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Genes involved in the biosynthesis of PQQ from Acinetobacter calcoaceticus.

From a gene bank of the Acinetobacter calcoaceticus genome a plasmid was isolated that complements four different classes of PQQ- mutants. Subclones of this plasmid revealed that the four corresponding PQQ genes are located on a fragment of 5 kilobases. The nucleotide sequence of this 5 kb fragment was determined and by means of Tn5 insertion mutants the reading frames of the PQQ genes could be identified. Three of the PQQ genes code for proteins of Mr 29700 (gene I), Mr 10800 (gene II) and Mr 43600 (gene III) respectively. In the DNA region where gene IV was mapped however the largest possible reading frame encodes for a polypeptide of only 24 amino acids. A possible role for this small polypeptide will be discussed. Finally we show that expression of the four PQQ genes in Acinetobacter 1woffi and Escherichia coli lead to the synthesis of the coenzyme in these organisms.

Acinetobacter↗

PQQ: biosynthetic studies in Methylobacterium AM1 and Hyphomicrobium X using specific 13C labeling and NMR.

Using 13C labeling and NMR spectroscopy we have determined biosynthetic precursors of pyrroloquinoline quinone (PQQ) in two closely related serine-type methylotrophs, Methylobacterium AM1 and Hyphomicrobium X. Analysis of the 13C-labeling data revealed that PQQ is constructed from two amino acids: the portion containing N-6,C-7, 8, 9 and the two carboxylic acid groups, C-7' and 9', is derived-intact -from glutamate. The remaining portion is derived from tyrosine; the phenol side chain provides the six carbons of the ring containing the orthoquinone, whereas internal cyclization of the amino acid backbone forms the pyrrole-2-carboxylic acid moiety. This is analogous to the cyclization of dopaquinone to form dopachrome. Dopaquinone is a product of the oxidation of tyrosine (via dopa) in reactions catalyzed by monophenol monooxygenase (EC 1.14.18.1). Starting with tyrosine and glutamate, we will discuss possible biosynthetic routes to PQQ.

Amino Acids↗

Mutants of Methylobacterium organophilum unable to synthesize PQQ.

The phenotype of mutants unable to synthesize PQQ is analyzed for different categories of methylotrophic bacteria. The advantages offered by strains dissimilating methylamine through methylated amino-acids are discussed. In M.organophilum, 40% of the mutants unable to grow in methanol medium but with normal methylamine utilization, were affected in PQQ metabolism. The genetic properties of M. organophilum useful to study PQQ mutants are discussed, mainly the use of pSUP106 to create insertion mutations in the bacterial chromosome and to replace wild-type genes by modified genes. An example is given of the possibility to create R' plasmids containing large fragments of M.organophilum DNA. Some physiological properties of a PQQ mutant are described, regarding growth kinetics, PQQ uptake and accumulation.

Coenzymes↗

Degradation pathway of 2-chloroethanol in Pseudomonas stutzeri strain JJ under denitrifying conditions.

The pathway of 2-chloroethanol degradation in the denitrifying Pseudomonas stutzeri strain JJ was investigated. In cell-free extracts, activities of a phenazine methosulfate (PMS)-dependent chloroethanol dehydrogenase, an NAD-dependent chloroacetaldehyde dehydrogenase, and a chloroacetate dehalogenase were detected. This suggested that the 2-chloroethanol degradation pathway in this denitrifying strain is the same as found in aerobic bacteria that degrade chloroethanol. Activity towards primary alcohols, secondary alcohols, diols, and other chlorinated alcohols could be measured in cell-free extracts with chloroethanol dehydrogenase (CE-DH) activity. PMS and phenazine ethosulfate (PES) were used as primary electron acceptors, but not NAD, NADP or ferricyanide. Cells of strain JJ cultured in a continuous culture under nitrate limitation exhibited chloroethanol dehydrogenase activity that was a 12 times higher than in cells grown in batch culture. However, under chloroethanol-limiting conditions, CE-DH activity was in the same range as in batch culture. Cells grown on ethanol did not exhibit CE-DH activity. Instead, NAD-dependent ethanol dehydrogenase (E-DH) activity and PMS-dependent E-DH activity were detected.

Aldehyde Oxidoreductases↗

Detection of V-type nerve agent degradation products at electrodes modified by PPy/PQQ using CaCl2 as supporting electrolyte.

Electrochemical detection without derivatization was used to detect thiol-containing degradation products of V-type nerve agents. Electropolymerization of pyrrole was used for entrapment of the biocatalyst PQQ to produce a sensor. Various parameters which affect the detection processes such as the type of the supporting electrolyte used during electrodeposition and the thickness of the polypyrrole film were examined and optimized. Electocatalytic oxidation of thiols by the PPy/PQQ electrode was strongly affected by the presence of Ca2+ cations during electrodeposition of the PPy/PQQ. Cyclic voltammetry, linear sweep voltammetry and amperometry have been used for electrode characterization. Amperometric detection of the V-type nerve agent thiol degradation products 2-(dimethylamino)ethanethiol (DMAET) and 2-(diethylamino)ethanethiol (DEAET) was performed at 0.38 V. Linear calibration plots were observed for these compounds. The detection limits of 4.5 and 3 microM were obtained for DMAET and DEAET respectively, with sensitivities of 1.18 and 1.37 nA microM(-1) cm(-2).

Biosensing Techniques↗

New developments in oxidative fermentation.

Oxidative fermentations have been well established for a long time, especially in vinegar and in L-sorbose production. Recently, information on the enzyme systems involved in these oxidative fermentations has accumulated and new developments are possible based on these findings. We have recently isolated several thermotolerant acetic acid bacteria, which also seem to be useful for new developments in oxidative fermentation. Two different types of membrane-bound enzymes, quinoproteins and flavoproteins, are involved in oxidative fermentation, and sometimes work with the same substrate but produce different oxidation products. Recently, there have been new developments in two different oxidative fermentations, D-gluconate and D-sorbitol oxidations. Flavoproteins, D-gluconate dehydrogenase, and D-sorbitol dehydrogenase were isolated almost 2 decades ago, while the enzyme involved in the same oxidation reaction for D-gluconate and D-sorbitol has been recently isolated and shown to be a quinoprotein. Thus, these flavoproteins and a quinoprotein have been re-assessed for the oxidation reaction. Flavoprotein D-gluconate dehydrogenase and D-sorbitol dehydrogenase were shown to produce 2-keto- D-gluconate and D-fructose, respectively, whereas the quinoprotein was shown to produce 5-keto- D-gluconate and L-sorbose from D-gluconate and D-sorbitol, respectively. In addition to the quinoproteins described above, a new quinoprotein for quinate oxidation has been recently isolated from Gluconobacter strains. The quinate dehydrogenase is also a membrane-bound quinoprotein that produces 3-dehydroquinate. This enzyme can be useful for the production of shikimate, which is a convenient salvage synthesis system for many antibiotics, herbicides, and aromatic amino acids synthesis. In order to reduce energy costs of oxidative fermentation in industry, several thermotolerant acetic acid bacteria that can grow up to 40 degrees C have been isolated. Of such isolated strains, some thermotolerant Acetobacter species were found to be useful for vinegar fermentation at a high temperature such 38-40 degrees C, where mesophilic strains showed no growth. They oxidized higher concentrations of ethanol up to 9% without any appreciable lag time, while alcohol oxidation with mesophilic strains was delayed or became almost impossible under such conditions. Several useful Gluconobacter species of thermotolerant acetic acid bacteria are also found, especially L-erythrulose-producing strains and cyclic alcohol-oxidizing strains. Gluconobacter frateurii CHM 43 is able to rapidly oxidize meso-erythritol at 37 degrees C leading to the accumulation of L-erythrulose, which may replace dihydroxyacetone in cosmetics. G. frateuriiCHM 9 is able to oxidize cyclic alcohols to their corresponding cyclic ketones or aliphatic ketones, which are known to be useful for preparing many different physiologically active compounds such as oxidized steroids or oxidized bicyclic ketones. The enzymes involved in these meso-erythritol and cyclic alcohol oxidations have been purified and shown to be a similar type of membrane-bound quinoproteins, consisting of a high molecular weight single peptide. This is completely different from another quinoprotein, alcohol dehydrogenase of acetic acid bacteria, which consists of three subunits including hemoproteins.

Acetic Acid↗

Correlation between acetic acid resistance and characteristics of PQQ-dependent ADH in acetic acid bacteria.

In this study, we compared the growth properties and molecular characteristics of pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) among highly acetic acid-resistant strains of acetic acid bacteria. Gluconacetobacter europaeus exhibited the highest resistance to acetic acid (10%), whereas Gluconacetobacter intermedius and Acetobacter pasteurianus resisted up to 6% of acetic acid. In media with different concentrations of acetic acid, the maximal acetic acid production rate of Ga. europaeus slowly increased, but specific growth rates decreased concomitant with increased concentration of acetic acid in medium. The lag phase of A. pasteurianus was twice and four times longer in comparison to the lag phases of Ga. europaeus and Ga. intermedius, respectively. PQQ-dependent ADH activity was twice as high in Ga. europaeus and Ga. intermedius as in A. pasteurinus. The purified enzymes showed almost the same specific activity to each other, but in the presence of acetic acid, the enzyme activity decreased faster in A. pasteurianus and Ga. intermedius than in Ga. europaeus. These results suggest that high ADH activity in the Ga. europaeus cells and high acetic acid stability of the purified enzyme represent two of the unique features that enable this species to grow and stay metabolically active at extremely high concentrations of acetic acid.

Acetic Acid↗

Periplasmic PQQ-dependent glucose oxidation in free-living and symbiotic rhizobia.

The expression of the pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH) of Rhizobium tropici CIAT899 and Sinorhizobium meliloti RCR2011 was investigated under different nutrient-limiting conditions in continuous cultures, under different conditions of phosphate availability, and in S. meliloti bacteroids. The presence of free PQQ in alfalfa root exudates has also been assayed. It was shown that apo-GDH or holoenzyme was actively synthesized by these rhizobia, with the concomitant production of gluconate from glucose, under certain environmental conditions. GDH activity was also detected in bacteroids from alfalfa root nodules inoculated with either S. meliloti RCR2011 or 102F34. It was also shown that free PQQ was present in root exudates of alfalfa, but its production is ascribed to the activity of Erwinia sp., a normal contaminant of these seeds.

Culture Media↗

Cloning and characterization of a novel human homolog* of mouse U26, a putative PQQ-dependent AAS dehydrogenase.

Mouse U26 has been defined as a 2-aminoadipic 6-semialdehyde dehydrogenase. It was speculated to be a PQQ-dependent AAS dehydrogenase due to the research of demonstrating PQQ as a new B vitamin. We isolated a novel human cDNA from the human fetal brain cDNA library we constructed. Its deduced protein was most related to mouse U26. Thus, we termed it human U26. This putative protein contains an AMP-binding domain, a Phosphopantetheine-binding domain and six PQQ-binding motifs. Human U26 mRNA is ubiquitously expressed in adult tissues and is highly expressed in colon adenocarcinoma (CX-1) and colon adenocarcinoma (GI-112) cell lines. Further study should be made to clarify the precise function of human U26.

Aldehyde Dehydrogenase↗

Detection and determination of pyrroloquinoline quinone, the coenzyme of quinoproteins.

A convenient determination of pyrroloquinoline quinone (PQQ) in extracts of purified enzymes is possible with ion-pair chromatography on a HPLC reverse-phase column and with uv detection. However, when culture supernatants have to be analyzed, a fluorescence detection system is more appropriate. Proof for the presence of PQQ can be obtained by treating such a sample with butyraldehyde, which converts the coenzyme into a stable adduct having a suitable retention time in the system. The sensitivity and selectivity of the analysis can be further enhanced by reducing the sample with NaBH4, which produces the dihydrodiol form of the coenzyme (PQQH4) and oxidizing PQQH4 with NaIO4 to a strongly fluorescing compound. A procedure is described for the easy preparation of an apoenzyme from the quinoprotein glucose dehydrogenase of Pseudomonas aeruginosa strains. With this biological test system, very low amounts of PQQ can be detected. However, when PQQ is present in the form of adducts, the analysis has to be performed via reduction to PQQH4, oxidation with NaIO4, and HPLC.

Apoenzymes↗

Method of enzymatic determination of pyrroloquinoline quinone.

An improved enzymatic method for the determination of pyrroloquinoline quinone, a novel prosthetic group of some important oxidoreductases, has been developed with cytoplasmic membrane of Escherichia coli K-12, in which D-glucose dehydrogenase (EC 1.1.99.17) was completely resolved to apo-enzyme by EDTA treatment. Incubation of the EDTA-treated membrane with exogenous pyrroloquinoline quinone in the presence of magnesium ions gave a quantitative determination of pyrroloquinoline quinone by assaying the restored D-glucose dehydrogenase activity. This novel enzymatic method was confirmed to be highly reproducible up to 10 ng of pyrroloquinoline quinone and could be applied to a routine assay of pyrroloquinoline quinone.

Cell Membrane↗

Enzymatic determination of pyrroloquinoline quinone using crude membranes from Escherichia coli.

Escherichia coli K-12 (ATCC 10 798) contains only the apoform of membrane-bound D-glucose dehydrogenase (pyrroloquinoline quinone-dependent). Crude membrane preparations or even crude cell extracts are suitable for a sensitive determination of pyrroloquinoline quinone under appropriate conditions. The apoform of D-glucose dehydrogenase from E. coli is reconstituted with pyrroloquinoline quinone to the holoenzyme in the presence of Mg2+ and assayed for glucose dehydrogenase activity. By increasing the time of reconstitution it is possible to detect minute amounts of about 20 pg pyrroloquinoline quinone.

Apoenzymes↗

Amperometric electrochemical detection of pyrroloquinoline quinone in high-performance liquid chromatography.

The electrochemistry of pyrroloquinoline quinone (PQQ) enables its reduction and oxidation at a variety of electrode surfaces. Two methods of PQQ quantitation are described using amperometric electrochemical detection after HPLC separation. In one method a single electrode is used to reduce PQQ in the eluant stream and is sensitive to as little as 10 pmol of material. The second method is a dual electrode method that takes advantage of the reversible nature of the PQQ redox cycle and though only half as sensitive as the single electrode method, it is more specific. The advantages of the method lie in its simplicity, sensitivity, and low cost.

Chromatography, High Pressure Liquid↗

An electron-nuclear double-resonance study of methanol dehydrogenase and its coenzyme radical.

Proton as well as deuteron ENDOR (electron-nuclear double resonance) spectroscopy were performed of methanol dehydrogenase and pyrrolo-quinoline semiquinone (PQQH). Samples were examined in H2O- and 2H2O-containing buffers at 4.2 degrees K with Ka-band (33.5 GHz) frequency. Measurements of the enzyme in 2H2O revealed that the signals observed around the proton free-precession frequency belong to exchangeable protons. Therefore, our earlier assumption (R. de Beer et al. (1979) J. Chem. Phys. 70, 4491-4495) that these signals originate from protons in the aromatic ring of PQQH is incorrect. The proton matrix signal of the enzyme in H2O and 2H2O are nearly similar, while a deuteron matrix signal is not observed in the latter case. It is concluded, therefore, that the coenzyme is situated in a hydrophobic site of the enzyme.

Alcohol Oxidoreductases↗

Failure to verify the presence of pyrroloquinoline quinone (PQQ) in bovine plasma amine oxidase by gas chromatography/mass spectrometry.

The presence of covalently bound pyrroloquinoline quinone (PQQ) in bovine plasma amine oxidase (BPAO) was examined by the use of gas chromatography/mass spectrometry. The enzyme was subjected to proteolysis with proteinase in the presence of [U-13C]PQQ as an internal standard. After isolation and 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 (SIM). In the SIM profile, although the sample extract obtained from BPAO treated with proteinase clearly showed the peak at m/z 462 for the internal standard, there were no peaks detectable at m/z 448, showing the absence of PQQ in the proteolysis digest of BPAO. Thus, our results do not support the claim that BPAO contains covalently bound PQQ in its structure.

Amine Oxidase (Copper-Containing)↗

Nitrile hydratase is a quinoprotein. A possible new function of pyrroloquinoline quinone: activation of H2O in an enzymatic hydration reaction.

Nitrile hydratase has been proved to be a quinoprotein with pyrroloquinoline quinone (PQQ) as a prosthetic group. The broad shoulder from 300 to 500 nm in the absorption spectrum of Brevibacterium nitrile hydratase suggested the presence of PQQ. Since PQQ was attached to the enzyme through a covalent linkage, the chromophores were isolated by acid hydrolysis, protease digestion and successive chromatographic separation. The isolated chromophores showed the similar spectroscopic characteristics to those of obtained from the amine oxidase of Aspergillus niger, in which PQQ is covalently linked. The isolated chromophores potently activated apo-D-glucose dehydrogenase (EC 1.1.99.17), supporting the presence of PQQ or a PQQ-like compound in nitrile hydratase. The finding of PQQ in nitrile hydratase strongly suggests a new function of PQQ, i.e., the activation of H2O in the enzymatic hydration reaction.

Brevibacterium↗

Significant interaction between low-spin iron(III) site and pyrroloquinoline quinone in active center of nitrile hydratase.

Nitrile hydratase, a unique non-heme iron enzyme, contains low-spin Fe(III) site and pyrroloquinoline quinone in the active center. The enzymatic activity of nitrile hydratase is strongly inhibited by typical carbonyl reagents such as phenylhydrazine and semicarbazide. Of special interest is the fact that these carbonyl reagents induced significant alteration of the ESR features of the native iron(III) enzyme at pH 7.2. In addition, the isobutyronitrile(inhibitor)-bound enzyme was also remarkably affected by phenylhydrazine and then the transformed ESR characteristics were different from those of phenylhydrazine-treated enzyme. The present results reveal the first evidence for an important interaction between the low-spin Fe(III) site and pyrroloquinoline quinone which is essential for the activity of nitrile hydratase.

Binding Sites↗