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At least 19 recordsLinked to original sources
PubMed1988

Protein lysine 6-oxidase (lysyl oxidase) cofactor: methoxatin (PQQ) or pyridoxal?

1. Treatment of chick embryos with two lathyrogens lowered lysyl oxidase and increased collagen extractability. 2. Subsequent treatment with pyridoxal restored both parameters towards normal, whereas PQQ treatment was less effective. 3. These results suggest the requirement of a pyridoxal derivative for the formation of the enzyme, acting either as cofactor or because its formation requires some pyridoxal-dependent enzyme. The cochromatography of the enzyme with [3H]pyridoxine-derived radioactivity supports the cofactor role. 4. The conclusions of other authors that lysyl oxidase contains PQQ relates to enzymes from other species or to amine oxidases not characterised as lysyl oxidase.

Amino Acid Oxidoreductases↗
PubMed1988

Evidence for PQQ as cofactor in 3,4-dihydroxyphenylalanine (dopa) decarboxylase of pig kidney.

Pig kidney 3,4-dihydroxyphenylalanine (dopa) decarboxylase (EC 4.1.1.28) was purified to homogeneity. Treatment of the enzyme with phenylhydrazine (PH) according to a procedure developed for analysis of quinoproteins gave products which were identified as the hydrazone of pyridoxal phosphate (PLP) and the C(5)-hydrazone of pyrroloquinoline quinone (PQQ). This method failed, however, in quantifying the amounts of cofactor. Direct hydrolysis of the enzyme by refluxing with hexanol and concentrated HCl led to detachment of PQQ from the protein in a quantity of 1 PQQ per enzyme molecule. In view of the reactivity of PQQ towards amines and amino acids, we postulate that it participates as a covalently bound cofactor in the catalytic cycle of the enzyme, in interplay with PLP. Since several other enzymes have been reported to show the atypical behaviour of dopa decarboxylase, it seems that the PLP-containing group of enzymes can be subdivided into pyridoxoproteins and pyridoxo-quinoproteins.

Animals↗
PubMed1987

Phenylhydrazine as probe for cofactor identification in amine oxidoreductases. Evidence for PQQ as the cofactor in methylamine dehydrogenase.

Homogeneous methylamine dehydrogenase (primary-amine:(acceptor) oxidoreductase (deaminating), EC 1.4.99.3, MADH) from the bacterium Thiobacillus versutus was treated with the inhibitor phenylhydrazine (PH). Derivatization of the cofactor in MADH took place in a fast reaction to give compound I. A different product, compound II, was formed in a slow reaction at high O2 concentrations. The compounds I and II could be removed from the protein by proteolysis with pronase and purified to homogeneity. Products showing identical absorption spectra and chromatographic behaviour were isolated from the reaction mixture after incubating pyrroloquinoline quinone (PQQ) with PH. Upon dissolving in dimethyl sulphoxide, both the enzyme-derived as well as the model-system-derived compounds I and II were nearly quantitatively transformed into PQQ. The conclusion is, therefore, that MADH from T. versutus contains covalently bound PQQ, removable from the protein with pronase, and not a structural analogue of this cofactor without the carboxylic acid groups, as was recently proposed for MADH from Bacterium W3A1 [(1986) Biochem. Biophys. Res. Commun. 141, 562-568]. The properties of compounds I and II suggest that they are the 'azo adduct' and the 'hydrazone adduct' of PH and PQQ at the C(5)-position, respectively. The finding that the reaction of a hydrazine with PQQ can lead to two different products, in enzymes as well as in a model system, has important implications for the interpretation of recent comparative studies aimed at detection of PQQ in amine oxidoreductases with Raman spectroscopy.

Oxidoreductases Acting on CH-NH Group Donors↗
PubMed1992

The pyrroloquinoline quinone (PQQ) coenzymes: a case of mistaken identity.

Recent evidence has failed to support the claim for a pyrroloquinoline quinone (PQQ) cofactor in mammalian enzymes previously reported to have PQQ. The validity of the original analysis now has been questioned, and a second cofactor, topa quinone, has been identified in at least one enzyme.

Animals↗
PubMed1996

Increased production of recombinant pyrroloquinoline quinone (PQQ) glucose dehydrogenase by metabolically engineered Escherichia coli strain capable of PQQ biosynthesis.

We have previously shown that the production of recombinant Escherichia coli PQQGDH was greatly improved by using a medium supplemented with the cofactor PQQ, which is not synthesized in E. coli. We show here that the increase in the accumulated PQQGDH is due to the increased stability of the holo-enzyme over apo-enzyme, using recombinant Acinetobacter calcoaceticus PQQGDH. In order to achieve cost-effective PQQGDH production, we incorporated the genes for PQQ biosynthetic pathway from Klebsiella pneumoniae into E. coli, which as a result allowed E. coli to produce PQQ. Using this metabolically engineered E. coli strain as a host, a 10-fold increase in the production of recombinant A. calcoaceticus PQQGDH was achieved, compared to the condition without PQQ and MgCl2.

Acinetobacter calcoaceticus↗
PubMed1992

Apparent oxygen-dependent inhibition by superoxide dismutase of the quinoprotein methanol dehydrogenase.

Methanol dehydrogenase activity, when assayed with phenazine ethosulfate (PES) as an electron acceptor, was inhibited by superoxide dismutase (SOD) and by Mn2+ only under aerobic conditions. Catalase, formate, and other divalent cations did not inhibit the enzyme. The enzyme also exhibited significantly higher levels of activity when assayed with PES under anaerobic conditions relative to aerobic conditions. The oxygen- and superoxide-dependent effects on methanol dehydrogenase were not observed when either Wurster's Blue or cytochrome c-55li was used as an electron acceptor. Another quinoprotein, methylamine dehydrogenase, which possesses tryptophan tryptophylquinone (TTQ) rather than pyrroloquinoline quinone (PQQ) as a prosthetic group, was not inhibited by SOD or Mn2+ when assayed with PES as an electron acceptor. Spectroscopic analysis of methanol dehydrogenase provided no evidence for any oxygen- or superoxide-dependent changes in the redox state of the enzyme-bound PQQ cofactor of methanol dehydrogenase. To explain these data, a model is presented in which this cofactor reacts reversibly with oxygen and superoxide, and in which oxygen is able to compete with PES as an electron acceptor for the reduced species.

Alcohol Oxidoreductases↗
PubMed1999

The 1.7 A crystal structure of the apo form of the soluble quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus reveals a novel internal conserved sequence repeat.

The crystal structure of a dimeric apo form of the soluble quinoprotein glucose dehydrogenase (s-GDH) from Acinetobacter calcoaceticus has been solved by multiple isomorphous replacement followed by density modification, and was subsequently refined at 1. 72 A resolution to a final crystallographic R-factor of 16.5% and free R-factor of 20.8% [corrected]. The s-GDH monomer has a beta-propeller fold consisting of six four-stranded anti-parallel beta-sheets aligned around a pseudo 6-fold symmetry axis. The enzyme binds three calcium ions per monomer, two of which are located in the dimer interface. The third is bound in the putative active site, where it may bind and functionalize the pyrroloquinoline quinone (PQQ) cofactor. A data base search unexpectedly showed that four uncharacterized protein sequences are homologous to s-GDH with many residues in the putative active site absolutely conserved. This indicates that these homologs may have a similar structure and that they may catalyze similar PQQ-dependent reactions.A structure-based sequence alignment of the six four-stranded beta-sheets in s-GDH's beta-propeller fold shows an internally conserved sequence repeat that gives rise to two distinct conserved structural motifs. The first structural motif is found at the corner of the short beta-turn between the inner two beta-strands of the beta-sheets, where an Asp side-chain points back into the beta-sheet to form a hydrogen-bond with the OH/NH of a Tyr/Trp side-chain in the same beta-sheet. The second motif involves an Arg/Lys side-chain in the C beta-strand of one beta-sheet, which forms a bidentate salt-bridge with an Asp/Glu in the CD loop of the next beta-sheet. These intra and inter-beta-sheet hydrogen-bonds are likely to contribute to the stability of the s-GDH beta-propeller fold.

Acinetobacter calcoaceticus↗
PubMed1995

A pAO1-encoded molybdopterin cofactor gene (moaA) of Arthrobacter nicotinovorans: characterization and site-directed mutagenesis of the encoded protein.

A gene homologous to moaA, the gene responsible for the expression of a protein involved in an early step in the synthesis of the molybdopterin cofactor of Escherichia coli, was found to be located 2.7-kb upstream of the nicotine dehydrogenase (ndh) operon on the catabolic plasmid pAO1 of Arthrobacter nicotinovorans. The MoaA protein, containing 354 amino acids, migrated on an SDS-polyacrylamide gel with an apparent molecular weight of 40,000, in good agreement with the predicted molecular weight of 38,880. The pAO1-encoded moaA gene from A. nicotinovorans was expressed in E. coli as an active protein that functionally complemented moaA mutants. Its deduced amino acid sequence shows 43% identity to the E. coli MoaA, 44% to the NarAB gene product from Bacillus subtilis, and 42% to the gene product of two contiguous ORFs from Methanobacterium formicicum. N-terminal sequences, including the motif CxxxCxYC, are conserved among the MoaA and NarAB proteins. This motif is also present in proteins involved in PQQ cofactor synthesis in almost all the NifB proteins reported so far and in the fixZ gene product from Rhizobium leguminosarum. Mutagenesis of any of these three conserved cysteine residues to serine abolished the biological activity of MoaA, while substitution of the tyrosine by either serine, phenylalanine, or alanine did not alter the capacity of the protein to complement the moaA mutation in E. coli. A second Cys-rich domain with the motif FCxxC(13x)C is found close to the C-terminus of MoaA and NarAB proteins. These two Cys-rich sequences may be involved in the coordination of a metal ions. The pAO1 copy of moaA may not be unique in the A. nicotinovorans genome since the molybdopterin cofactor oxidation products were detected in cell extracts from a plasmidless strain.

Amino Acid Sequence↗
PubMed1989

The influence of the culture pH value on the direct glucose oxidative pathway in Klebsiella pneumoniae NCTC 418.

Klebsiella pneumoniae NCTC 418 was cultured aerobically in chemostat cultures (D = 0.3 h-1; 35 degrees C) under respectively carbon-, phosphate-, potassium-, sulphate-, and ammonia-limited conditions with glucose as the sole carbon and energy source. The effect of the external pH value on glucose metabolism and on the enzymes of the direct glucose oxidative pathway was examined. The pH value of the medium had a profound influence on both the activity and the synthesis of the glucose dehydrogenase and the gluconate dehydrogenase. At pH values ranging from pH 5.5 to pH 6.0 maximal activity and synthesis of these enzymes resulted in a more than 80% conversion of the glucose consumed into gluconate and 2-ketogluconate under potassium- or phosphate-limited conditions. On the other hand, no gluconate and/or 2-ketogluconate production could be detected when K. pneumoniae was cultured at pH 8.0. Whereas the synthesis of gluconate dehydrogenase seemingly was completely repressed, still some glucose dehydrogenase was present. The lack of glucose dehydrogenase activity at pH 8.0 was shown not to be due to the dissociation of the cofactor PQQ from the enzyme.

Aerobiosis↗
PubMed1988

Kinetic and spectral studies on the redox forms of methanol dehydrogenase from Hyphomicrobium X.

Several reaction rate constants in the catalytic cycle of methanol dehydrogenase (EC 1.1.99.8) in vitro were determined with stopped-flow spectrophotometry. The studies revealed that the high pH required for adequate activity of the enzyme is related to the strong pH dependency of the oxidation rates of the reduced and semiquinone enzyme forms, MDHred and MDHsem, with the artificial electron acceptor Wurster's blue. The rate-limiting step in the catalytic cycle is associated with the conversion of oxidized enzyme-substrate complex (MDHox.S) into reduced enzyme (MDHred) and product. The effect of activator (ammonium salts) was also confined to this step, but even saturating concentrations were unable to remove the limitation completely. Making use of the large deuterium isotope effect associated with substrate oxidation in the absence of activator, the transient MDHox.C2H3OH complex could be isolated and its slow decomposition into MDHred and formaldehyde could be demonstrated. Further evidence is presented to support the view that the different absorption spectra observed originate from genuine redox forms of methanol dehydrogenase with different redox states of the cofactor PQQ and not from enzyme-electron acceptor complexes. The results confirm and extend our original view on the mechanism of action and contradict the mechanism proposed by others [Parkes, C. & Abeles, R. H. (1984) Biochemistry 23, 6355-6363].

Alcohol Oxidoreductases↗
PubMedDate not supplied

Quantitative aspects of glucose metabolism by Escherichia coli B/r, grown in the presence of pyrroloquinoline quinone.

Escherichia coli B/r was grown in chemostat cultures under various limitations with glucose as carbon source. Since E. coli only synthesized the glucose dehydrogenase (GDH) apo-enzyme and not the appropriate cofactor, pyrroloquinoline quinone (PQQ), no gluconate production could be observed. However, when cell-saturating amounts of PQQ (nmol to mumol range) were pulsed into steady state glucose-excess cultures of E. coli, the organisms responded with an instantaneous formation of gluconate and an increased oxygen consumption rate. This showed that reconstitution of GDH in situ was possible. Hence, in order to examine the influence on glucose metabolism of an active GDH, E. coli was grown aerobically in chemostat cultures under various limitations in the presence of PQQ. It was found that the presence of PQQ indeed had a sizable effect: at pH 5.5 under phosphate- or sulphate-limited conditions more than 60% of the glucose consumed was converted to gluconate, which resulted in steady state gluconate concentrations up to 80 mmol/l. The specific rate of gluconate production (0.3-7.6 mmol.h-1.(g dry wt cells)-1) was dependent on the growth rate and the nature of the limitation. The production rate of other overflow metabolites such as acetate, pyruvate, and 2-oxoglutarate, was only slightly altered in the presence of PQQ. The fact that the cells were now able to use an active GDH apparently did not affect apo-enzyme synthesis.

Carbon↗
PubMed2000

Separation of the enzyme cofactor pyrroloquinoline quinone and three isomeric analogues by capillary electrophoresis with ion-pairing media.

The enzyme cofactor pyrroloquinoline quinone (PQQ) was successfully separated from three closely related isomeric analogues by capillary electrophoresis with ultraviolet detection. Rapid and efficient separation of all four negatively charged isomers with baseline resolution was achieved by the addition of low concentrations (1-5 mM) of short chain tetraalkylammonium (TAA) salts to the capillary buffer. The TAA cations act as ion-pairing agents and promote differential migration of the isomers with only a minimal reduction in the electroosmotic flow. The effects of the TAA salt concentration and the alkyl chain length were examined. Detection limits of PQQ and its isomers were in the range of 7-15 microM with mass detection limits of 98-210 fmol.

Chromatography, High Pressure Liquid↗
PubMed1999

Detailed active site configuration of a new crystal form of methanol dehydrogenase from Methylophilus W3A1 at 1.9 A resolution.

The three-dimensional structure of a new crystal form of methanol dehydrogenase from Methylophilus W3A1 has been obtained in the presence of substrate using data recorded at a synchrotron. The structure of this approximately 140 kDa heterotetramer, refined at 1. 9 A resolution, reveals the detailed configuration of its redox cofactor, pyrroloquinoline quinone (PQQ). C4, one of the oxygen-bearing atoms of this orthoquinone is in a planar configuration while C5, which bears the other quinone oxygen, is tetrahedral, suggesting that the PQQ is in the semiquinone redox state. The substrate binding site has been identified close to PQQ and to the side chain of Asp297, the putative active site base. The proximity of the hydroxyl of methanol to C5 of PQQ compared to the greater separation of the substrate methyl group from C5 supports the addition-elimination reaction mechanism involving a hemiketal intermediate.

Alcohol Oxidoreductases↗
PubMed2000

Functions of amino acid residues in the active site of Escherichia coli pyrroloquinoline quinone-containing quinoprotein glucose dehydrogenase.

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).

Binding Sites↗
PubMed1992

The Entner-Doudoroff pathway in Escherichia coli is induced for oxidative glucose metabolism via pyrroloquinoline quinone-dependent glucose dehydrogenase.

The Entner-Doudoroff pathway was shown to be induced for oxidative glucose metabolism when Escherichia coli was provided with the periplasmic glucose dehydrogenase cofactor pyrroloquinoline quinone (PQQ). Induction of the Entner-Doudoroff pathway by glucose plus PQQ was established both genetically and biochemically and was shown to occur in glucose transport mutants, as well as in wild-type E. coli. These data complete the body of evidence that proves the existence of a pathway for oxidative glucose metabolism in E. coli. PQQ-dependent oxidative glucose metabolism provides a metabolic branch point in the periplasm; the choices are either oxidation to gluconate followed by induction of the Entner-Doudoroff pathway or phosphotransferase-mediated transport. The oxidative glucose pathway might be important for survival of enteric bacteria in aerobic, low-phosphate, aquatic environments.

Biological Transport, Active↗
PubMed1992

Cloning of an Erwinia herbicola gene necessary for gluconic acid production and enhanced mineral phosphate solubilization in Escherichia coli HB101: nucleotide sequence and probable involvement in biosynthesis of the coenzyme pyrroloquinoline quinone.

Escherichia coli is capable of synthesizing the apo-glucose dehydrogenase enzyme (GDH) but not the cofactor pyrroloquinoline quinone (PQQ), which is essential for formation of the holoenzyme. Therefore, in the absence of exogenous PQQ, E. coli does not produce gluconic acid. Evidence is presented to show that the expression of an Erwinia herbicola gene in E. coli HB101(pMCG898) resulted in the production of gluconic acid, which, in turn, implied PQQ biosynthesis. Transposon mutagenesis showed that the essential gene or locus was within a 1.8-kb region of a 4.5-kb insert of the plasmid pMCG898. This 1.8-kb region contained only one apparent open reading frame. In this paper, we present the nucleotide sequence of this open reading frame, a 1,134-bp DNA fragment coding for a protein with an M(r) of 42,160. The deduced sequence of this protein had a high degree of homology with that of gene III (M(r), 43,600) of a PQQ synthase gene complex from Acinetobacter calcoaceticus previously identified by Goosen et al. (J. Bacteriol. 171:447-455, 1989). In minicell analysis, pMCG898 encoded a protein with an M(r) of 41,000. These data indicate that E. coli HB101(pMCG898) produced the GDH-PQQ holoenzyme, which, in turn, catalyzed the oxidation of glucose to gluconic acid in the periplasmic space. As a result of the gluconic acid production, E. coli HB101(pMCG898) showed an enhanced mineral phosphate-solubilizing phenotype due to acid dissolution of the hydroxyapatite substrate.

Acinetobacter calcoaceticus↗