Cellular Uptake of Pyrroloquinoline Quinone in Its Intact and Derivatized Forms from the Cell Culture Medium of 3T3-L1.

Following a growing interest in the physiological effects of pyrroloquinoline quinone (PQQ), more cell culture experiments have begun to elucidate its mechanism of action. However, to our knowledge, no reports have used instrumental analysis, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), to study cellular uptake of PQQ. In addition, despite the propensity of PQQ to react with amino acids and other compounds, only a handful of cell culture experiments have been conducted on PQQ derivatives. In the present study, we prepared PQQ derivatives by reacting PQQ with various amino acids and used them as reference standards for optimizing the LC-MS/MS analysis conditions to detect PQQ and its derivatives. Using this method, we evaluated the uptake of PQQ into mouse 3T3-L1 cells and found that most PQQ added to the medium was taken up by the cells in its unchanged form, while some PQQ reacted with amino acids in the medium and was taken up by the cells as PQQ derivatives. These results suggest that PQQ derivatives may contribute to the physiological effects of PQQ. To further elucidate the function of PQQ, it is necessary for future studies to clarify the activity of PQQ derivatives and to evaluate the types of PQQ present in food, animal, and cell samples in more detail.

Insights into Pyrroloquinoline Quinone (PQQ) Effects on Soil Nutrients and Pathogens from Pepper Monocropping Soil under Anaerobic and Aerobic Conditions.

Imbalances of soil available nutrients and soilborne diseases have seriously restricted the productivity of crops and jeopardized food security worldwide. Pyrroloquinoline quinone (PQQ), a redox cofactor in some bacteria involved in glucose metabolism and phosphorus mineralization, could be anticipated to alter soil ecosystems to a certain extent. However, there is limited information on PQQ defending soilborne pathogens and regulating soil main nutrients. Here, a pot experiment based on mono-cropping soils of pepper was conducted to examine the effects of PQQ amendment on reconstructing soil microbial communities and soil nutrients under aerobic/anaerobic conditions comprising three treatments, namely, control, PQQ (aerobic), and FL-PQQ (anaerobic). The results revealed that soil microbial community composition and soil nutrients were distinctly altered by PQQ regimes. Compared to control, PQQ treatment significantly increased the content of soil available phosphorus (AP), while FL_PQQ treatment strongly improved the content of soil available nitrogen (AN). In terms of pathogens, relative to control, both PQQ treatments suppressed the abundances of pathogens, of which FL_PQQ treatment significantly decreased the abundance of the pathotrophic fungal by 64% and the abundance of Fusarium oxysporum by 57%, largely attributed to the increase of organic acid generators (Oxobacter, Hydrogenispora) and potential antagonists (Bacillus, Talaromyces). Structural equation modeling (SEM) showed that PQQ regimes suppressed pathogens by indirectly regulating soil physicochemical properties and microbial communities. Overall, we proposed that PQQ application both in aerobic/anaerobic conditions could improve soil available nutrients and suppress soil pathogens in pepper monocropping soils. IMPORTANCE The attention to PQQ (pyrroloquinoline quinone) effect on soil nutrients and pathogens was less paid in monocropping soils. However, the underlying microbial interacting mechanism remains unclear. Adopting a novel external bio-additive, the effects of PQQ on soil main nutrients and the pathotrophic fungal under aerobic and anaerobic regimes will be investigated, which would help to improve soil quality health. Our main conclusion was that PQQ would help to remediate monocropping obstacle soils in terms of soil nutrients and soil pathogens by associating with the microbial community, and anaerobic PQQ application more favored amelioration of continuous obstacle soils. These results will benefit the health and sustainable development of pepper production as well as other greenhouse vegetable production.

Studies of pyrroloquinoline quinone species in solution and in lanthanide-dependent methanol dehydrogenases.

Pyrroloquinoline quinone (PQQ) is a redox cofactor in calcium- and lanthanide-dependent alcohol dehydrogenases that has been known and studied for over 40 years. Despite its long history, many questions regarding its fluorescence properties, speciation in solution and in the active site of alcohol dehydrogenase remain open. Here we investigate the effects of pH and temperature on the distribution of different PQQ species (H3PQQ to PQQ3- in addition to water adducts and in complex with lanthanides) with NMR and UV-Vis spectroscopy as well as time-resolved laser-induced fluorescence spectroscopy (TRLFS). Using a europium derivative from a new, recently-discovered class of lanthanide-dependent methanol dehydrogenase (MDH) enzymes, we utilized two techniques to monitor Ln binding to the active sites of these enzymes. Employing TRLFS, we were able to follow Eu(III) binding directly to the active site of MDH using its luminescence and could quantify three Eu(III) states: Eu(III) in the active site of MDH, but also in solution as PQQ-bound Eu(III) and in the aquo-ion form. Additionally, we used the antenna effect to study PQQ and simultaneously Eu(III) in the active site.

Structure-Function Analysis of a Quinone-Dependent Dehydrogenase Capable of Deoxynivalenol Detoxification.

The pyrroloquinoline quinone (PQQ)-dependent dehydrogenase DepA detoxifies deoxynivalenol (DON) by converting the C3-OH into a keto group. Herein, two crystal structures of DepA and its complex with PQQ were determined, together with biochemical evidence confirming the interactions of DepA with PQQ and DON and revealing a unique tyrosine residue important for substrate selection. Furthermore, four loops over the active site essential for DepA activity were identified, of which three loops were stabilized by PQQ, and the fourth loop invisible in both structures was considered important for binding DON, together constituting a lid for the active site. Preliminary engineering of the loop showed its potential for enzyme improvement. This study provides structural insights into how a PQQ-dependent dehydrogenase is equipped with the function of DON conversion and for the first time shows the necessity of a lid structure for PQQ-dependent dehydrogenase activity, laying foundation for structure-based design to enhance catalysis efficiency.

Ion Mobility Studies of Pyrroloquinoline Quinone Aza-Crown Ether-Lanthanide Complexes.

Lanthanide-dependent enzymes and their biomimetic complexes have arisen as an interesting target of research in the past decade. These enzymes, specifically, pyrroloquinoline quinone (PQQ)-bearing methanol dehydrogenases, efficiently convert alcohols to the respective aldehydes. To rationally design bioinspired alcohol dehydrogenation catalysts, it is imperative to understand the species involved in catalysis. However, given the extremely flexible coordination sphere of lanthanides, it is often difficult to assess the number and nature of the active species. Here, we show how such questions can be addressed by using a combination of ion mobility spectrometry, mass spectrometry, and quantum-chemical calculations to study the test systems PQQ and lanthanide-PQQ-crown ether ligand complexes. Specifically, we determine the gas-phase structures of [PQQH2]-, [PQQH2+H2O]-, [PQQH2+MeOH]-, [PQQ-15c5+H]+, and [PQQ-15c5+Ln+NO3]2+ (Ln = La to Lu, except Pm). In the latter case, a trend to smaller collision cross sections across the lanthanide series is clearly observable, in line with the well-known lanthanide contraction. We hope that in the future such investigations will help to guide the design and understanding of lanthanide-based biomimetic complexes optimized for catalytic function.

pH-Triggered Transition from Micellar Aggregation to a Host-Guest Complex Accompanied by a Color Change.

A pH-triggered transition from micellar aggregation to a host-guest complex was achieved based on the supramolecular interactions between calixpyridinium and pyrroloquinoline quinone disodium salt (PQQ-2Na) accompanied by a color change. Our design has the following three advantages: (1) a regular spherical micellar assembly is fabricated by the supramolecular interactions between calixpyridinium and PQQ-2Na at pH 6 in an aqueous solution, (2) increasing the pH can lead to a transition from micellar aggregation to a host-guest complex due to the deprotonation of calixpyridinium, and at the same time (3) increasing the pH can lead to a color change owing to the deprotonation of calixpyridinium and the complexation of deprotonated calixpyridinium with PQQ-2Na. Benefitting from the low toxicity of calixpyridinium and PQQ-2Na, this pH-induced transition from micellar aggregation to a host-guest complex was further studied as a controllable-release model.

Understanding the biological role of PqqB in Pseudomonas stutzeri using molecular dynamics simulation approach.

Phosphate solubilization is an important and widely studied plant growth promoting trait exhibited by many bacteria. Pyrroloquinoline quinone (PQQ), a redox cofactor of methanol and glucose dehydrogenases has been well established as essential for phosphate solubilization. PQQ operon has been well studied in growth promoting rhizobacteria like Pseudomonas spp., Gluconobacter oxydans, Klebsiella pneumoniae, etc. However, the role of PqqB is quite ambiguous as its functional role has been contradicted in many studies. In the present study, we selected Pseudomonas stutzeri - a well-known P solubilizing bacterium as a representative species of the Pseudomonas genus on the basis of phylogenetic and statistical analyses of PqqB proteins. A 3 D model was generated for this protein. Docking of PqqB with PQQ showed good interaction with a theoretical binding affinity of -7.4 kcal/mol. On the other hand, docking of PqqC with 3a-(2-amino-2-carboxy-ethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydro-quinoline-7,9-dicarboxylic acid (AHQQ, immediate precursor of PQQ) showed strong interaction (-10.4 kcal/mol) but the same was low with PQQ (-6.4 kcal/mol). Molecular dynamic simulation of both the complexes showed stable conformation. The binding energy of PqqB-PQQ complex (-182.710 ± 16.585 kJ/mol) was greater than PqqC-PQQ complex (-166.114 ± 12.027 kJ/mol). The results clearly indicated that kinetically there is a possibility that after cyclization of AHQQ to PQQ by PqqC, PQQ can be taken up by PqqB and transported to periplasm for the oxidation of glucose. To the best of our knowledge, this is the first attempt to understand the biological role of PqqB on the basis of molecular interactions and dynamics.Communicated by Ramaswamy H. Sarma.

Effects of Pyrroloquinoline Quinone on Lipid Metabolism and Anti-Oxidative Capacity in a High-Fat-Diet Metabolic Dysfunction-Associated Fatty Liver Disease Chick Model.

Metabolic dysfunction-associated fatty liver disease (MAFLD) and its interaction with many metabolic pathways raises global public health concerns. This study aimed to determine the therapeutic effects of Pyrroloquinoline quinone (PQQ, provided by PQQ.Na2) on MAFLD in a chick model and primary chicken hepatocytes with a focus on lipid metabolism, anti-oxidative capacity, and mitochondrial biogenesis. The MAFLD chick model was established on laying hens by feeding them a high-energy low-protein (HELP) diet. Primary hepatocytes isolated from the liver of laying hens were induced for steatosis by free fatty acids (FFA) and for oxidative stress by hydrogen peroxide (H2O2). In the MAFLD chick model, the dietary supplementation of PQQ conspicuously ameliorated the negative effects of the HELP diet on liver biological functions, suppressed the progression of MAFLD mainly through enhanced lipid metabolism and protection of liver from oxidative injury. In the steatosis and oxidative stress cell models, PQQ functions in the improvement of the lipid metabolism and hepatocytes tolerance to fatty degradation and oxidative damage by enhancing mitochondrial biogenesis and then increasing the anti-oxidative activity and anti-apoptosis capacity. At both the cellular and individual levels, PQQ was demonstrated to exert protective effects of hepatocyte and liver from fat accumulation through the improvement of mitochondrial biogenesis and maintenance of redox homeostasis. The key findings of the present study provide an in-depth knowledge on the ameliorative effects of PQQ on the progression of fatty liver and its mechanism of action, thus providing a theoretical basis for the application of PQQ, as an effective nutrient, into the prevention of MAFLD.

Epimerization of Deoxynivalenol by the Devosia Strain A6-243 Assisted by Pyrroloquinoline Quinone.

Deoxynivalenol (DON) is a secondary metabolite produced by several Fusarium species that is hazardous to humans and animals after entering food chains. In this study, by adding cofactors, the Devosia strain A6-243 is identified as the DON-transforming bacteria from a bacterial consortium with the ability to biotransform DON of Pseudomonas sp. B6-24 and Devosia strain A6-243, and its effect on the biotransformation process of DON is studied. The Devosia strain A6-243 completely biotransformed 100 µg/mL of DON with the assistance of the exogenous addition of PQQ (pyrroloquinoline quinone) within 48 h and produced non-toxic 3-epi-DON (3-epi-deoxynivalenol), while Pseudomonas sp. B6-24 was not able to biotransform DON, but it had the ability to generate PQQ. Moreover, the Devosia strain A6-243 not only degraded DON, but also exhibited the ability to degrade 3-keto-DON (3-keto-deoxynivalenol) with the same product 3-epi-DON, indicating that DON epimerization by the Devosia strain A6-243 is a two-step enzymatic reaction. The most suitable conditions for the biodegradation process of the Devosia strain A6-243 were a temperature of 16-37 °C and pH 7.0-10, with 15-30 µM PQQ. In addition, the Devosia strain A6-243 was found to completely remove DON (6.7 µg/g) from DON-contaminated wheat. The results presented a reference for screening microorganisms with the ability of biotransform DON and laid a foundation for the development of enzymes for the detoxification of mycotoxins in grain and its products.

Bioconversion of recombinantly produced precursor peptide pqqA into pyrroloquinoline quinone (PQQ) using a cell-free in vitro system.

Pyrroloquinoline quinone (PQQ) has been recognized as the third class of redox cofactors in addition to the well-known nicotinamides (NAD(P)+) and flavins (FAD, FMN). It plays important physiological roles in various organisms and has strong antioxidant properties. The biosynthetic pathway of PQQ involves a gene cluster composed of 4-7 genes, named pqqA-G, among which pqqA is a key gene for PQQ synthesis, encoding the precursor peptide PqqA. To produce recombinant PqqA in E. coli, fusion tags were used to increase the stability and solubility of the peptide, as well simplify the scale-up of the fermentation process. In this paper, pqqA from Gluconobacter oxydans 621H was expressed in E. coli BL21 (DE3) as a fusion protein with SUMO and purified using a hexahistidine (His6) tag. The SUMO fusion protein and His6 tag were specifically recognized and cleaved by the SUMO specific ULP protease, and immobilized-metal affinity chromatography was used to obtain high-purity precursor peptide PqqA. Expression and purification of target proteins was confirmed by Tricine-SDS-PAGE. Finally, the synthesis of PQQ in a cell-free enzymatic reaction in vitro was confirmed by LC-MS.

Crystal structure of a calcium(II)-pyrroloquinoline quinone (PQQ) complex outside a protein environment.

Pyrroloquinoline quinone (PQQ) is an important cofactor of calcium- and lanthanide-dependent alcohol dehydrogenases, and has been known for over 30 years. Crystal structures of Ca-MDH enzymes (MDH is methanol dehydrogenase) have been known for some time; however, crystal structures of PQQ with biorelevant metal ions have been lacking in the literature for decades. We report here the first crystal structure analysis of a Ca-PQQ complex outside the protein environment, namely, poly[[undecaaquabis(µ-4,5-dioxo-4,5-dihydro-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylato)tricalcium(II)] dihydrate], {[Ca3(C14H3N2O8)2(H2O)11]·2H2O}n. The complex crystallized as Ca3PQQ2·13H2O with Ca2+ in three different positions and PQQ3-, including an extensive hydrogen-bond network. Similarities and differences to the recently reported structure with biorelevant europium (Eu2PQQ2) are discussed.

Ameliorate effect of pyrroloquinoline quinone against cyclophosphamide-induced nephrotoxicity by activating the Nrf2 pathway and inhibiting the NLRP3 pathway.

AIMS: Cyclophosphamide (CTX) is an effective anti-tumor and immunosuppressive agent, but it induces nephrotoxicity in clinical applications. The present study aimed to evaluate the protective effect of pyrroloquinoline quinone (PQQ) on CTX-induced nephrotoxicity. MAIN METHODS: We injected male ICR mice with CTX (80 mg/kg/day), and determined nephrotoxicity indices, MDA and antioxidant defenses, inflammatory cytokines, and the levels of main proteins in the Nrf2-HO-1 and NLRP3 signaling pathways. KEY FINDINGS: PQQ has significantly decreased the serum levels of creatinine and urea compared to Model group. When treated with PQQ, MDA, IL-1ß, IL-6, and TNF-α levels have decreased, and SOD, GSH-Px, and CAT activity have increased in the kidney tissues of CTX-induced mice. PQQ activated the Nrf2-mediated signaling pathway, as indicated by the increased expression of Nrf2, HO-1, GCLM, and NQO1. Moreover, PQQ inhibited the NLRP3 inflammatory pathway, as indicated by the reduced expression of NLRP3, ASC, and Caspase-1. SIGNIFICANCE: Our results suggest that PQQ protects against CTX-induced nephrotoxicity, probably by activating the Nrf2-mediated antioxidant pathway and inhibiting the NLRP3 inflammatory pathway.

A Microelectronic Sensor Device Powered by a Small Implantable Biofuel Cell.

Biocatalytic buckypaper electrodes modified with pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase and bilirubin oxidase for glucose oxidation and oxygen reduction, respectively, were prepared for their use in a biofuel cell. A small (millimeter-scale; 2×3×2 mm3 ) enzyme-based biofuel cell was tested in a model glucose-containing aqueous solution, in human serum, and as an implanted device in a living gray garden slug (Deroceras reticulatum), producing electrical power in the range of 2-10 µW (depending on the glucose source). A microelectronic temperature-sensing device equipped with a rechargeable supercapacitor, internal data memory and wireless data downloading capability was specifically designed for activation by the biofuel cell. The power management circuit in the device allowed the optimized use of the power provided by the biofuel cell dependent on the sensor operation activity. The whole system (power-producing biofuel cell and power-consuming sensor) operated autonomously by extracting electrical energy from the available environmental source, as exemplified by extracting power from the glucose-containing hemolymph (blood substituting biofluid) in the slug to power the complete temperature sensor system and read out data wirelessly. Other sensor systems operating autonomously in remote locations based on the concept illustrated here are envisaged for monitoring different environmental conditions or can be specially designed for homeland security applications, particularly in detecting bioterrorism threats.

Crystal Structure of the Catalytic and Cytochrome b Domains in a Eukaryotic Pyrroloquinoline Quinone-Dependent Dehydrogenase.

Pyrroloquinoline quinone (PQQ) was discovered as a redox cofactor of prokaryotic glucose dehydrogenases in the 1960s, and subsequent studies have demonstrated its importance not only in bacterial systems but also in higher organisms. We have previously reported a novel eukaryotic quinohemoprotein that exhibited PQQ-dependent catalytic activity in a eukaryote. The enzyme, pyranose dehydrogenase (PDH), from the filamentous fungus Coprinopsis cinerea (CcPDH) of the Basidiomycete division, is composed of a catalytic PQQ-dependent domain classified as a member of the novel auxiliary activity family 12 (AA12), an AA8 cytochrome b domain, and a family 1 carbohydrate-binding module (CBM1), as defined by the Carbohydrate-Active Enzymes (CAZy) database. Here, we present the crystal structures of the AA12 domain in its apo- and holo-forms and the AA8 domain of this enzyme. The crystal structures of the holo-AA12 domain bound to PQQ provide direct evidence that eukaryotes have PQQ-dependent enzymes. The AA12 domain exhibits a six-blade ß-propeller fold that is also present in other known PQQ-dependent glucose dehydrogenases in bacteria. A loop structure around the active site and a calcium ion binding site are unique among the known structures of bacterial quinoproteins. The AA8 cytochrome domain has a positively charged area on its molecular surface, which is partly due to the propionate group of the heme interacting with Arg181; this feature differs from the characteristics of cytochrome b in the AA8 domain of the fungal cellobiose dehydrogenase and suggests that this difference may affect the pH dependence of electron transfer.IMPORTANCE Pyrroloquinoline quinone (PQQ) is known as the "third coenzyme" following nicotinamide and flavin. PQQ-dependent enzymes have previously been found only in prokaryotes, and the existence of a eukaryotic PQQ-dependent enzyme was in doubt. In 2014, we found an enzyme in mushrooms that catalyzes the oxidation of various sugars in a PQQ-dependent manner and that was a PQQ-dependent enzyme found in eukaryotes. This paper presents the X-ray crystal structures of this eukaryotic PQQ-dependent quinohemoprotein, which show the active site, and identifies the amino acid residues involved in the binding of the cofactor PQQ. The presented X-ray structures reveal that the AA12 domain is in a binary complex with the coenzyme, clearly proving that PQQ-dependent enzymes exist in eukaryotes as well as prokaryotes. Because no biosynthetic system for PQQ has been reported in eukaryotes, future research on the symbiotic systems is expected.

Lanthanide-induced conformational change of methanol dehydrogenase involving coordination change of cofactor pyrroloquinoline quinone.

There is emerging interest in the role of lanthanides as cofactors for XoxF-type methanol dehydrogenase (MDH). Here, classical molecular dynamics simulations combined with fragment molecular orbital calculations were employed to rationalize the enzymatic activities of MDH (both XoxF- and MxaF-types) carrying different lanthanides. In XoxF-type MDH, lanthanide binding to cofactor pyrroloquinoline quinone was found to switch from tridentate to unidentate fashion as it switches from lighter to heavier lanthanides. This fact possibly plays a crucial role in the enzymatic activity exclusive to XoxF-type MDH incorporating lighter lanthanides.

Electrocatalysis of a Europium-Dependent Bacterial Methanol Dehydrogenase with Its Physiological Electron-Acceptor Cytochrome†cGJ.

We report the first electrochemical study of a lanthanoid-dependent methanol dehydrogenase (Eu-MDH) from the acidophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV with its own physiological cytochrome cGJ electron acceptor. Eu-MDH harbours a redox active 2,7,9-tricarboxypyrroloquinoline quinone (PQQ) cofactor which is non-covalently bound but coordinates trivalent lanthanoid elements including Eu3+ . Eu-MDH and the cytochrome were co-adsorbed with the biopolymer chitosan and cast onto a mercaptoundecanol (MU) monolayer modified Au working electrode. Cyclic voltammetry of cytochrome cGJ reveals a well-defined quasi-reversible FeIII/II redox couple at +255 mV vs. NHE at pH 7.5 and this response is pH independent. The reversible one-electron response of the cytochrome cGJ transforms into a sigmoidal catalytic wave in the presence of Eu-MDH and its substrates (methanol or formaldehyde). The catalytic current was pH-dependent and pH 7.3 was found to be optimal. Kinetic parameters (pH dependence, activation energy) obtained by electrochemistry show the same trends as those obtained from an artificial phenazine ethosulfate/dichlorophenol indophenol assay.

Determination of pyrroloquinoline quinone by enzymatic and LC-MS/MS methods to clarify its levels in foods.

Pyrroloquinoline quinone (PQQ) is believed to be a new B vitamin-like compound, and PQQ supplementation has received attention as a possible treatment for diseases including dementia and diabetes. However, the distribution of PQQ in foods is unclear, due to the difficulty in analyzing the compound. Therefore, in this study, enzymatic and LC-MS/MS methods were optimized to enable an accurate analysis of PQQ in foods. The optimized methods were applied to the screening of foods, in which PQQ contents were identified in ng/g or ng/mL levels. Furthermore, we newly found that some foods related to acetic acid bacteria contain PQQ at 1.94~5.59 ng/mL higher than beer, which is known to contain relatively high amounts of PQQ. These results suggest that the optimized methods are effective for the screening of foods containing PQQ. Such foods with high PQQ content may be valuable as functional foods effective towards the treatment of certain diseases.

The PedS2/PedR2 Two-Component System Is Crucial for the Rare Earth Element Switch in Pseudomonas putida KT2440.

In Pseudomonas putida KT2440, two pyrroloquinoline quinone-dependent ethanol dehydrogenases (PQQ-EDHs) are responsible for the periplasmic oxidation of a broad variety of volatile organic compounds (VOCs). Depending on the availability of rare earth elements (REEs) of the lanthanide series (Ln3+), we have recently reported that the transcription of the genes encoding the Ca2+-utilizing enzyme PedE and the Ln3+-utilizing enzyme PedH are inversely regulated. With adaptive evolution experiments, site-specific mutations, transcriptional reporter fusions, and complementation approaches, we now demonstrate that the PedS2/PedR2 (PP_2671/PP_2672) two-component system (TCS) plays a central role in the observed REE-mediated switch of PQQ-EDHs in P. putida We provide evidence that in the absence of lanthanum (La3+), the sensor histidine kinase PedS2 phosphorylates its cognate LuxR-type response regulator PedR2, which in turn not only activates pedE gene transcription but is also involved in repression of pedH Our data further suggest that the presence of La3+ lowers kinase activity of PedS2, either by the direct binding of the metal ions to the periplasmic region of PedS2 or by an uncharacterized indirect interaction, leading to reduced levels of phosphorylated PedR2. Consequently, the decreasing pedE expression and concomitant alleviation of pedH repression causes-in conjunction with the transcriptional activation of the pedH gene by a yet unknown regulatory module-the Ln3+-dependent transition from PedE- to PedH-catalyzed oxidation of alcoholic VOCs.IMPORTANCE The function of lanthanides for methanotrophic and methylotrophic bacteria is gaining increasing attention, while knowledge about the role of rare earth elements (REEs) in nonmethylotrophic bacteria is still limited. The present study investigates the recently described differential expression of the two PQQ-EDHs of P. putida in response to lanthanides. We demonstrate that a specific TCS is crucial for their inverse regulation and provide evidence for a dual regulatory function of the LuxR-type response regulator involved. Thus, our study represents the first detailed characterization of the molecular mechanism underlying the REE switch of PQQ-EDHs in a nonmethylotrophic bacterium and stimulates subsequent investigations for the identification of additional genes or phenotypic traits that might be coregulated during REE-dependent niche adaptation.

Aqueous polythiophene electrosynthesis: A new route to an efficient electrode coupling of PQQ-dependent glucose dehydrogenase for sensing and bioenergetic applications.

In this study, polythiophene copolymers have been used as modifier for electrode surfaces in order to allow the immobilization of active pyrroloquinoline quinone dependent glucose dehydrogenase (PQQ-GDH) and to simultaneously improve the direct electrical connection of the enzyme with the electrode. Polymer films are electrosynthesized in aqueous solution without the need of surfactants onto carbon nanotubes modified gold electrodes from mixtures of 3-thiopheneacetic acid (ThCH2CO2H) and 3-methoxythiophene (ThOCH3) using a potentiostatic pulse method. Polythiophene deposition significantly improves the bioelectrocatalysis of PQQ-GDH: the process starts at - 200 mV vs. Ag/AgCl and allows well-defined glucose detection at 0 V vs. Ag/AgCl with high current density. Several parameters of the electro-polymerization method have been evaluated to maximize the anodic current output after enzyme coupling. The polymer deposited by this new procedure has been morphologically and chemically characterized by different methods (SEM, EDX, FT-IR, UV-Vis, XPS and Raman spectroscopy). The bioelectrocatalytic response towards increasing glucose concentrations exhibits a dynamic range extending from 1 µM to 2 mM. The low applied potential allows to avoid interferences from easily oxidizable substances such as uric acid and ascorbic acid. Short and long-term stability has been evaluated. Finally, the PQQ-GDH electrode has been coupled to a bilirubin oxidase (BOD)- and carbon nanotube-based cathode in order to test its performance as anode of a biofuel cell. The promising results suggest a further investigation of this kind of polymers and, in particular, the study of the interaction with other enzymes in order to employ them in building up biosensors and biofuel cells.

Pyrroloquinoline Quinone, a Redox-Active o-Quinone, Stimulates Mitochondrial Biogenesis by Activating the SIRT1/PGC-1α Signaling Pathway.

Pyrroloquinoline quinone (PQQ), a redox-active o-quinone found in various foods and mammalian tissues, has received an increasing amount of attention because of a number of health benefits that can be attributed to its ability to enhance mitochondrial biogenesis. However, its underlying molecular mechanism remains incompletely understood. We have now established that the exposure of mouse NIH/3T3 fibroblasts to a physiologically relevant concentration of PQQ significantly stimulates mitochondrial biogenesis. The exposure of NIH/3T3 cells to 10-100 nM PQQ for 48 h resulted in increased levels of Mitotracker staining, mitochondrial DNA content, and mitochondrially encoded cytochrome c oxidase subunit 1 (MTCO1) protein. Moreover, we observed that PQQ treatment induces deacetylation of the peroxisome proliferator-activated receptor-γ-coactivator 1α (PGC-1α) and facilitates its nuclear translocation and target gene expression but does not affect its protein levels, implying increased activity of the NAD+-dependent protein deacetylase sirtuin 1 (SIRT1). Indeed, treatment with a SIRT1 selective inhibitor, EX-527, hampered the ability of PQQ to stimulate PGC-1α-mediated mitochondrial biogenesis. We also found that the PQQ treatment caused a concentration-dependent increase in the cellular NAD+ levels, but not the total NAD+ and NADH levels. Our results suggest that PQQ-inducible mitochondrial biogenesis can be attributed to activation of the SIRT1/PGC-1α signaling pathway by enhancing cellular NAD+ formation.