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PubMed1998

pqqA is not required for biosynthesis of pyrroloquinoline quinone in Methylobacterium extorquens AM1.

Methylobacterium extorquens AM1 is a facultative methylotroph that oxidizes methanol via the pyrroloquinoline quinone (PQQ)-linked enzyme methanol dehydrogenase. In M. extorquens AM1 and other PQQ-synthesizing bacteria, several genes are involved in the synthesis of PQQ and one of these, pqqA, has been proposed to encode a peptide precursor of PQQ. In other PQQ-synthesizing bacteria, pqqA is required for PQQ production. In this study, it is shown that both deletion and insertion mutants of pqqA in M. extorquens AM1 grow normally on methanol and produce PQQ. The level of PQQ production is reduced in the insertion mutant, but it is sufficient to allow normal growth on methanol. These results suggest either that a different peptide in M. extorquens AM1 can substitute for PqqA in pqqA mutants, or that PqqA-like peptides may not be obligatory precursors of PQQ. In addition, it is shown that the methanol oxidation transcriptional regulator gene, mxbM, is required for normal methanol induction of PQQ synthesis.

Amino Acid Sequence↗
PubMed2003

Promoters and transcripts for genes involved in methanol oxidation in Methylobacterium extorquens AM1.

Twenty-five genes are involved in methanol oxidation to formaldehyde by the methanol dehydrogenase system in the facultative methylotroph Methylobacterium extorquens AM1 organized in five gene clusters. RT-PCR was used to assess the transcripts for the main gene clusters that encode methanol dehydrogenase and proteins required for its activity (mxaFGJIRSACKLDEHB), and the enzymes that are required for the synthesis of the methanol dehydrogenase prosthetic group, pyrroloquinoline quinone (pqqABC/DE and the pqqFG cluster). In both cases, positive bands were obtained corresponding to mRNA spanning each of the genes in the cluster, but not across the first and last genes and the gene immediately upstream or downstream of the cluster, respectively. These results suggest that these three gene clusters are each transcribed as a single operon. Confirmation was obtained by cloning a number of intergenic regions into a promoter probe vector. None of these regions showed significant promoter activity. Promoter regions were analysed for mxaF, pqqA, orf181 upstream of pqqFG, and mxaW, a gene located upstream of mxaF and divergently transcribed. The promoter regions for these genes were defined to within 100, 46, 124 and 146 bp, respectively, and the two unknown transcriptional start sites were determined, for mxaW and orf181. Alignment of these promoter regions suggests that they all may be transcribed by the sigma(70) orthologue in M. extorquens AM1.

Alcohol Oxidoreductases↗
PubMed1999

Crystallization and preliminary diffraction studies of two quinoprotein alcohol dehydrogenases (ADHs): a soluble monomeric ADH from Pseudomonas putida HK5 (ADH-IIB) and a heterotrimeric membrane-bound ADH from Gluconobacter suboxydans (ADH-GS).

Crystals of a soluble monomeric quinocytochrome alcohol dehydrogenase (ADH-IIB) and of a trimeric membrane-associated quinocytochrome alcohol dehydrogenase (ADH-GS) have been obtained. The ADH-IIB crystals are triclinic, with one monomer in the unit cell, and were obtained in the presence of PEG 8000, sodium citrate, HEPES buffer and 2-propanol. X-ray data were collected at 110 K to 1. 9 A resolution (R(merge) = 6.4%) and the orientation of a methanol dehydrogenase search molecule (from Methylophilus methylotrophus W3A1) was obtained by molecular replacement. Preliminary refinement of this model (10.0-3.0 A resolution, R = 0.37, R(free) = 0.40) led to tentative identification of the two highest peaks in a native anomalous difference Fourier map as the Fe atom of the heme and a calcium ion interacting with the PQQ prosthetic group. The ADH-GS crystals are tetragonal, displaying six similar lattices, both primitive and centered, and were grown by the sitting-drop method after replacement of Triton X-100 by dodecylmaltoside or octaethylene glycol monododecyl ether in the presence of ammonium sulfate and sodium acetate buffer, with and without PEG 3500 and calcium ion. The best diffraction is obtained at 110 K where the resolution extends to about 4 A in the a and b directions and about 3 A in the c direction.

Alcohol Oxidoreductases↗
PubMed1992

Detergent-shock response in enteric bacteria.

Our work on bacterial detergent resistance started with the realization that bacteria growing in a sink full of soap must be resistant to the detergents in that soap. We chose sodium dodecyl sulphate (SDS) as a model detergent and decided to see how much SDS the bacterium growing in the sink could tolerate. The research program thus initiated has shown that bacteria such as Enterobacter cloacae can grow in up to 25% SDS and that SDS-shock proteins constitute c. 8% of the proteins synthesized by SDS-grown Escherichia coli. It has also provided explanations why enteric bacteria are oxidase negative, and how pyrroloquinoline quinone (PQQ) enters the periplasmic space. Finally, for E. coli, it has provided evidence for an alternate, phosphate-limited, aquatic life style which places greater emphasis on the Entner-Doudoroff pathway. Detergent resistance is important both medically and ecologically, e.g. entry of pathogens via bile-salt-containing intestinal tracts and biodegradation of detergent-like pollutants such as those resulting from oil spills. Our current research is focused on SDS-induced modifications of the cytoplasmic membrane and the presence of SDS in the periplasm.

Detergents↗
PubMed1980

Structure and activity of the prosthetic group of methanol dehydrogenase.

The reconstructive ability of the isolated prosthetic group of methanol dehydrogenase with the apoenzyme of glucose dehydrogenase and the results of electron spin resonance measurements suggest that the prosthetic group has not been modified during the isolation. This result, and the properties of the directly isolated prosthetic group and derivatives, confirm the suggestion that its structure is 2,7,9-tricarboxy-1H-pyrrolo[2,3-f]quinoline-4,5-dione. From the activity shown by derivatives of the prosthetic group and of structural analogues in the apoenzyme test it is concluded that the o-quinone structure is essential for activity. Hence the trivial name pyrrolo-quinoline quinone would be appropriate. The testing of the analogues also shows that the pyrrolo ring and the 9-carboxylic acid group are not essential for activity as they can be replaced by a pyridinol ring and a 9-hydroxy group respectively. The determination of the molar absorption coefficient of the prosthetic group (18 400 M-1 cm-1 at 249 nm) enables its quantitative anaysis. Thus it could be established that methanol dehydrogenase contains one prosthetic group per enzyme molecule. The consequences of this result in relation to already known properties of this 'quinoprotein' dehydrogenase are discussed.

Alcohol Oxidoreductases↗
PubMed1981

Characterization of the second prosthetic group in methanol dehydrogenase from hyphomicrobium X.

Procedures are described for preparing 2,7,9-tricarboxy-1H-pyrrolo [2, 3-f]quinoline-4,5-diol (pyrrolo-quinoline quinol) from 2.7,9-tricarboxy-1 H-pyrrolo[2,3-f]quinoline-4,5-dione (pyrrolo-quinoline quinone). When methanol dehydrogenase is denatured, two compounds are liberated which have the same properties as the quinone and quinol mentioned above. On analysing the extract by high-performance liquid chromatography, one molecule of the quinone and one molecule of the quinol per enzyme molecule are found. Mixtures of pyrrolo-quinoline quinone and pyrrolo-quinoline quinol at high pH produce the semiquinone form and, under certain conditions, a diamagnetic complex. Since electron spin resonance (ESR) shows that methanol dehydrogenase contains the semiquinone and the absorption spectrum suggests the presence of a diamagnetic dimer, it is tentatively concluded that the two prosthetic group molecules in the enzyme interact with each other. NMR experiments of pyrrolo-quinoline quinone in 2H2O demonstrate that it is partly hydrated, most probably at the C-5 position. Although methanol adds in the same way, it is still questionable whether the product of this addition plays a role in the mechanism of the enzymic reaction. Potentiometric titrations show a midpoint potential of the quinone/quinol couple of + 90 mV at pH 7.0 and the formation of the semiquinone as an intermediate in the titration at pH 13.0.

Alcohol Oxidoreductases↗
PubMed1982

Covalent addition of H2O, enzyme substrates and activators to pyrrolo-quinoline quinone, the coenzyme of quinoproteins.

The fluorescence excitation and the adsorption spectrum of 2,7,9-tricarboxy-1H-pyrrolo [2,3-f]quinoline-4,5-dione (pyrrolo-quinoline quinone, PQQ), measured in the pH range 7.0-10.0, are quite different. However, when the temperature of the solution is lowered, the shape and maxima of these spectra become more similar. 1H-NMR in 2H2O revealed a temperature-dependent equilibrium between PQQ and a hydrated form. Evidence is presented that low temperature favours the formation of the fluorescing species which is PQQ, hydrated at the C-5 position. Even further hydration is possible since absorption, fluorescence and NMR spectroscopy of PQQ in borate buffer pH 10.0 reveal additional hydration at the C-4 position, pointing to a dihydrate. PQQ also reacts with quinoprotein enzyme substrates and activators. Spectroscopic measurements showed the existence of 5-alkoxy-5-hydroxy-PQQ and 5-amino-5-hydroxy-PQQ in the presence of alcohols and 2 M NH4Cl, pH 9.0, respectively. In the latter case, the existence of 5-imino-PQQ could also be demonstrated. Addition compounds with amines appear to be unstable. The amines become probably oxidized because pyrrolo-quinoline quinol (PQQH2) was found as the reaction product. On the other hand, an addition compound containing an imino bond could be isolated after addition of urea to a PQQ solution. Spectral characteristics of PQQ and its addition compounds are presented since these data are necessary for the spectral analysis of quinoproteins and the quantitative estimation of coenzyme.

Chemical Phenomena↗
PubMed1989

On the mechanism of inhibition of methanol dehydrogenase by cyclopropane-derived inhibitors.

Extraction of cyclopropanol-inactivated methanol dehydrogenase (MDH) gave a mixture of two interconverting compounds. The same compounds could be prepared from 2,7,9-tricarboxy-1H-pyrrolo[2,3-f]quinoline-4,5-dione (PQQ) and cyclopropanol using a metal oxide (e.g. Ag2O) as a catalyst. Structure elucidation revealed that a C5 3-propanal adduct of PQQ is formed which is present in the extract as a diastereoisomeric mixture of the ring-closed form. Cyclopropanone gave an analogous product, while cyclopropylmethanol behaved as a substrate and was oxidized by the enzyme without ring-opening. From the work described, several arguments can be derived to reject the idea that inactivation proceeds via formation of a pair of free radicals. The mechanism probably consists of a concerted proton abstraction, rearrangement of the cyclopropoxy anion to a ring-opened carbanion and attack of the latter on the electrophilic C5 of PQQ. The measured rate of inactivation (3.7 s-1) is in agreement with such a mechanism. The role of the metal oxide and the enzyme in this process is the catalysis of the addition step and possibly a positioning of the reactants. As only a sole type of quinoprotein alcohol dehydrogenase becomes inhibited, the cyclopropane derivatives studied here can be regarded as mechanism-based inhibitors. The modified PQQ in cyclopropanone-inactivated MDH is fluorescent. A fluorescent intermediate was also observed in the catalytic cycle of MDH with methanol as a substrate. Its rate of formation and decay and the strongly decreased level of fluorescence in the presence of activator are in accordance with the view that the fluorescing species is the previously found oxidized-MDH.substrate (MDHox.S) complex. Since the decomposition of this complex requires activator and model studies have failed so far to mimic the enzyme, it seems that the combination of enzyme and activator is essential for the oxidation of the alcohol substrate.

Alcohol Oxidoreductases↗
PubMed1995

Quinohaemoprotein ethanol dehydrogenase from Comamonas testosteroni. Purification, characterization, and reconstitution of the apoenzyme with pyrroloquinoline quinone analogues.

Pyrroloquinoline-quinone(PQQ)-free quinohaemoprotein ethanol dehydrogenase (QH-EDH) apoenzyme was isolated from ethanol-grown Comamonas testosteroni. The purified apoenzyme, showing a single band of 71 kDa on native gel electrophoresis, could be only partially converted into active holoenzyme by addition of PQQ in the presence of calcium ions. In addition to a band with a molecular mass of 71 kDa, additional bands of 51 kDa and 25 kDa were observed with SDS/PAGE. Analysis of the N-terminal sequences of the bands and comparison with the DNA sequence of the gene, suggested that the latter two originate from the former one, due to scission occurring at a specific site between two vicinal residues in the protein chain. The extent of scission appeared to increase during growth of the organism. After addition of PQQ to apoenzyme, holoenzyme and nicked, inactive enzyme could be separated. Holoenzyme prepared in this way was found to contain equimolar amounts of PQQ, Ca2+ and covalently bound haem. EPR spectra of fully oxidized apo-QH-EDH and holo-QH-EDH showed g values typical for low-spin haem c proteins. In partially oxidized holo-QH-EDH an organic radical signal attributed to the semiquinone form of PQQ was observed. Binding of PQQ leads to conformational changes, as reflected by changes of spectral and chromatographic properties. Reconstitution of apoenzyme with PQQ analogues resulted in a decreased activity and enantioselectivity for the oxidation of chiral alcohols. Compared with PQQ, analogues with a large substituent had a lower affinity for the apoenzyme. Results with other analogues indicated that possession of the o-quinone/o-quinol moiety is not essential for binding but it is for activity.

Alcohol Oxidoreductases↗
PubMed1997

Ca2+ and its substitutes have two different binding sites and roles in soluble, quinoprotein (pyrroloquinoline-quinone-containing) glucose dehydrogenase.

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 Ca2+ 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 Ca2+ can be removed from M* with chelator, transforming it into M. Reassociation of M* to D occurs spontaneously at 20 degrees 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 Mg2+, Ba2+, Co2+, Ni2+, Zn2+, 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 Ca2+ 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.

Acinetobacter calcoaceticus↗
PubMedDate not supplied

The protective effect of pyrroloquinoline quinone and its derivatives against carbon tetrachloride-induced liver injury of rats.

Pyrroloquinoline quinone (PQQ) and its derivative, oxazo pyrroloquinoline (OPQ-G), protected rats from experimental liver injury induced by carbon tetrachloride (CCl4) in vivo. This effect was observed after an intraperitoneal injection of 5 mg/kg PQQ or OPQ-G, which was given twice, 10 min and 1 h before CCl4 administration. Pyrroloquinoline quinone protected primary cultured rat hepatocytes from CCl4 toxicity in vitro. This protection was most effective at a concentration of 3 mumol/L PQQ. Pyrroloquinoline quinone derivatives (oxazo pyrroloquinoline, methyl-thioethyl oxazo pyrroloquinoline and PQQ-allylester) also protected the hepatocytes from CCl4 toxicity. Pyrroloquinoline quinone and its derivatives inhibited the lucigenin-enhanced chemiluminescence from isolated hepatocytes initiated by CCl4. These results suggest that eliminating free radicals is one of the protective mechanisms of PQQ and its derivatives against CCl4-induced liver injury.

Acridines↗
PubMed1993

Energy conservation by pyrroloquinoline quinol-linked xylose oxidation in Pseudomonas putida NCTC 10936 during carbon-limited growth in chemostat culture.

When grown in carbon source-limited chemostat cultures with lactate or glucose as the carbon and energy source and xylose as an additional source of reducing equivalents. Pseudomonas putida NCTC 10936 oxidized xylose to xylonolactone and xylonate. No other products were formed from this pentose, nor was it incorporated into biomass. The presence of xylose in these cultures resulted in higher Yglucose and Ylactate values as compared to cultures without xylose indicating that biologically useful energy was conserved during the periplasmic oxidation of xylose. As the Y0 values for growth on glucose or on lactate alone were equal to the Y0 values for growth with xylose as co-substrate, it is concluded that for glucose- or lactate-limited growth energy conservation by PQQH2 oxidation is as efficient as by NADH2 oxidation.

Bacterial Proteins↗
PubMed1994

New unified nomenclature for genes involved in the oxidation of methanol in gram-negative bacteria.

The system involving the oxidation of methanol to formaldehyde in Gram-negative methylotrophic bacteria is complex. A total of 32 genes have been reported, termed mox, for methanol oxidation, and it is possible that more will be identified. Some mox genes carrying out completely different functions have been given the same designations by different laboratories and others have been given separate designations that were later discovered to be the same. It is now important to change the mox nomenclature to remedy this confusing situation. This communication proposes a new nomenclature for genes involved in methanol oxidation based on currently known linkage groups.

Genes, Bacterial↗
PubMed1996

Identification and characterization of the pqqDGC gene cluster involved in pyrroloquinoline quinone production in an obligate methylotroph Methylobacillus flagellatum.

Pyrroloquinoline quinone is a prosthetic group of bacterial methanol dehydrogenases as well as some alcohol and glucose dehydrogenases. Genes involved in pyrroloquinoline quinone production have previously been cloned from the representatives of the alpha and gamma subdivisions of the Proteobacteria. We report identification and the sequence of the pqqDGC gene cluster in the obligate methylotroph, Methylobacillus flagellatum, which belongs to the beta subdivision. The deduced products of the pqq genes from M. flagellatum appear to be more similar to their counterparts from non-methylotrophic species of the gamma subdivision than to a facultative methylotroph of the alpha subdivision. A non-polar mutation in pqqG was constructed and resulted in a strain impaired in growth on methanol. This mutant accumulated a detectable amount of intracellular pyrroloquinoline quinone, but in contrast to the wild type, did not excrete pyrroloquinoline quinone into the culture medium. The possible role of PqqG is discussed.

Amino Acid Sequence↗
PubMed1998

Expression of genes from Rahnella aquatilis that are necessary for mineral phosphate solubilization in Escherichia coli.

Rahnella aquatilis is a Gram-negative bacterium that can fix atmospheric nitrogen and also has the ability to solubilize mineral phosphate. We have cloned the genes that confer the mineral phosphate solubilizing (Mps) trait from this organism by mobilizing a cosmid library of R. aquatilis into Escherichia coli HB101. A 7.0-kb EcoRI fragment from a cosmid, when transferred into E. coli strains HB101 and DH5 alpha, conferred the ability to solubilize hydroxyapatite and the production of gluconic acid to E. coli. The relative amounts of soluble phosphate and gluconic acid produced by the cloned 7.0-kb EcoRI fragment in E. coli were significantly higher than those of R. aquatilis. Nucleotide sequence analysis revealed two complete open reading frames (ORF1 and ORF2) and a partial ORF, ORF1 and ORF2 encoded proteins of molecular mass 10 kDa and 44 kDa. The 44-kDa protein showed extensive sequence similarity to pqqE of Erwinia herbicola, Klebsiella pneumoniae and A. Acinetobacter calcoaceticus. The 10-kDa protein revealed strong similarity to the pqqD of K. pneumoniae and A. calcoaceticus.

Chemotactic Factors↗