQQGDHs can be completely altered by substituting each corresponding region. The EDTA tolerance of A. calcoaceticus PQQGDH is mostly within a region composed of about 90 amino acid residues located between 45 and 56% of the distance from the N-terminal region.

Acinetobacter calcoaceticus↗
PubMed1995

High performance liquid chromatographic separation and pH-dependent electrochemical properties of pyrroloquinoline quinone and three closely related isomeric analogues.

Pyrroloquinoline quinone 1 (PQQ, methoxatin, 4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid) is a novel coenzyme. Closely related isomeric analogues of PQQ may be formed in nature and may possess PQQ-like functions. Here, the electrochemical pH-dependent properties and the high performance liquid chromatographic (HPLC) separation protocols are reported for three major isomeric PQQ analogues likely to be formed in nature. The electrochemical data (cyclic voltammetry) provided the pKas of the five ionizable groups in each isomeric PQQ. The HPLC protocols provide the means of investigating the formation of PQQ and the PQQ isomers reported here during the normal turnover of eukaryotic amine oxidases containing integral topaquinone residues, as well as from direct enzyme-mediated reactions which remain to be characterized.

Chromatography, High Pressure Liquid↗
PubMed2000

X-ray structure of the quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa: basis of substrate specificity.

The homodimeric enzyme form of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa ATCC 17933 crystallizes readily with the space group R3. The X-ray structure was solved at 2.6 A resolution by molecular replacement. Aside from differences in some loops, the folding of the enzyme is very similar to the large subunit of the quinoprotein methanol dehydrogenases from Methylobacterium extorquens or Methylophilus W3A1. Eight W-shaped beta-sheet motifs are arranged circularly in a propeller-like fashion forming a disk-shaped superbarrel. No electron density for a small subunit like that in methanol dehydrogenase could be found. The prosthetic group is located in the centre of the superbarrel and is coordinated to a calcium ion. Most amino acid residues found in close contact with the prosthetic group pyrroloquinoline quinone and the Ca(2+) are conserved between the quinoprotein ethanol dehydrogenase structure and that of the methanol dehydrogenases. The main differences in the active-site region are a bulky tryptophan residue in the active-site cavity of methanol dehydrogenase, which is replaced by a phenylalanine and a leucine side-chain in the ethanol dehydrogenase structure and a leucine residue right above the pyrrolquinoline quinone group in methanol dehydrogenase which is replaced by a tryptophan side-chain. Both amino acid exchanges appear to have an important influence, causing different substrate specificities of these otherwise very similar enzymes. In addition to the Ca(2+) in the active-site cavity found also in methanol dehydrogenase, ethanol dehydrogenase contains a second Ca(2+)-binding site at the N terminus, which contributes to the stability of the native enzyme.

Alcohol Oxidoreductases↗
PubMed1999

An unusual role of tryptophan in PQQ-containing quinoproteins.

Methanol dehydrogenase is a bacterial quinoprotein containing PQQ at its active site, which is in the centre of a superbarrel structure made up of eight beta-sheets arranged radially as the blades of a propeller. A series of novel tryptophan-docking interactions between the beta-sheets make planar, stabilizing girdles around the periphery of the protein. The tryptophan residues form stacking interactions, and hydrogen bonds through their indole ring NH groups.

Alcohol Oxidoreductases↗
PubMed1991

Kinetic properties of human liver tryptophan pyrrolase.

Human liver tryptophan pyrrolase (TPO) activity exhibited substrate level regulation. TPO showed biphasic activity to tryptophan when low ascorbate was used as an activator. The high affinity form (Km for tryptophan: 0.05 mM) was promoted by low ascorbate and low tryptophan. The low affinity form (Km for tryptophan: 0.4 mM) was induced by high concentrations of tryptophan or ascorbate. Both high and low affinity forms showed the same affinity to oxygen. The high affinity form was also induced by pyrroloquinoline quinone, but this effect was decreased by catalase, suggesting the participation of H2O2.

Ascorbic Acid↗
PubMed1994

X-ray structure of PQQ-dependent methanol dehydrogenase.

The three-dimensional structure of the PQQ-dependent quinoprotein, methanol dehydrogenase from Methylobacterium extorquens AM1, has been determined at 3A resolution. The a2b2 tetrameric enzyme has a large a-chain of almost spherical form with a chain fold in which eight 4-stranded antiparallel b-sheets segments are arranged radially around a pseudo 8-fold molecular symmetry axis. The much smaller b-chain is surprisingly not globular, but has an extended conformation running across the surface of the alpha-subunit. The PQQ prosthetic group is buried within the large a-subunit located on the pseudo 8-fold molecular symmetry axis. It is surrounded by protein side-chains but not covalently bound. Associated with the PQQ are two unexpected features: a vicinal disulphide bridge formed between Cys103 and Cys104, and a calcium ion bound between the protein and the PQQ. Vicinal disulphide bridges forming highly distorted structures containing a cis peptide bond, have been proposed to be present in one or two enzymes but have not previously been available for detailed structural investigation. Activity studies have indicated that the ability of the enzyme to transfer electrons derived from the reduction of the alcohols to the specific cytochrome CL receptor is lost when the vicinal disulphide bridge is reduced. The roles of the calcium ions and the b-chain in the enzyme's activity remain to be determined.

Alcohol Oxidoreductases↗
PubMed1990

The role of magnesium and calcium ions in the glucose dehydrogenase activity of Klebsiella pneumoniae NCTC 418.

Magnesium-limited chemostat cultures of Klebsiella pneumoniae NCTC 418 with 20 microM CaCl2 in the medium showed a low rate of gluconate plus 2-ketogluconate production relative to potassium- or phosphate-limited cultures. However, when the medium concentration of CaCl2 was increased to 1 mM, the glucose dehydrogenase (GDH) activities also increased and became similar to those observed in potassium- or phosphate limited cultures. It is concluded that this is due to Mg2+ and Ca2+ ions being involved in the binding of pyrroloquinoline quinone (PQQ) to the GDH apoenzyme. There seems to be an absolute requirement of divalent cations for proper enzyme functioning and in this respect Ca2+ ions could replace Mg2+ ions. The high GDH activity which has been found in cells grown under Mg2(+)-limited conditions in the presence of higher concentrations of Ca2+ ions, is compatible with the earlier proposal that GDH functions as an auxiliary energy generating system involved in the maintenance of high transmembrane ion gradients.

Calcium↗
PubMed1992

Nucleotide sequence and structure of the Klebsiella pneumoniae pqq operon.

A 6940 bp Klebsiella pneumoniae chromosomal DNA fragment, containing genes involved in pyrroloquinoline quinone (PQQ) biosynthesis, was sequenced. Six open reading frames, pqqA, pqqB, pqqC, pqqD, pqqE and pqqF were identified in the pqq operon, which coded for polypeptides of 2764 (23 amino acids), 33,464, 28,986, 10,436, 42,881 and 83,616 Da, respectively. The transcription startpoint was mapped by primer extension analysis, upstream of pqqA, and promoter boxes could be identified. The gene products of pqqB, pqqC, pqqE and pqqF were detected in maxi-cells and the molecular weights of the proteins corresponded with the molecular weights deduced from the nucleotide sequence. The gene products of pqqA, pqqB, pqqC, pqqD and pqqE show 49%-64% identity in amino acid sequence with those of pqqIV, pqqV, pqqI, pqqII and pqqIII respectively in the cloned pqq cluster of Acinetobacter calcoaceticus. The 84 kDa protein encoded by pqqF, which is not present in the cloned pqq cluster of A. calcoaceticus but which is essential for PQQ biosynthesis in K. pneumoniae and Escherichia coli, seems to belong to a family of proteases.

Amino Acid Sequence↗
PubMed1989

The separate roles of PQQ and apo-enzyme syntheses in the regulation of glucose dehydrogenase activity in Klebsiella pneumoniae NCTC 418.

No holoenzyme pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase and only very low apoenzyme levels could be detected in cells of Klebsiella pneumoniae, growing anaerobically, or carrying out a fumarate or nitrate respiration. Low glucose dehydrogenase activity in some aerobic glucose-excess cultures of K. pneumoniae (ammonia or sulphate limitation) was increased significantly by addition of PQQ, whereas in cells already possessing a high glucose dehydrogenase activity (phosphate or potassium limitation) extra PQQ had almost no effect. These observations indicate that the glucose dehydrogenase activity in K. pneumoniae is modulated by both PQQ synthesis and synthesis of the glucose dehydrogenase apo-enzyme.

Aerobiosis↗
PubMed1989

Physiological significance and bioenergetic aspects of glucose dehydrogenase.

The regulation of the PQQ-linked glucose dehydrogenase in different organisms is reviewed. It is concluded that this enzyme functions as an auxiliary energy-generating mechanism, because it is maximally synthesized under conditions of energy stress. It is now definitively established that the oxidation of glucose to gluconate generates metabolically useful energy. The magnitude of the contribution of the oxidation of glucose to gluconate via this enzyme to the growth yield of organisms such as Acinetobacter calcoaceticus is not yet clear.

Bacteria↗
PubMed1989

Quinoprotein D-glucose dehydrogenases in Acinetobacter calcoaceticus LMD 79.41: purification and characterization of the membrane-bound enzyme distinct from the soluble enzyme.

Acinetobacter calcoaceticus is known to contain soluble and membrane-bound quinoprotein D-glucose dehydrogenases while other oxidative bacteria such as Pseudomonas or Gluconobacter contain only membrane-bound enzyme. The two different forms were believed to be the same enzyme or interconvertible. Present results show that the two different forms of glucose dehydrogenase are distinct from each other in their enzymatic and immunological properties as well as in their molecular size. The soluble and membrane-bound glucose dehydrogenases were separated after French press-disruption by repeated ultracentrifugation, and then purified to nearly homogeneous state. The soluble enzyme was a polypeptide of 55 Kdaltons, while the membrane-bound enzyme was a polypeptide of 83 Kdaltons which is mainly monomeric in detergent solution. Both enzymes showed different enzymatic properties including substrate specificity, optimum pH, kinetics for glucose, and reactivity for ubiquinone-homologues. Furthermore, the two enzymes could be distinguished immunochemically; the membrane-bound enzyme is cross-reactive with an antibody raised against membrane-bound enzyme purified from Pseudomonas but not with antibody elicited against the soluble enzyme, while the soluble enzyme is not cross-reactive with the antibody of membrane-bound enzyme. Data also suggest that the membrane-bound enzyme functions by linking to the respiratory chain via ubiquinone though the function of the soluble enzyme remains unclear.

Acinetobacter↗