Absorbance-based assay for membrane disruption by antimicrobial peptides and synthetic copolymers using pyrroloquinoline quinone-loaded liposomes

A simple homogeneous assay for the detection of membrane permeabilization by antimicrobial peptides and synthetic copolymers is described. Liposomes encapsulating pyrroloquinoline quinone (PQQ), the prosthetic group of the apoenzyme glucose dehydrogenase (GDH), are used to detect membrane permeabilization by the antimicrobial peptides MSI-594 and MSI-78 as well as various synthetic antimicrobial copolymers in an optical microwell assay. PQQ-loaded liposomes and the peptide or copolymer are added to wells of a 96-well microtiter plate. If the integrity of the liposome is compromised, the PQQ encapsulated in the liposomes is released and available for activating the apoenzyme. The release of PQQ catalyzes a color change in the presence of apo-GDH, glucose, and the redox dye 1,6-dichlorophenol indophenol (DCPIP) that can be evaluated through a visual color change. For more quantitative measurements, the absorbance change over a 30 min period was measured. The absorbance change is related to the activity and concentration for a given antimicrobial agent. Furthermore, by varying liposome compositions to include cholesterol, the potential toxicity of the peptide or polymer toward mammalian cells can be readily evaluated. The assay is simple and sensitive and will be useful for analyzing the membrane permeation/disruption properties of a host of antimicrobial peptides and synthetic polymers.     

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.Abbreviations PQQ 2, 7, 9-tricarboxy-1H-pyrrolo-(2,3-f)quinoline-4,5-dione - WB Wurster's Blue (1,4-bis-(dimethylamino)-benzene perchlorate)     

The quinoprotein dehydrogenases for methanol and glucose

This review summarises our current understanding of two of the main types of quinoprotein dehydrogenase in which pyrroloquinoline quinone (PQQ) is the only prosthetic group. These are the soluble methanol dehydrogenase and the membrane glucose dehydrogenase (mGDH). The membrane GDH has an additional N-terminal domain by which it is tightly anchored to the membrane, and a periplasmic domain whose structure has been modelled on the X-ray structure of the alpha-subunit of MDH which contains PQQ in the active site. This review discusses their structures and mechanisms, concentrating particularly on the pathways for electron transfer from the reduced PQQ, through the protein, to their electron acceptors. In MDH, this is the specific cytochrome c(L), the electron transfer pathway probably involving the unique disulphide ring in the active site. By contrast, mGDH contains a permanently bound ubiquinone, which acts as a single electron carrier, mediating electron transfer through the protein to the membrane ubiquinone.     

Fluorescence resonance energy transfer (FRET) using ssDNA binding fluorescent dye

There is a need for simple and inexpensive methods for genotyping single nucleotide polymorphisms (SNPs) and short insertion/deletion variations (InDels). In this work, I demonstrate that a single-stranded DNA (ssDNA) binding dye can be used as a donor fluorophore for fluorescence resonance energy transfer (FRET). The method presented is a homogenous assay in which detection is based on the FRET from the fluorescence of the ssDNA dye bound to the unmodified detection primer to the fluorescent nucleotide analog incorporated into this detection primer during cyclic template directed primer extension reaction. Collection of the FRET emission spectrum with a scanning fluorescence spectrophotometer allows powerful data analysis. The fluorescence emission signal is modified by the optical properties of the assay vessel. This seems to be a completely neglected parameter. By proper selection of the optical properties of the assay plate one can improve the detection of the fluorescence emission signal.     

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 mol 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.Abbreviations HEPES N-2-hydroxy-ethylpiperazine-N-2-ethane sulphonic acid - MES 2-morpholinoethane sulphonic acid - PQQ pyrroloquinoline quinone (systematic name: 2,7,9-tricarboxy-1H-pyrrolo-(2,3-f)-quinoline-4,5-dione) - WB Wurster's Blue (systematic name: 1,4-bis-(dimethylamino)-benzene perchlorate     

Non-coordinated synthesis of glucose dehydrogenase and its prosthetic group PQQ in Acinetobacter and Pseudomonas species

Abstract The activity of pyrrolo-quinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH) was determined in Acinetobacter and Pseudomonas species, grown under different conditions. In Acinetobacter lwoffi which, in contrast to Acinetobacter calcoaceticus , is unable to oxidize glucose to gluconic acid, the absence of GDH activity was not due to the absence of GDH protein (apoenzyme) but to the absence of its prosthetic group, PQQ. GDH activity could be restored by addition of PQQ to cell suspensions. Taxonomic implication of these results are discussed. Pseudomonas aeruginosa , strain PAO1 is known to contain active GDH when grown aerobically on glucose, but to lack this activity when grown anaerobically with nitrate. Also in this organism the absence of active GDH was due to lack of PQQ synthesis under these conditions, since GDH activity could be reconstituted by addition of PQQ to cell-free extracts.
Similar observations were made with cultures of Pseudomonas acidovorans and Rhodopseudomonas sphaeroides , indicating that control of GDH activity by PQQ synthesis maybe widespread among bacteria.     

Amperometric DNA sensor using the pyrroquinoline quinone glucose dehydrogenase-avidin conjugate

A new amperometric DNA sensor was constructed using a pyrroquinoline quinone glucose dehydrogenase ((PQQ)GDH) conjugated with avidin. Our aim was to specifically detect the DNA sequence of the invA virulence gene from the pathogenic bacterium Salmonella. Probe DNA with a sequence complementary to that of a specific fragment of the invA gene was immobilized onto a carbon paste electrode. After hybridization with biotinylated target DNA, (PQQ)GDH-avidin conjugate was added and the resulting electric current was measured. The electric current is generated from glucose oxidization catalyzed by (PQQ)GDH via 1-methoxyphenazine methosulphate (m-PMS) electron mediator. The sensor response increased with the addition of glucose and in the presence of 6.3 mM glucose the response increased with increasing DNA in the range 5.0x10(-8)-1.0x10(-5) M.     

Wiring of PQQ-dehydrogenases

The performance of pyrroloquinoline quinone (PQQ) dependent alcohol dehydrogenase (ADH) and two types of PQQ-glucose dehydrogenases in solution and when immobilized on the carbon paste electrodes modified with ferrocene derivatives is investigated. The immobilization of ADH consisting of PQQ and four hemes improves its stability up to 10 times. Both PQQ and heme moieties are involved in the electron transport from substrate to electrode. The ferrocene derivatives improve the electron transport 10-fold. Membrane-bound alcohol dehydrogenase from Gluconobacter sp. 33, intracellular soluble glucose dehydrogenase from Acinetobacter calcoaceticus L.M.D. 79.41 (s-GDH), and the membrane-bound enzyme (m-GDH) from Erwinia sp. 34-1 were purified and investigated. Soluble and membrane-bound PQQ-glucose dehydrogenases display different behavior during the immobilization on the modified carbon electrodes. The immobilization of s-GDH leads to a decrease in both stability and substrate specificity of the enzyme. This suggests that PQQ dissociates from the enzyme active center and operates as a free-diffusing mediator. The rate-limiting step of the process is likely the loading of PQQ onto the apo-enzyme. The immobilization of m-GDH leads to its substantial stabilization and improves the substrate specificity. The nature of m-GDH binding to the electrode surface is presumably similar to the binding to the cell membrane through its anchor-subunit. The enzyme operates as an enzyme and mediator complex.     

Interaction of ethidium bromide with DNA. Optical and electrooptical study

The binding of ethidium bromide to DNA has been studied by various optical methods. From fluorescence polarization studies, and film, electric linear dichroism, and circular dichroism spectra, we propose assignments of the absorption bands of the dye, which are discussed in connection with wave-mechanical calculations recently reported. The optical activity induced in the dye absorption bands upon binding to DNA was attributed to various origins depending on the electronic transition considered. The visible absorption band displayed a circular dichroism due to the asymmetry of the binding site and independent of the amount of binding. The transition identified at 378 nm from the circular dichroism and electric dichroism observations was thought to be due to a magnetic-dipole transition. It remained constant with increasing amounts of dye bound. The main ultraviolet band showed circular dichroism characteristics corresponding to exciton interactions between dye molecules bound to neighboring sites. The electric dichroism observed for the strongly bound dye molecules indicated that the phenanthridinium ring of ethidium bromide was probably not perfectly parallel to the DNA base planes. When the amount of dye bound to DNA exceeded the maximum amount compatible with the exclusion of adjacent binding sites, the electric dichroism decreased owing to the appearance of externally bound dye molecules with no contribution to the dichroism. Sonicated DNA was used to study the lengthening of the DNA molecule upon complexation. Although the viscosity of the complexes increased with the amount of binding, the rotational diffusion coefficient measured by the electric birefringence relaxation was not detectably affected. The absence of variation in the electric birefringence with the binding indicated that the DNA base stacking remained unaltered.     

Fluorometric determination of deoxyribonuclease I activity with PicoGreen

A rapid and sensitive assay for the detection of deoxyribonuclease I (DNase I) activity is described. This method is based on the ability of PicoGreen dye to enhance its fluorescence when bound to double-stranded DNA. In the standard assay, reaction mixtures containing the DNase I sample and 0.2 microg of the substrate DNA were prepared in a fluorescence microtiter plate and incubated at 37 degrees C. At the end of the reaction, the diluted PicoGreen reagent was added to each well and fluorescence intensity was measured with a fluorescence plate reader. By this assay, it was possible to determine precisely as little as 5 pg of DNase I within an hour. Moreover, using a small amount of the substrate DNA, the method was shown to be suitable for the sensitive detection of DNase I inhibitor activity.     

Evidence for a membrane-bound pyrroloquinoline quinone-linked glucose dehydrogenase in Acetobacter diazotrophicus

Acetobacter diazotrophicus possesses a pyrroloquinoline quinone-linked glucose dehydrogenase (PQQ-GDH). The enzyme seemingly belongs to the type II PQQ-GDH enzymes and, at least under the culture conditions tested, the organism synthesizes enough PQQ to saturate the apo-enzyme. The synthesis of this enzyme is stimulated when the organism is grown under N₂-fixing conditions. It is proposed that this enzyme may play an important role in providing extra energy in N₂-fixing cells.     

The consequence of stimulating glucose dehydrogenase activity by the addition of PQQ on metabolite production by Agrobacterium radiobacter NCIB 11883

Summary Agrobacterium radiobacter NCIB 11 883 does not produce gluconate under conditions of glucose excess in batch or continuous culture. However, the addition of micromolar concentrations of pyrrolo quinoline quinone (PQQ) to fermentation media resulted in rapid excretion of gluconate by batch and continuous cultures. This rapid dehydrogenation of glucose was found in cells grown under carbon and nitrogen limitation and is constitutive which suggests that the only reason why this activity is not normally expressed is due to the inability of the organism to synthesize the prosthetic group (PQQ) of the glucose dehydrogenase enzyme.Although the addition of PQQ to batch and continuous cultures caused a very rapid specific rate of gluconate production (0.6–1.1 g gluconate g^-1 dry wt. h^-1) the rate of exopolysaccharide production remained unaltered. Indeed, when the rates of substrate and oxygen uptake are corrected for the rate of gluconate production in the presence of PQQ there appears to be little physiological consequence as a result of this oxidation.     

Use of bromthymol blue for studying the thermotropic properties of the liposomes from egg lecithin

The dependence of the turbidity changes at 615 nm monobilayers liposomes from egg yolk lecithin in the presence of bromothymol blue on temperature and storage conditions has been investigated. It is established that the thermotropic properties of liposomes change irregularly and depend on the storage conditions. Sharp release of the bound dye at temperature above 35-37 degrees C is associated with thermotropic change in liposomes and the detected effects, with the change of orientation of the phosphorylcholine group.     

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.     

吡咯喹啉醌（PQQ）的研究进展

吡咯喹啉醌是一种与烟酰胺核苷酸、黄素核苷酸不同的新型辅基.近年来,荷兰、日本等学者对它进行了初步研究,而国内研究起步较晚,文章综述了吡咯喹啉醌的发现、分离纯化、鉴定、理化性质以及生理功能,这有利于进一步研究吡咯喹啉醌的分布、产生机理、生物学性质、生理功能及其应用.这将对促进酶学学科的发展具有重要的理论和实践意义.     

On the biosynthesis of free and covalently bound PQQ. Glutamic acid decarboxylase from Escherichia coli is a pyridoxo-quinoprotein

Analysis of glutamic acid decarboxylase (GDC) (EC 4.1.1.15) from Escherichia coli ATCC 11246 revealed the presence of six pyridoxal phosphates (PLPs) as well as six covalently bound pyrroloquinoline quinones (PQQs) per hexameric enzyme molecule. This is the second example of a pyridoxo-quinoprotein, suggesting that other atypical pyridoxoproteins (PLP-containing enzymes) have similar cofactor composition. Since the organism did not produce free PQQ and its quinoprotein glucose dehydrogenase was present in the apo form, free PQQ is not used in the assemblage of GDC. Most probably, biosynthesis of covalently bound cofactor occurs in situ via a route which is different from that of free PQQ. Thus, organisms previously believed to be unable to synthesize (free) PQQ could in fact be able to produce quinoproteins with covalently bound cofactor. Implications for the role of PQQ in eukaryotic cells are discussed.     

The enzymatic reaction-induced configuration change of the prosthetic group PQQ of methanol dehydrogenase

Methanol dehydrogenase is a heterotetrameric enzyme containing the prosthetic group pyrroloquinoline quinone (PQQ), which catalyzes the oxidation of methanol to formaldehyde. The crystal structure of methanol dehydrogenase from Methylophilus W3A1, previously determined at high resolution, exhibits a non-planar configuration of the PQQ ring system and lends support for a hydride transfer mechanism of the enzymatic reaction catalyzed by the enzyme. To investigate why PQQ is in the C5-reduced form and to better understand the catalytic mechanism of the enzyme, three structures of this enzyme in a new crystal form have been determined at higher resolution. Two of the three crystals were grown in the presence of 1 and 50 mM methanol, respectively, both structures of which show non-planar configurations of the PQQ ring system, confirming the previous conclusion; the other was crystallized in the presence of 50 mM ethanol, the structure of which displays a planar ring system for PQQ. Comparison of these structures reveals that the configuration change of PQQ is induced by the enzymatic reaction. The reaction takes place and the C5-reduced PQQ intermediate is produced when the enzyme co-crystallizes with methanol, but the enzymatic reaction does not take place and the PQQ ring retains a planar configuration of the oxidized orthoquinone form when ethanol instead of methanol is present in the crystallization solution.     

Selection of glucose-assimilating variants ofAcinetobacter calcoaceticus LMD 79.41 in chemostat culture

Glucose metabolism has been studied in two strains ofAcinetobacter calcoaceticus. Strain LMD 82.3, was able to grow on glucose and possessed glucose dehydrogenase (EC 1.1.99.17). Glucose oxidation by whole cells was stimulated by PQQ, the prosthetic group of glucose dehydrogenase. PQQ not only increased the rate of glucose oxidation and gluconic acid production but also shortened the lag phase for growth on glucose. Strain LMD 79.41 also possessed glucose dehydrogenase but was unable to grow on glucose. Batch cultures and carbon-limited chemostat cultures growing on acetate in the presence of glucose oxidized the sugar to gluconic acid, which was not further metabolized. However, after prolonged cultivation on mixtures of acetate and glucose, carbon-limited chemostat cultures suddenly acquired the capacity to utilize gluconate. This phenomenon was accompanied by the appearance of gluconate kinase and a repression of isocitrate lyase synthesis. In contrast to the starter culture, cells from chemostats which had been fully adapted to gluconate utilization, were able to utilize glucose as a sole carbon and energy source in liquid and solid media.     

The structure and mechanism of methanol dehydrogenase

This is a review of recent work on methanol dehydrogenase (MDH), a pyrroloquinoline quinone (PQQ)-containing enzyme catalysing the oxidation of methanol to formaldehyde in methylotrophic bacteria. Although it is the most extensively studied of this class of dehydrogenases, it is only recently that there has been any consensus about its mechanism. This is partly due to recent structural studies on normal and mutant enzymes and partly due to more definitive work on the mechanism of related alcohol and glucose dehydrogenases. This work has also led to conclusions about the subsequent path of electrons and protons during the reoxidation of the reduced quinol form of the prosthetic group.     

Adduct formation of pyrroloquinoline quinone and amino acid

When pyrroloquinoline quinone (PQQ) is mixed with an amino acid, a corresponding Schiff base PQQ adduct is readily formed between carbonyl groups of PQQ and the primary amino group. A potent growth stimulating effect for microorganisms was observed with the PQQ adduct when it was administered in a culture medium. Although PQQ itself shows a marked growth stimulating effect, PQQ adducts appeared to be more active than authentic PQQ when compared on a molar basis. Conversely, unlike authentic PQQ, PQQ adducts were shown to be less active (greater than or equal to 100-fold) as the prosthetic group for a quinoprotein apo-glucose dehydrogenase when examined by holoenzyme formation by exogenous addition of PQQ or PQQ adducts. These observations suggested that PQQ adduct formation readily occurs during isolation procedures for PQQ from biological materials or PQQ - chromophore from quinoproteins. Therefore, the presence of such adducts gives a PQQ estimation much lower than theoretically expected. As an example, formation, isolation and characterization of PQQ - serine are described.