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Sample records for adenine dinucleotide flavin

  1. Flavin adenine dinucleotide content of quinone reductase 2: analysis and optimization for structure-function studies.

    PubMed

    Leung, Kevin Ka Ki; Litchfield, David W; Shilton, Brian H

    2012-01-01

    Quinone reductase 2 (NQO2) is a broadly expressed enzyme implicated in responses to a number of compounds, including protein kinase inhibitors, resveratrol, and antimalarial drugs. NQO2 includes a flavin adenine dinucleotide (FAD) cofactor, but X-ray crystallographic analysis of human NQO2 expressed in Escherichia coli showed that electron density for the isoalloxazine ring of FAD was weak and there was no electron density for the adenine mononucleotide moiety. Reversed-phase high-performance liquid chromatography (HPLC) of the NQO2 preparation indicated that FAD was not present and only 38% of the protomers contained flavin mononucleotide (FMN), explaining the weak electron density for FAD in the crystallographic analysis. A method for purifying NQO2 and reconstituting with FAD such that the final content approaches 100% occupancy with FAD is presented here. The enzyme prepared in this manner has a high specific activity, and there is strong electron density for the FAD cofactor in the crystal structure. Analysis of NQO2 crystal structures present in the Protein Data Bank indicates that many may have sub-stoichiometric cofactor content and/or contain FMN rather than FAD. This method of purification and reconstitution will help to optimize structural and functional studies of NQO2 and possibly other flavoproteins.

  2. Surface enhanced Raman scattering investigation of protein-bound flavin adenine dinucleotide structure

    NASA Astrophysics Data System (ADS)

    Maskevich, S. A.; Strekal, N. D.; Artsukevich, I. M.; Kivach, L. N.; Chernikevich, I. P.

    1995-04-01

    The SERS spectra of alcohol oxidase from Pichia pastoris adsorbed on a silver electrode were obtained. The similarities and differences of these spectra with the SERS spectrum of free flavin adenine dinucleiotide were considered. The dependence of relative intensity of 1258 cm -1 band from the electrode potential in the protein SERS spectra differed from that of free flavin. From the data on this band being sensitive to the protein-flavin interaction a suggestion was made about incomplete dissociation of flavin from the protein. This conclusion is confirmed both by the fluorescence data and the SERS data on alcohol oxidase purified from Candida boidinii. The results of the SERS investigation of the interaction between the substrate, ethanol and the cofactor, FAD, as well as between protein-bound cofactor with the substrate are presented. The problem of retaining the protein enzyme activity is discussed.

  3. Biochemical characterization of a flavin adenine dinucleotide-dependent monooxygenase, ornithine hydroxylase from Pseudomonas aeruginosa, suggests a novel reaction mechanism.

    PubMed

    Meneely, Kathleen M; Lamb, Audrey L

    2007-10-23

    Pyoverdin is the hydroxamate siderophore produced by the opportunistic pathogen Pseudomonas aeruginosa under the iron-limiting conditions of the human host. This siderophore includes derivatives of ornithine in the peptide backbone that serve as iron chelators. PvdA is the ornithine hydroxylase, which performs the first enzymatic step in preparation of these derivatives. PvdA requires both flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) for activity; it was found to be a soluble monomer most active at pH 8.0. The enzyme demonstrated Michaelis-Menten kinetics in an NADPH oxidation assay, but a hydroxylation assay indicated substrate inhibition at high ornithine concentration. PvdA is highly specific for both substrate and coenzyme, and lysine was shown to be a nonsubstrate effector and mixed inhibitor of the enzyme with respect to ornithine. Chloride is a mixed inhibitor of PvdA with respect to ornithine but a competitive inhibitor with respect to NADPH, and a bulky mercurial compound (p-chloromercuribenzoate) is a mixed inhibitor with respect to ornithine. Steady-state experiments indicate that PvdA/FAD forms a ternary complex with NADPH and ornithine for catalysis. PvdA in the absence of ornithine shows slow substrate-independent flavin reduction by NADPH. Biochemical comparison of PvdA to p-hydroxybenzoate hydroxylase (PHBH, from Pseudomonas fluorescens) and flavin-containing monooxygenases (FMOs, from Schizosaccharomyces pombe and hog liver microsomes) leads to the hypothesis that PvdA catalysis proceeds by a novel reaction mechanism. PMID:17900176

  4. Role of key residues at the flavin mononucleotide (FMN):adenylyltransferase catalytic site of the bifunctional riboflavin kinase/flavin adenine dinucleotide (FAD) Synthetase from Corynebacterium ammoniagenes.

    PubMed

    Serrano, Ana; Frago, Susana; Velázquez-Campoy, Adrián; Medina, Milagros

    2012-11-08

    In mammals and in yeast the conversion of Riboflavin (RF) into flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) is catalysed by the sequential action of two enzymes: an ATP:riboflavin kinase (RFK) and an ATP:FMN adenylyltransferase (FMNAT). However, most prokaryotes depend on a single bifunctional enzyme, FAD synthetase (FADS), which folds into two modules: the C-terminal associated with RFK activity and the N-terminal associated with FMNAT activity. Sequence and structural analysis suggest that the 28-HxGH-31, 123-Gx(D/N)-125 and 161-xxSSTxxR-168 motifs from FADS must be involved in ATP stabilisation for the adenylylation of FMN, as well as in FAD stabilisation for FAD phyrophosphorolysis. Mutants were produced at these motifs in the Corynebacterium ammoniagenes FADS (CaFADS). Their effects on the kinetic parameters of CaFADS activities (RFK, FMNAT and FAD pyrophosphorilase), and on substrates and product binding properties indicate that H28, H31, N125 and S164 contribute to the geometry of the catalytically competent complexes at the FMNAT-module of CaFADS.

  5. The first step in polyethylene glycol degradation by sphingomonads proceeds via a flavoprotein alcohol dehydrogenase containing flavin adenine dinucleotide.

    PubMed

    Sugimoto, M; Tanabe, M; Hataya, M; Enokibara, S; Duine, J A; Kawai, F

    2001-11-01

    Several Sphingomonas spp. utilize polyethylene glycols (PEGs) as a sole carbon and energy source, oxidative PEG degradation being initiated by a dye-linked dehydrogenase (PEG-DH) that oxidizes the terminal alcohol groups of the polymer chain. Purification and characterization of PEG-DH from Sphingomonas terrae revealed that the enzyme is membrane bound. The gene encoding this enzyme (pegA) was cloned, sequenced, and expressed in Escherichia coli. The purified recombinant enzyme was vulnerable to aggregation and inactivation, but this could be prevented by addition of detergent. It is as a homodimeric protein with a subunit molecular mass of 58.8 kDa, each subunit containing 1 noncovalently bound flavin adenine dinucleotide but not Fe or Zn. PEG-DH recognizes a broad variety of primary aliphatic and aromatic alcohols as substrates. Comparison with known sequences revealed that PEG-DH belongs to the group of glucose-methanol-choline (GMC) flavoprotein oxidoreductases and that it is a novel type of flavoprotein alcohol dehydrogenase related (percent identical amino acids) to other, so far uncharacterized bacterial, membrane-bound, dye-linked dehydrogenases: alcohol dehydrogenase from Pseudomonas oleovorans (46%); choline dehydrogenase from E. coli (40%); L-sorbose dehydrogenase from Gluconobacter oxydans (38%); and 4-nitrobenzyl alcohol dehydrogenase from a Pseudomonas species (35%). PMID:11673442

  6. Electron-transfer studies with a new flavin adenine dinucleotide dependent glucose dehydrogenase and osmium polymers of different redox potentials.

    PubMed

    Zafar, Muhammad Nadeem; Wang, Xiaoju; Sygmund, Christoph; Ludwig, Roland; Leech, Dónal; Gorton, Lo

    2012-01-01

    A new extracellular flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase from Glomerella cingulata (GcGDH) was electrochemically studied as a recognition element in glucose biosensors. The redox enzyme was recombinantly produced in Pichia pastoris and homogeneously purified, and its glucose-oxidizing properties on spectrographic graphite electrodes were investigated. Six different Os polymers, the redox potentials of which ranged in a broad potential window between +15 and +489 mV versus the normal hydrogen electrode (NHE), were used to immobilize and "wire" GcGDH to the spectrographic graphite electrode's surface. The GcGDH/Os polymer modified electrodes were evaluated by chronoamperometry using flow injection analysis. The current response was investigated using a stepwisely increased applied potential. It was observed that the ratio of GcGDH/Os polymer and the overall loading of the enzyme electrode significantly affect the performance of the enzyme electrode for glucose oxidation. The best-suited Os polymer [Os(4,4'-dimethyl-2,2'-bipyridine)(2)(PVI)Cl](+) had a potential of +309 mV versus NHE, and the optimum GcGDH/Os polymer ratio was 1:2 yielding a maximum current density of 493 μA·cm(-2) at a 30 mM glucose concentration.

  7. Electron-transfer studies with a new flavin adenine dinucleotide dependent glucose dehydrogenase and osmium polymers of different redox potentials.

    PubMed

    Zafar, Muhammad Nadeem; Wang, Xiaoju; Sygmund, Christoph; Ludwig, Roland; Leech, Dónal; Gorton, Lo

    2012-01-01

    A new extracellular flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase from Glomerella cingulata (GcGDH) was electrochemically studied as a recognition element in glucose biosensors. The redox enzyme was recombinantly produced in Pichia pastoris and homogeneously purified, and its glucose-oxidizing properties on spectrographic graphite electrodes were investigated. Six different Os polymers, the redox potentials of which ranged in a broad potential window between +15 and +489 mV versus the normal hydrogen electrode (NHE), were used to immobilize and "wire" GcGDH to the spectrographic graphite electrode's surface. The GcGDH/Os polymer modified electrodes were evaluated by chronoamperometry using flow injection analysis. The current response was investigated using a stepwisely increased applied potential. It was observed that the ratio of GcGDH/Os polymer and the overall loading of the enzyme electrode significantly affect the performance of the enzyme electrode for glucose oxidation. The best-suited Os polymer [Os(4,4'-dimethyl-2,2'-bipyridine)(2)(PVI)Cl](+) had a potential of +309 mV versus NHE, and the optimum GcGDH/Os polymer ratio was 1:2 yielding a maximum current density of 493 μA·cm(-2) at a 30 mM glucose concentration. PMID:22091984

  8. Redox State of Flavin Adenine Dinucleotide Drives Substrate Binding and Product Release in Escherichia coli Succinate Dehydrogenase

    PubMed Central

    Cheng, Victor W.T.; Piragasam, Ramanaguru Siva; Rothery, Richard A.; Maklashina, Elena; Cecchini, Gary; Weiner, Joel H.

    2016-01-01

    The Complex II family of enzymes, comprising the respiratory succinate dehydrogenases and fumarate reductases, catalyze reversible interconversion of succinate and fumarate. In contrast to the covalent flavin adenine dinucleotide (FAD) cofactor assembled in these enzymes, the soluble fumarate reductases (e.g. that from Shewanella frigidimarina) that assemble a noncovalent FAD cannot catalyze succinate oxidation but retain the ability to reduce fumarate. In this study, an SdhA-H45A variant that eliminates the site of the 8α-N3-histidyl covalent linkage between the protein and the FAD was examined. The variants SdhA-R286A/K/Y and -H242A/Y, that target residues thought to be important for substrate binding and catalysis were also studied. The variants SdhA-H45A and -R286A/K/Y resulted in assembly of a noncovalent FAD cofactor, which led to a significant decrease (−87 mV or more) in its reduction potential. The variant enzymes were studied by electron paramagnetic resonance spectroscopy following stand-alone reduction and potentiometric titrations. The “free” and “occupied” states of the active site were linked to the reduced and oxidized states of the FAD, respectively. Our data allows for a proposed model of succinate oxidation that is consistent with tunnel diode effects observed in the succinate dehydrogenase enzyme and a preference for fumarate reduction catalysis in fumarate reductase homologues that assemble a noncovalent FAD. PMID:25569225

  9. Electrochemical behavior of flavin adenine dinucleotide adsorbed onto carbon nanotube and nitrogen-doped carbon nanotube electrodes.

    PubMed

    Goran, Jacob M; Stevenson, Keith J

    2013-11-01

    Flavin adenine dinucleotide (FAD) is a cofactor for many enzymes, but also an informative redox active surface probe for electrode materials such as carbon nanotubes (CNTs) and nitrogen-doped CNTs (N-CNTs). FAD spontaneously adsorbs onto the surface of CNTs and N-CNTs, displaying Langmuir adsorption characteristics. The Langmuir adsorption model provides a means of calculating the electroactive surface area (ESA), the equilibrium constant for the adsorption and desorption processes (K), and the Gibbs free energy of adsorption (ΔG°). Traditional ESA measurements based on the diffusional flux of a redox active molecule to the electrode surface underestimate the ESA of porous materials because pores are not penetrated. Techniques such as gas adsortion (BET) overestimate the ESA because it includes both electroactive and inactive areas. The ESA determined by extrapolation of the Langmuir adsorption model with the electroactive surface probe FAD will penetrate pores and only include electroactive areas. The redox activity of adsorbed FAD also displays a strong dependency on pH, which provides a means of determining the pKa of the surface confined species. The pKa of FAD decreases as the nitrogen content in the CNTs increases, suggesting a decreased hydrophobicity of the N-CNT surface. FAD desorption at N-CNTs slowly transforms the main FAD surface redox reaction with E1/2 at -0.84 V into two new, reversible, surface confined redox reactions with E1/2 at -0.65 and -0.76 V (vs Hg/Hg2SO4), respectively (1.0 M sodium phosphate buffer pH = 6.75). This is the first time these redox reactions have been observed. The new surface confined redox reactions were not observed during FAD desorption from nondoped CNTs.

  10. Urea induced unfolding dynamics of flavin adenine dinucleotide (FAD): spectroscopic and molecular dynamics simulation studies from femto-second to nanosecond regime.

    PubMed

    Sengupta, Abhigyan; Singh, Reman K; Gavvala, Krishna; Koninti, Raj Kumar; Mukherjee, Arnab; Hazra, Partha

    2014-02-20

    Here, we investigate the effect of urea in the unfolding dynamics of flavin adenine dinucleotide (FAD), an important enzymatic cofactor, through steady state, time-resolved fluorescence spectroscopic and molecular dynamics (MD) simulation studies. Steady state results indicate the possibility of urea induced unfolding of FAD, inferred from increasing emission intensity of FAD with urea. The TCSPC and up-conversion results suggest that the stack-unstack dynamics of FAD severely gets affected in the presence of urea and leads to an increase in the unstack conformation population from 15% in pure water to 40% in 12 M urea. Molecular dynamics simulation was employed to understand the nature of the interaction between FAD and urea at the molecular level. Results depict that urea molecules replace many of the water molecules around adenine and isoalloxazine rings of FAD. However, the major driving force for the stability of this unstack conformations arises from the favorable stacking interaction of a significant fraction of the urea molecules with adenine and isoalloxazine rings of FAD, which overcomes the intramolecular stacking interaction between themselves observed in pure water.

  11. Electrochemical synthesis and characterization of TiO2 nanoparticles and their use as a platform for flavin adenine dinucleotide immobilization and efficient electrocatalysis

    NASA Astrophysics Data System (ADS)

    Ashok Kumar, S.; Lo, Po-Hsun; Chen, Shen-Ming

    2008-06-01

    Here, we report the electrochemical synthesis of TiO2 nanoparticles (NPs) using the potentiostat method. Synthesized particles have been characterized by using x-ray diffraction (XRD) studies, atomic force microscopy (AFM) and scanning electron microscopy (SEM). The results revealed that the TiO2 film produced was mainly composed of rutile and that the particles are of a size in the range of 100 ± 50 nm. TiO2 NPs were used for the modification of a screen printed carbon electrode (SPE). The resulting TiO2 film coated SPE was used to immobilize flavin adenine dinucleotide (FAD). The flavin enzyme firmly attached onto the metal oxide surface and this modified electrode showed promising electrocatalytic activities towards the reduction of hydrogen peroxide (H2O2) in physiological conditions. The electrochemistry of FAD confined in the oxide film was investigated. The immobilized FAD displayed a pair of redox peaks with a formal potential of -0.42 V in pH 7.0 oxygen-free phosphate buffers at a scan rate of 50 mV s-1. The FAD in the nanostructured TiO2 film retained its bioactivity and exhibited excellent electrocatalytic response to the reduction of H2O2, based on which a mediated biosensor for H2O2 was achieved. The linear range for the determination of H2O2 was from 0.15 × 10-6 to 3.0 × 10-3 M with the detection limit of 0.1 × 10-6 M at a signal-to-noise ratio of 3. The stability and repeatability of the biosensor is also discussed.

  12. Influence of surface adsorption on the interfacial electron transfer of flavin adenine dinucleotide and glucose oxidase at carbon nanotube and nitrogen-doped carbon nanotube electrodes.

    PubMed

    Goran, Jacob M; Mantilla, Sandra M; Stevenson, Keith J

    2013-02-01

    The adsorption of flavin adenine dinucleotide (FAD) and glucose oxidase (GOx) onto carbon nanotube (CNT) and nitrogen-doped CNT (N-CNT) electrodes was investigated and found to obey Langmuir adsorption isotherm characteristics. The amount adsorbed and adsorption maximum are dependent on exposure time, the concentration of adsorbate, and the ionic strength of the solution. The formal potentials measured for FAD and GOx are identical, indicating that the observed electroactivity is from FAD, the redox reaction center of GOx. When glucose is added to GOx adsorbed onto CNT/N-CNT electrodes, direct electron transfer (DET) from enzyme-active FAD is not observed. However, efficient mediated electron transfer (MET) occurs if an appropriate electron mediator is placed in solution, or the natural electron mediator oxygen is used, indicating that GOx is adsorbed and active on CNT/N-CNT electrodes. The observed surface-confined redox reaction at both CNT and N-CNT electrodes is from FAD that either specifically adsorbs from solution or adsorbs from the holoprotein subsequently inactivating the enzyme. The splitting of cathodic and anodic peak potentials as a function of scan rate provides a way to measure the heterogeneous electron-transfer rate constant (k(s)) using Laviron's method. However, the measured k(s) was found to be under ohmic control, not under the kinetic control of an electron-transfer reaction, suggesting that k(s) for FAD on CNTs is faster than the measured value of 7.6 s(-1).

  13. New insights into the analysis of the electrode kinetics of flavin adenine dinucleotide redox center of glucose oxidase immobilized on carbon electrodes.

    PubMed

    Simonov, Alexandr N; Grosse, Willo; Mashkina, Elena A; Bethwaite, Blair; Tan, Jeff; Abramson, David; Wallace, Gordon G; Moulton, Simon E; Bond, Alan M

    2014-03-25

    New insights into electrochemical kinetics of the flavin adenine dinucleotide (FAD) redox center of glucose-oxidase (GlcOx) immobilized on reduced graphene oxide (rGO), single- and multiwalled carbon nanotubes (SW and MWCNT), and combinations of rGO and CNTs have been gained by application of Fourier transformed AC voltammetry (FTACV) and simulations based on a range of models. A satisfactory level of agreement between experiment and theory, and hence establishment of the best model to describe the redox chemistry of FAD, was achieved with the aid of automated e-science tools. Although still not perfect, use of Marcus theory with a very low reorganization energy (≤0.3 eV) best mimics the experimental FTACV data, which suggests that the process is gated as also deduced from analysis of FTACV data obtained at different frequencies. Failure of the simplest models to fully describe the electrode kinetics of the redox center of GlcOx, including those based on the widely employed Laviron theory is demonstrated, as is substantial kinetic heterogeneity of FAD species. Use of a SWCNT support amplifies the kinetic heterogeneity, while a combination of rGO and MWCNT provides a more favorable environment for fast communication between FAD and the electrode. PMID:24571209

  14. Role of the C-terminal extension stacked on the re-face of the isoalloxazine ring moiety of the flavin adenine dinucleotide prosthetic group in ferredoxin-NADP(+) oxidoreductase from Bacillus subtilis.

    PubMed

    Seo, Daisuke; Asano, Tomoya; Komori, Hirofumi; Sakurai, Takeshi

    2014-08-01

    Ferredoxin-NADP(+) oxidoreductase [EC 1.18.1.2] from Bacillus subtilis (BsFNR) is homologous to the bacterial NADPH-thioredoxin reductase, but possesses a unique C-terminal extension that covers the re-face of the isoalloxazine ring moiety of the flavin adenine dinucleotide (FAD) prosthetic group. In this report, we utilize BsFNR mutants depleted of their C-terminal residues to examine the importance of the C-terminal extension in reactions with NADPH and ferredoxin (Fd) from B. subtilis by spectroscopic and steady-state reaction analyses. The depletions of residues Y313 to K332 (whole C-terminal extension region) and S325 to K332 (His324 intact) resulted in significant increases in the catalytic efficiency with NADPH in diaphorase assay with ferricyanide, whereas Km values for ferricyanide were increased. In the cytochrome c reduction assay in the presence of B. subtilis ferredoxin, the S325-K332 depleted mutant displayed a significant decrease in the turnover rate with an Fd concentration range of 1-10 μM. The Y313-K332 depleted mutant demonstrated an increase in the rate of the direct reduction of horse heart cytochrome c in the absence of Fd. These data indicated that depletion of the C-terminal extension plays an important role in the reaction of BsFNR with ferredoxin.

  15. The role of Val-265 for flavin adenine dinucleotide (FAD) binding in pyruvate oxidase: FTIR, kinetic, and crystallographic studies on the enzyme variant V265A.

    PubMed

    Wille, Georg; Ritter, Michaela; Weiss, Manfred S; König, Stephan; Mäntele, Werner; Hübner, Gerhard

    2005-04-01

    In pyruvate oxidase (POX) from Lactobacillus plantarum, valine 265 participates in binding the cofactor FAD and is responsible for the strained conformation of its isoalloxazine moiety that is visible in the crystal structure of POX. The contrasting effects of the conservative amino acid exchange V265A on the enzyme's catalytic properties, cofactor affinity, and protein structure were investigated. The most prominent effect of the exchange was observed in the 2.2 A crystal structure of the mutant POX. While the overall structures of the wild-type and the variant are similar, flavin binding in particular is clearly different. Local disorder at the isoalloxazine binding site prevents modeling of the complete FAD cofactor and two protein loops of the binding site. Only the ADP moiety shows well-defined electron density, indicating an "anchor" function for this part of the molecule. This notion is corroborated by competition experiments where ADP was used to displace FAD from the variant enzyme. Despite the fact that the affinity of FAD binding in the variant is reduced, the catalytic properties are very similar to the wild-type, and the redox potential of the bound flavin is the same for both proteins. The rate of electron transfer toward the flavin during turnover is reduced to one-third compared to the wild-type, but k(cat) remains unchanged. Redox-triggered FTIR difference spectroscopy of free FAD shows the nu(C(10a)=N(1)) band at 1548 cm(-)(1). In POX-V265A, this band is found at 1538 cm(-)(1) and thus shifted less strongly than in wild-type POX where it is found at 1534 cm(-)(1). Taking these observations together, the conservative exchange V265A in POX has a surprisingly small effect on the catalytic properties of the enzyme, whereas the effect on the three-dimensional structure is rather big.

  16. Alkaloid cluster gene ccsA of the ergot fungus Claviceps purpurea encodes chanoclavine I synthase, a flavin adenine dinucleotide-containing oxidoreductase mediating the transformation of N-methyl-dimethylallyltryptophan to chanoclavine I.

    PubMed

    Lorenz, Nicole; Olsovská, Jana; Sulc, Miroslav; Tudzynski, Paul

    2010-03-01

    Ergot alkaloids are indole-derived secondary metabolites synthesized by the phytopathogenic ascomycete Claviceps purpurea. In wild-type strains, they are exclusively produced in the sclerotium, a hibernation structure; for biotechnological applications, submerse production strains have been generated by mutagenesis. It was shown previously that the enzymes specific for alkaloid biosynthesis are encoded by a gene cluster of 68.5 kb. This ergot alkaloid cluster consists of 14 genes coregulated and expressed under alkaloid-producing conditions. Although the role of some of the cluster genes in alkaloid biosynthesis could be confirmed by a targeted knockout approach, further functional analyses are needed, especially concerning the early pathway-specific steps up to the production of clavine alkaloids. Therefore, the gene ccsA, originally named easE and preliminarily annotated as coding for a flavin adenine dinucleotide-containing oxidoreductase, was deleted in the C. purpurea strain P1, which is able to synthesize ergot alkaloids in axenic culture. Five independent knockout mutants were analyzed with regard to alkaloid-producing capability. Thin-layer chromatography (TLC), ultrapressure liquid chromatography (UPLC), and mass spectrometry (MS) analyses revealed accumulation of N-methyl-dimethylallyltryptophan (Me-DMAT) and traces of dimethylallyltryptophan (DMAT), the first pathway-specific intermediate. Since other alkaloid intermediates could not be detected, we conclude that deletion of ccsA led to a block in alkaloid biosynthesis beyond Me-DMAT formation. Complementation with a ccsA/gfp fusion construct restored alkaloid biosynthesis. These data indicate that ccsA encodes the chanoclavine I synthase or a component thereof catalyzing the conversion of N-methyl-dimethylallyltryptophan to chanoclavine I. PMID:20118373

  17. RESPIRATORY PATHWAYS IN THE MYCOPLASMA. II. PATHWAY OF ELECTRON TRANSPORT DURING OXIDATION OF REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDE BY MYCOPLASMA HOMINIS.

    PubMed

    VANDEMARK, P J; SMITH, P F

    1964-07-01

    VanDemark, P. J. (University of South Dakota, Vermillion), and P. F. Smith. Respiratory pathways in the Mycoplasma. II. Pathway of electron transport during oxidation of reduced nicotinamide adenine dinucleotide by Mycoplasma hominis. J. Bacteriol. 88:122-129. 1964.-Unlike the flavin-terminated respiratory pathway of the fermentative Mycoplasma, the respiratory chain of the nonfermentative M. hominis strain 07 appears to be more complex, involving quinones and cytochromes in addition to flavins. In addition to reduction by reduced nicotine adenine dinucleotide (NADH) and reduced nicotine adenine dinucleotide phosphate, nonpyridine nucleotide-linked reduction of the respiratory chain of this organism occurred with succinate, lactate, and short-chained acyl coenzyme A derivatives as electron donors. Enzymes catalyzing the oxidation of NADH included an NADH oxidase, a diaphorase, a quinone reductase, and a cytochrome c reductase. The oxidation of NADH was sensitive to a variety of inhibitors, including 10(-4)m Atabrine, 10(-3)m sodium amytal, 10(-5)mp-chloromercuribenzoate, 10(-4)m antimycin A, and 10(-4)m potassium cyanide. The oxidase was resolved by the addition of 5% trichloroacetic acid and reactivated by the addition of flavin adenine dinucleotide but not flavin mononucleotide. The M. hominis sonic extract contained an NADH-coenzyme Q reductase. The oxidation of NADH was stimulated by the addition of either menadione or vitamin K(2) (C(35)). The oxidase was inactivated by extraction with ether or irradiation at 360 mmu. The ether-inactivated enzyme was partially reactivated by the addition of "lipid" extract of the enzyme and coenzyme Q(6). Difference spectra of the cell extracts revealed the presence of "b" and "a" type cytochromes. These cell extracts were found to contain a cyanide-and azide-sensitive cytochrome oxidase and catalase. PMID:14197876

  18. Ultra-performance liquid chromatography tandem mass-spectrometry (uplc-ms/ms) for the rapid, simultaneous analysis of thiamin, riboflavin, flavin adenine dinucleotide, nicotinamide and pyridoxal in human milk

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A novel, rapid and sensitive Ultra Performance Liquid-Chromatography tandem Mass-Spectrometry (UPLC-MS/MS) method for the simultaneous determination of several B-vitamins in human milk was developed. Resolution by retention time or multiple reaction monitoring (MRM) for thiamin, riboflavin, flavin a...

  19. Mutants of Neurospora deficient in nicotinamide adenine dinucleotide (phosphate) glycohydrolase.

    PubMed Central

    Nelson, R E; Selitrennikoff, C P; Siegel, R W

    1975-01-01

    A new screening technique has been developed for the rapid identification of Neurospora crassa mutants that are deficient in nicotinamide adenine dinucleotide glycohydrolase (NADase) and nicotinamide adenine dinucleotide phosphate glycohydrolase (NADPase) activities. Using this procedure, five single-gene mutants were isolated whose singular difference from wild type appeared to be the absence of NAD(P)ase (EC 3.2.2.6). All five mutants were found to be genetically allelic and did not complement in heterocaryons. This gene, nada [NAD(P)ase], was localized in linkage group IV. One of the nada alleles was found to specify an enzyme that was critically temperature sensitive and had altered substrate affinity. Mutations at the nada locus did not affect the genetic program for the expression of NAD(P)ase during cell differentiation, nor did they have a general effect on NAD catabolism. Nada mutations did not have simultaneous effects on other glycohydrolase activities. Tests of dominance (in heterocaryons) and in vitro mixing experiments did not provide evidence that nada mutations alter activators or inhibitors of NAD(P)ase. Thus, the nada gene appears to specify only the structure of N. crassa NAD(P)ase. Images PMID:165174

  20. Nicotinamide adenine dinucleotide-dependent and nicotinamide adenine dinucleotide-independent lactate dehydrogenases in homofermentative and heterofermentative lactic acid bacteria.

    PubMed

    Doelle, H W

    1971-12-01

    Three homofermentative (Lactobacillus plantarum B38, L. plantarum B33, Pediococcus pentosaceus B30) and three heterofermentative (Leuconostoc mesenteroides 39, L. oenos B70, Lactobacillus brevis) lactic acid bacteria were examined for the presence or absence of nicotinamide adenine dinucleotide (NAD)-dependent and NAD-independent d- and l-lactate dehydrogenases. Two of the six strains investigated, P. pentosaceus and L. oenos, did not exhibit an NAD-independent enzyme activity capable of reducing dichlorophenol indophenol. The pH optima of the lactic dehydrogenases were determined. The NAD-dependent enzymes from homofermentative strains exhibited optima at pH 7.8 to 8.8, whereas values from 9.0 to 10.0 were noted for these enzymes from heterofermentative organisms. The optima for the NAD-independent enzymes were between 5.8 and 6.6. The apparent Michaelis-Menten constants determined for both NAD and the substrates demonstrated the existence of a greater affinity for d- than l-lactic acid. A comparison of the specific NAD-dependent and NAD-independent lactate dehydrogenase activities revealed a direct correlation of the d/l ratios of these activities with the type of lactic acid produced during the growth of the organism.

  1. Oxidation of Reduced Nicotinamide Adenine Dinucleotide Phosphate by Isolated Corn Mitochondria 1

    PubMed Central

    Koeppe, D. E.; Miller, Raymond J.

    1972-01-01

    Isolated corn (Zea mays L.) mitochondria were found to oxidize reduced nicotinamide adenine dinucleotide phosphate in a KCl reaction medium. This oxidation was dependent on the presence of calcium or phosphate or both. Strontium and manganese substituted for calcium, but magnesium or barium did not. The oxidation of NADPH produced contraction of mitochondria swollen in KCl. Further evidence that the oxidation of NADPH was coupled was observed in respiratory control and adenosine diphosphate-oxygen ratios that were comparable to those reported for reduced nicotinamide adenine dinucleotide. The pathways of electron flow from NADH and NADPH were compared through the addition of electron transport inhibitors. The only difference between the two dinucleotides was that amytal was found to inhibit almost totally the state 3 oxidation of NADPH, but had little effect on the state 3 oxidation of NADH. The hypothetical pathways for electron flow from NADPH are discussed, as are the possible sites of calcium and phosphate stimulation. PMID:16657960

  2. Affinity chromatography of nicotinamide-adenine dinucleotide-linked dehydrogenases on immobilized derivatives of the dinucleotide.

    PubMed

    Barry, S; O'Carra, P

    1973-12-01

    1. Three established methods for immobilization of ligands through primary amino groups promoted little or no attachment of NAD(+) through the 6-amino group of the adenine residue. Two of these methods (coupling to CNBr-activated agarose and to carbodi-imide-activated carboxylated agarose derivatives) resulted instead in attachment predominantly through the ribosyl residues. Other immobilized derivatives were prepared by azolinkage of NAD(+) (probably through the 8 position of the adenine residue) to a number of different spacer-arm-agarose derivatives. 2. The effectiveness of these derivatives in the affinity chromatography of a variety of NAD-linked dehydrogenases was investigated, applying rigorous criteria to distinguish general or non-specific adsorption effects from truly NAD-specific affinity (bio-affinity). The ribosyl-attached NAD(+) derivatives displayed negligible bio-affinity for any of the NAD-linked dehydrogenases tested. The most effective azo-linked derivative displayed strong bio-affinity for glycer-aldehyde 3-phosphate dehydrogenase, weaker bio-affinity for lactate dehydrogenase and none at all for malate dehydrogenase, although these three enzymes have very similar affinities for soluble NAD(+). Alcohol dehydrogenase and xanthine dehydrogenase were subject to such strong non-specific interactions with the hydrocarbon spacer-arm assembly that any specific affinity was completely eclipsed. 3. It is concluded that, in practice, the general effectiveness of a general ligand may be considerably distorted and attenuated by the nature of the immobilization linkage. However, this attenuation can result in an increase in specific effectiveness, allowing dehydrogenases to be separated from one another in a manner unlikely to be feasible if the general effectiveness of the ligand remained intact. 4. The bio-affinity of the various derivatives for lactate dehydrogenase is correlated with the known structure of the NAD(+)-binding site of this enzyme. Problems

  3. YeeO from Escherichia coli exports flavins

    PubMed Central

    McAnulty, Michael J; Wood, Thomas K

    2014-01-01

    Multidrug and toxic compound extrusion (MATE) proteins help maintain cellular homeostasis by secreting metabolic wastes. Flavins may occur as cellular waste products, with their production and secretion providing potential benefit for industrial applications related to biofuel cells. Here we find that MATE protein YeeO from Escherichia coli exports both flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Significant amounts of flavins were trapped intracellularly when YeeO was produced indicating transport limits secretion of flavins. Wild-type E. coli secreted 3 flavins (riboflavin, FMN, and FAD), so E. coli likely produces additional flavin transporters. PMID:25482085

  4. YeeO from Escherichia coli exports flavins.

    PubMed

    McAnulty, Michael J; Wood, Thomas K

    2014-01-01

    Multidrug and toxic compound extrusion (MATE) proteins help maintain cellular homeostasis by secreting metabolic wastes. Flavins may occur as cellular waste products, with their production and secretion providing potential benefit for industrial applications related to biofuel cells. Here we find that MATE protein YeeO from Escherichia coli exports both flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Significant amounts of flavins were trapped intracellularly when YeeO was produced indicating transport limits secretion of flavins. Wild-type E. coli secreted 3 flavins (riboflavin, FMN, and FAD), so E. coli likely produces additional flavin transporters. PMID:25482085

  5. Conformational change in cytochrome P450 reductase adsorbed at a Au(110)—phosphate buffer interface induced by interaction with nicotinamide adenine dinucleotide phosphate

    NASA Astrophysics Data System (ADS)

    Smith, C. I.; Convery, J. H.; Harrison, P.; Khara, B.; Scrutton, N. S.; Weightman, P.

    2014-08-01

    Changes observed in the reflection anisotropy spectroscopy (RAS) profiles of monolayers of cytochrome P450 reductase adsorbed at Au(110)-electrolyte interfaces at 0.056 V following the addition of nicotinamide adenine dinucleotide phosphate (NADP+) are explained in terms of a simple model as arising from changes in the orientation of an isoalloxazine ring located in the flavin mononucleotide binding domain of the protein. The model also accounts for the changes observed in the RAS as the potential applied to the Au(110) surface is varied and suggests that differences in the dependence of the RAS profile of the adsorbed protein on the potential applied to the electrode in the absence and presence of NADP+ are explicable as arising from a competition between the applied potential acting to reduce the protein and the NADP+ to oxidize it.

  6. Nicotinic acid adenine dinucleotide phosphate-mediated calcium signalling in effector T cells regulates autoimmunity of the central nervous system

    PubMed Central

    Cordiglieri, Chiara; Odoardi, Francesca; Zhang, Bo; Nebel, Merle; Kawakami, Naoto; Klinkert, Wolfgang E. F.; Lodygin, Dimtri; Lühder, Fred; Breunig, Esther; Schild, Detlev; Ulaganathan, Vijay Kumar; Dornmair, Klaus; Dammermann, Werner; Potter, Barry V. L.; Guse, Andreas H.

    2010-01-01

    Nicotinic acid adenine dinucleotide phosphate represents a newly identified second messenger in T cells involved in antigen receptor-mediated calcium signalling. Its function in vivo is, however, unknown due to the lack of biocompatible inhibitors. Using a recently developed inhibitor, we explored the role of nicotinic acid adenine dinucleotide phosphate in autoreactive effector T cells during experimental autoimmune encephalomyelitis, the animal model for multiple sclerosis. We provide in vitro and in vivo evidence that calcium signalling controlled by nicotinic acid adenine dinucleotide phosphate is relevant for the pathogenic potential of autoimmune effector T cells. Live two photon imaging and molecular analyses revealed that nicotinic acid adenine dinucleotide phosphate signalling regulates T cell motility and re-activation upon arrival in the nervous tissues. Treatment with the nicotinic acid adenine dinucleotide phosphate inhibitor significantly reduced both the number of stable arrests of effector T cells and their invasive capacity. The levels of pro-inflammatory cytokines interferon-gamma and interleukin-17 were strongly diminished. Consecutively, the clinical symptoms of experimental autoimmune encephalomyelitis were ameliorated. In vitro, antigen-triggered T cell proliferation and cytokine production were evenly suppressed. These inhibitory effects were reversible: after wash-out of the nicotinic acid adenine dinucleotide phosphate antagonist, the effector T cells fully regained their functions. The nicotinic acid derivative BZ194 induced this transient state of non-responsiveness specifically in post-activated effector T cells. Naïve and long-lived memory T cells, which express lower levels of the putative nicotinic acid adenine dinucleotide phosphate receptor, type 1 ryanodine receptor, were not targeted. T cell priming and recall responses in vivo were not reduced. These data indicate that the nicotinic acid adenine dinucleotide phosphate

  7. Two-dimensional infrared spectroscopy of azido-nicotinamide adenine dinucleotide in water

    NASA Astrophysics Data System (ADS)

    Dutta, Samrat; Rock, William; Cook, Richard J.; Kohen, Amnon; Cheatum, Christopher M.

    2011-08-01

    Mid-IR active analogs of enzyme cofactors have the potential to be important spectroscopic reporters of enzyme active site dynamics. Azido-nicotinamide adenine dinucleotide (NAD+), which has been recently synthesized in our laboratory, is a mid-IR active analog of NAD+, a ubiquitous redox cofactor in biology. In this study, we measure the frequency-frequency time correlation function for the antisymmetric stretching vibration of the azido group of azido-NAD+ in water. Our results are consistent with previous studies of pseudohalides in water. We conclude that azido-NAD+ is sensitive to local environmental fluctuations, which, in water, are dominated by hydrogen-bond dynamics of the water molecules around the probe. Our results demonstrate the potential of azido-NAD+ as a vibrational probe and illustrate the potential of substituted NAD+-analogs as reporters of local structural dynamics that could be used for studies of protein dynamics in NAD-dependent enzymes.

  8. Conducting polymer and its composite materials based electrochemical sensor for Nicotinamide Adenine Dinucleotide (NADH).

    PubMed

    Omar, Fatin Saiha; Duraisamy, Navaneethan; Ramesh, K; Ramesh, S

    2016-05-15

    Nicotinamide Adenine Dinucleotide (NADH) is an important coenzyme in the human body that participates in many metabolic reactions. The impact of abnormal concentrations of NADH significantly causes different diseases in human body. Electrochemical detection of NADH using bare electrode is a challenging task especially in the presence of main electroactive interferences such as ascorbic acid (AA), uric acid (UA) and dopamine (DA). Modified electrodes have been widely explored to overcome the problems of poor sensitivity and selectivity occurred from bare electrodes. This review gives an overview on the progress of using conducting polymers, polyelectrolyte and its composites (co-polymer, carbonaceous, metal, metal oxide and clay) based modified electrodes for the sensing of NADH. In addition, developments on the fabrication of numerous conducting polymer composites based modified electrodes are clearly described.

  9. Production and characterization of reduced NAADP (nicotinic acid-adenine dinucleotide phosphate).

    PubMed Central

    Billington, Richard A; Thuring, Jan W; Conway, Stuart J; Packman, Len; Holmes, Andrew B; Genazzani, Armando A

    2004-01-01

    The pyridine nucleotide NAADP (nicotinic acid-adenine dinucleotide phosphate) has been shown to act as a Ca2+-releasing intracellular messenger in a wide variety of systems from invertebrates to mammals and has been implicated in a number of cellular processes. NAADP is structurally very similar to its precursor, the endogenous coenzyme NADP and while much is known about the reduced form of NADP, NADPH, it is not known whether NAADP can also exist in a reduced state. Here we report that NAADP can be reduced to NAADPH by endogenous cellular enzymes and that NAADPH is functionally inert at the NAADP receptor. These data suggest that NAADPH could represent a mechanism for rapidly inactivating NAADP in cells. PMID:14606955

  10. Isotope effect studies of the chemical mechanism of nicotinamide adenine dinucleotide malic enzyme from Crassula

    SciTech Connect

    Grissom, C.B.; Willeford, O.; Wedding, R.T.

    1987-05-05

    The /sup 13/C primary kinetic isotope effect on the decarboxylation of malate by nicotinamide adenine dinucleotide malic enzyme from Crassula argentea is 1.0199 +/- 0.0006 with proteo L-malate-2-H and 1.0162 +/- 0.0003 with malate-2-d. The primary deuterium isotope effect is 1.45 +/- 0.10 on V/K and 1.93 +/- 0.13 on V/sub max/. This indicates a stepwise conversion of malate to pyruvate and CO/sub 2/ with hydride transfer preceding decarboxylation, thereby suggesting a discrete oxaloacetate intermediate. This is in agreement with the stepwise nature of the chemical mechanism of other malic enzymes despite the Crassula enzyme's inability to reduce or decarboxylate oxaloacetate. Differences in morphology and allosteric regulation between enzymes suggest specialization of the Crassula malic enzyme for the physiology of crassulacean and acid metabolism while maintaining the catalytic events founds in malic enzymes from animal sources.

  11. Deficiency of the iron-sulfur clusters of mitochondrial reduced nicotinamide-adenine dinucleotide-ubiquinone oxidoreductase (complex I) in an infant with congenital lactic acidosis.

    PubMed

    Moreadith, R W; Batshaw, M L; Ohnishi, T; Kerr, D; Knox, B; Jackson, D; Hruban, R; Olson, J; Reynafarje, B; Lehninger, A L

    1984-09-01

    We report the case of an infant with hypoglycemia, progressive lactic acidosis, an increased serum lactate/pyruvate ratio, and elevated plasma alanine, who had a moderate to profound decrease in the ability of mitochondria from four organs to oxidize pyruvate, malate plus glutamate, citrate, and other NAD+-linked respiratory substrates. The capacity to oxidize the flavin adenine dinucleotide-linked substrate, succinate, was normal. The most pronounced deficiency was in skeletal muscle, the least in kidney mitochondria. Enzymatic assays on isolated mitochondria ruled out defects in complexes II, III, and IV of the respiratory chain. Further studies showed that the defect was localized in the inner membrane mitochondrial NADH-ubiquinone oxidoreductase (complex I). When ferricyanide was used as an artificial electron acceptor, complex I activity was normal, indicating that electrons from NADH could reduce the flavin mononucleotide cofactor. However, electron paramagnetic resonance spectroscopy performed on liver submitochondrial particles showed an almost total loss of the iron-sulfur clusters characteristic of complex I, whereas normal signals were noted for other mitochondrial iron-sulfur clusters. This infant is presented as the first reported case of congenital lactic acidosis caused by a deficiency of the iron-sulfur clusters of complex I of the mitochondrial electron transport chain.

  12. Decrease in nicotinamide adenine dinucleotide dehydrogenase is related to skin pigmentation.

    PubMed

    Nakama, Mitsuo; Murakami, Yuhko; Tanaka, Hiroshi; Nakata, Satoru

    2012-03-01

    Skin pigmentation is caused by various physical and chemical factors. It might also be influenced by changes in the physiological function of skin with aging. Nicotinamide adenine dinucleotide (NADH) dehydrogenase is an enzyme related to the mitochondrial electron transport system and plays a key role in cellular energy production. It has been reported that the functional decrease in this system causes Parkinson's disease. Another study reports that the amount of NADH dehydrogenase in heart and skeletal muscle decreases with aging. A similar decrease in the skin would probably affect its physiological function. However, no reports have examined the age-related change in levels of NADH dehydrogenase in human skin. In this study, we investigated this change and its effect on skin pigmentation using cultured human epidermal keratinocytes. The mRNA expression of NDUFA1, NDUFB7, and NDUFS2, subunits of NADH dehydrogenase, and its activity were significantly decreased in late passage keratinocytes compared to early passage cells. Conversely, the mRNA expression of melanocyte-stimulating cytokines, interleukin-1 alpha and endothelin 1, was increased in late passage cells. On the other hand, the inhibition of NADH dehydrogenase upregulated the mRNA expression of melanocyte-stimulating cytokines. Moreover, the level of NDUFB7 mRNA was lower in pigmented than in nonpigmented regions of skin in vivo. These results suggest the decrease in NADH dehydrogenase with aging to be involved in skin pigmentation.

  13. Magnitude of malate-aspartate reduced nicotinamide adenine dinucleotide shuttle activity in intact respiring tumor cells.

    PubMed

    Greenhouse, W V; Lehninger, A L

    1977-11-01

    Measurements of respiration, CO2 and lactate production, and changes in the levels of various key metabolites of the glycolytic sequence and tricarboxylic acid cycle were made on five lines of rodent ascites tumor cells (two strains of Ehrlich ascites tumor cells, Krebs II carcinoma, AS-30D carcinoma, and L1210 cells) incubated aerobically in the presence of uniformly labeled D-[14C]glucose. From these data, as well as earlier evidence demonstrating that the reduced nicotinamide adenine dinucleotide (NADH) shuttle in these cells requires a transaminase step and is thus identified as the malate-aspartate shuttle (W.V.V. Greenhouse and A.L. Lehninger, Cancer Res., 36: 1392-1396, 1976), metabolic flux diagrams were constructed for the five cell lines. These diagrams show the relative rates of glycolysis, the tricarboxylic acid cycle, electron transport, and the malate-aspartate shuttle in these tumors. Large amounts of cytosolic NADH were oxidized by the mitochondrial respiratory chain via the NADH shuttle, comprising anywhere from about 20 to 80% of the total flow of reducing equivalents to oxygen in these tumors. Calculations of the sources of energy for adenosine triphosphate synthesis indicated that on the average about one-third of the respiratory adenosine triphosphate is generated by electron flow originating from cytosolic NADH via the malate-aspartate shuttle.

  14. Preclinical evidence of mitochondrial nicotinamide adenine dinucleotide as an effective alarm parameter under hypoxia

    NASA Astrophysics Data System (ADS)

    Shi, Hua; Sun, Nannan; Mayevsky, Avraham; Zhang, Zhihong; Luo, Qingming

    2014-01-01

    Early detection of tissue hypoxia in the intensive care unit is essential for effective treatment. Reduced nicotinamide adenine dinucleotide (NADH) has been suggested to be the most sensitive indicator of tissue oxygenation at the mitochondrial level. However, no experimental evidence comparing the kinetics of changes in NADH and other physiological parameters has been provided. The aim of this study is to obtain the missing data in a systematic and reliable manner. We constructed four acute hypoxia models, including hypoxic hypoxia, hypemic hypoxia, circulatory hypoxia, and histogenous hypoxia, and measured NADH fluorescence, tissue reflectance, cerebral blood flow, respiration, and electrocardiography simultaneously from the induction of hypoxia until death. We found that NADH was not always the first onset parameter responding to hypoxia. The order of responses was mainly affected by the cause of hypoxia. However, NADH reached its alarm level earlier than the other monitored parameters, ranging from several seconds to >10 min. As such, we suggest that the NADH can be used as a hypoxia indicator, although the exact level that should be used must be further investigated. When the NADH alarm is detected, the body still has a chance to recover if appropriate and timely treatment is provided.

  15. Studies of yeast cell oxygenation and energetics by laser fluorometry of reduced nicotinamide adenine dinucleotide

    NASA Astrophysics Data System (ADS)

    Pan, Fu-shih; Chen, Stephen; Mintzer, Robert A.; Chen, Chin-Tu; Schumacker, Paul

    1991-03-01

    It is of fundamental importance for biological scientists to assess cellular energetics. Under aerobic conditions, the tricarboxylic acid cycle (TCA cycle) is coupled with the mitochondrial electron cascade pathway to provide the cell with energy. The nicotinamide adenine dinucleotide-conjugated pair (NAD and NADH) is the coenzyme in numerous important biomedical reactions which include several important dehydrogenase reactions in the TCA cycle. Based on Le Chatelier's principle, NADH will accumulate when this energy production mechanism is impaired. The relative amounts of NAD and NADH in a cell are defined as the redox state of the cell (Williamson et.al. 1967) which provides a valuable index of cellular energetics. The sum of the amounts of NAD and NADH in a cell may be assumed to be constant during a finite time; therefore, a reliable means of measuring the NADH concentration would provide us with a useful indicator of tissue viability. Traditionally, the quantities of NADH and NAD may be measured by chemical assay methods. We can avoid these tediois analyses by exploiting the significant difference between the ultraviolet absorption spectra of this redox pair. However, because of the opacity of biological samples and the interference of other biochemicals that also absorb ultraviolet radiation, measurement of NADH and NAD+ concentrations in vivo by absorption spectroscopy is not feasible.

  16. Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes.

    PubMed Central

    Eng, J; Lynch, R M; Balaban, R S

    1989-01-01

    Nicotinamide adenine dinucleotide (NADH) plays a critical role in oxidative phosphorylation as the primary source of reducing equivalents to the respiratory chain. Using a modified fluorescence microscope, we have obtained spectra and images of the blue autofluorescence from single rat cardiac myocytes. The optical setup permitted rapid acquisition of fluorescence emission spectra (390-595 nm) or intensified digital video images of individual myocytes. The spectra showed a broad fluorescence centered at 447 +/- 0.2 nm, consistent with mitochondrial NADH. Addition of cyanide resulted in a 100 +/- 10% increase in fluorescence, while the uncoupler FCCP resulted in a 82 +/- 4% decrease. These two transitions were consistent with mitochondrial NADH and implied that the myocytes were 44 +/- 6% reduced under the resting control conditions. Intracellular fluorescent structures were observed that correlated with the distribution of a mitochondrial selective fluorescent probe (DASPMI), the mitochondrial distribution seen in published electron micrographs, and a metabolic digital subtraction image of the cyanide fluorescence transition. These data are consistent with the notion that the blue autofluorescence of rat cardiac myocytes originates from mitochondrial NADH. Images FIGURE 9 FIGURE 10 FIGURE 2 FIGURE 3 FIGURE 8 FIGURE 11 PMID:2720061

  17. Nicotinamide adenine dinucleotide: An essential factor in preserving hearing in cisplatin-induced ototoxicity.

    PubMed

    Kim, Hyung-Jin; Oh, Gi-Su; Shen, AiHua; Lee, Su-Bin; Khadka, Dipendra; Pandit, Arpana; Shim, Hyeok; Yang, Sei-Hoon; Cho, Eun-Young; Song, Jeho; Kwak, Tae Hwan; Choe, Seong-Kyu; Park, Raekil; So, Hong-Seob

    2015-08-01

    Ototoxicity is an important issue in patients receiving cisplatin chemotherapy. Numerous studies have demonstrated that several mechanisms, including oxidative stress, DNA damage, and inflammatory responses, are closely associated with cisplatin-induced ototoxicity. Although much attention has been directed at identifying ways to protect the inner ear from cisplatin-induced damage, the precise underlying mechanisms have not yet been elucidated. The cofactor nicotinamide adenine dinucleotide (NAD(+)) has emerged as an important regulator of cellular energy metabolism and homeostasis. NAD(+) acts as a cofactor for various enzymes including sirtuins (SIRTs) and poly(ADP-ribose) polymerases (PARPs), and therefore, maintaining adequate NAD(+) levels has therapeutic benefits because of its effect on NAD(+)-dependent enzymes. Recent studies demonstrated that disturbance in intracellular NAD(+) levels is critically involved in cisplatin-induced cochlear damage associated with oxidative stress, DNA damage, and inflammatory responses. In this review, we describe the importance of NAD(+) in cisplatin-induced ototoxicity and discuss potential strategies for the prevention or treatment of cisplatin-induced ototoxicity with a particular focus on NAD(+)-dependent cellular pathways. PMID:25891352

  18. Kinetic properties of nicotinic acid adenine dinucleotide phosphate-induced Ca2+ release.

    PubMed

    Genazzani, A A; Mezna, M; Summerhill, R J; Galione, A; Michelangeli, F

    1997-03-21

    Three endogenous molecules have now been shown to release Ca2+ in the sea urchin egg: inositol trisphosphate (InsP3), cyclic adenosine 5'-diphosphate ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP), a derivative of NADP. While the mechanism through which the first two molecules are able to release Ca2+ is established and well characterized with InsP3 and cADPR-activating InsP3 and ryanodine receptors, respectively, the newly described NAADP has been shown to release Ca2+ via an entirely different mechanism. The most striking feature of this novel Ca2+ release mechanism is its inactivation, since subthreshold concentrations of NAADP are able to fully and irreversibly desensitize the channel. In the present study we have investigated the fast kinetics of activation and inactivation of NAADP-induced Ca2+ release. NAADP was found to release Ca2+ in a biphasic manner, and such release was preceded by a pronounced latent period, which was inversely dependent on concentration. Moreover, the kinetic features of NAADP-induced Ca2+ release were not altered by pretreatment with low concentrations of NAADP, although the extent of Ca2+ release was greatly affected. Our data suggest that the inactivation of NAADP-induced Ca2+ release is an all-or-none phenomenon, and while some receptors have been fully inactivated, those that remain sensitive to NAADP do so without any change in kinetic features. PMID:9065423

  19. Reduced nicotinamide adenine dinucleotide-activated phosphoenolpyruvate carboxylase in Pseudomonas MA: potential regulation between carbon assimilation and energy production.

    PubMed Central

    Newaz, S S; Hersh, L B

    1975-01-01

    Comparison of enzyme activities in crude extracts of methylamine-grown Pseudomonas MA (ATCC 23319) to those in succinate-grown cells indicates the involvement of an acetyl coenzyme A-independent phosphoenolpyruvate carboxylase in one-carbon metabolism. The purified phosphoenolpyruvate carboxylase is activated specifically by reduced nicotinamide adenine dinucleotide (KA = 0.2 mM). The regulatory properties of this enzyme suggests that phosphoenolpyruvate serves as a focal point for both carbon assimilation and energy metabolism. PMID:171253

  20. Electrochemical oxidation of dihydronicotinamide adenine dinucleotide at nitrogen-doped carbon nanotube electrodes.

    PubMed

    Goran, Jacob M; Favela, Carlos A; Stevenson, Keith J

    2013-10-01

    Nitrogen-doped carbon nanotubes (N-CNTs) substantially lower the overpotential necessary for dihydronicotinamide adenine dinucleotide (NADH) oxidation compared to nondoped CNTs or traditional carbon electrodes such as glassy carbon (GC). We observe a 370 mV shift in the peak potential (Ep) from GC to CNTs and another 170 mV shift from CNTs to 7.4 atom % N-CNTs in a sodium phosphate buffer solution (pH 7.0) with 2.0 mM NADH (scan rate 10 mV/s). The sensitivity of 7.4 atom % N-CNTs to NADH was measured at 0.30 ± 0.04 A M(-1) cm(-2), with a limit of detection at 1.1 ± 0.3 μM and a linear range of 70 ± 10 μM poised at a low potential of -0.32 V (vs Hg/Hg2SO4). NADH fouling, known to occur to the electrode surface during NADH oxidation, was investigated by measuring both the change in Ep and the resulting loss of electrode sensitivity. NADH degradation, known to occur in phosphate buffer, was characterized by absorbance at 340 nm and correlated with the loss of NADH electroactivity. N-CNTs are further demonstrated to be an effective platform for dehydrogenase-based biosensing by allowing glucose dehydrogenase to spontaneously adsorb onto the N-CNT surface and measuring the resulting electrode's sensitivity to glucose. The glucose biosensor had a sensitivity of 0.032 ± 0.003 A M(-1) cm(-2), a limit of detection at 6 ± 1 μM, and a linear range of 440 ± 50 μM.

  1. Photoaffinity Labeling of High Affinity Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-Binding Proteins in Sea Urchin Egg*♦

    PubMed Central

    Walseth, Timothy F.; Lin-Moshier, Yaping; Jain, Pooja; Ruas, Margarida; Parrington, John; Galione, Antony; Marchant, Jonathan S.; Slama, James T.

    2012-01-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Recent studies have identified two-pore channels (TPCs) as endolysosomal channels that are regulated by NAADP; however, the nature of the NAADP receptor binding site is unknown. To further study NAADP binding sites, we have synthesized and characterized [32P-5-azido]nicotinic acid adenine dinucleotide phosphate ([32P-5N3]NAADP) as a photoaffinity probe. Photolysis of sea urchin egg homogenates preincubated with [32P-5N3]NAADP resulted in specific labeling of 45-, 40-, and 30-kDa proteins, which was prevented by inclusion of nanomolar concentrations of unlabeled NAADP or 5N3-NAADP, but not by micromolar concentrations of structurally related nucleotides such as NAD, nicotinic acid adenine dinucleotide, nicotinamide mononucleotide, nicotinic acid, or nicotinamide. [32P-5N3]NAADP binding was saturable and displayed high affinity (Kd ∼10 nm) in both binding and photolabeling experiments. [32P-5N3]NAADP photolabeling was irreversible in a high K+ buffer, a hallmark feature of NAADP binding in the egg system. The proteins photolabeled by [32P-5N3]NAADP have molecular masses smaller than the sea urchin TPCs, and antibodies to TPCs do not detect any immunoreactivity that comigrates with either the 45-kDa or the 40-kDa photolabeled proteins. Interestingly, antibodies to TPC1 and TPC3 were able to immunoprecipitate a small fraction of the 45- and 40-kDa photolabeled proteins, suggesting that these proteins associate with TPCs. These data suggest that high affinity NAADP binding sites are distinct from TPCs. PMID:22117077

  2. Reduction of nicotinamide adenine dinucleotide by pyruvate:lipoate oxidoreductase in anaerobic, dark-grown Rhodospirillum rubrum mutant C.

    PubMed Central

    Gorrell, T E; Uffen, R L

    1978-01-01

    Cell extracts from fermentatively grown Rhodospirillum rubrum reduced about 80 nmol of nicotinamide adenine dinucleotide (NAD) per mg of protein per min under anaerobic conditions with sodium pyruvate. The reaction was specific for pyruvate and NAD; NAD phosphate was not reduced. Results indicated that pyruvate-linked NAD reduction occurred via pyruvate:lipoate oxidoreductase. The reaction required catalytic amounts of both coenzyme A and thiamine pyrophosphate. Addition of sodium arsenite inhibited enzyme activity by 90%. Pyruvate:lipoate oxidoreductase was the only system detected in anaerobic, dark-grown R. rubrum cell extracts which operated to produce reduced NAD. The low activity of the enzyme system suggested that it was not quantitatively important in ATP formation. PMID:207677

  3. Nicotinic Acid Adenine Dinucleotide Phosphate Analogs Substituted on the Nicotinic Acid and Adenine Ribosides. Effects on Receptor-Mediated Ca2+ release

    PubMed Central

    Trabbic, Christopher J.; Zhang, Fan; Walseth, Timothy F.; Slama, James T.

    2015-01-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca2+ releasing intracellular second messenger in both mammals and echinoderms. We report that large functionalized substituents introduced at the nicotinic acid 5-position are recognized by the sea urchin receptor, albeit with a 20–500 fold loss in agonist potency. 5-(3-Azidopropyl)-NAADP was shown to release Ca2+ with an EC50 of 31 µM and to compete with NAADP for receptor binding with an IC50 of 56 nM. Attachment of charged groups to the nicotinic acid of NAADP is associated with loss of activity, suggesting that the nicotinate riboside moiety is recognized as a neutral zwitterion. Substituents (Br- and N3-) can be introduced at the 8-adenosyl position of NAADP while preserving high potency and agonist efficacy and an NAADP derivative substituted at both the 5-position of the nicotinic acid and at the 8-adenosyl position was also recognized although the agonist potency was significantly reduced. PMID:25826221

  4. Application of nicotin amide-adenine dinucleotide analogs for clinical enzymology: alcohol dehydrogenase activity in liver injury.

    PubMed

    Fujisawa, K; Kimura, A; Minato, S; Tamaoki, H; Mizushima, H

    1976-06-01

    The activities of alcohol dehydrogease(ADH) in serum and in the subcellular fractions of rat liver were determined with n-amyl alcohol or ethanol as substrate and thionicotinamide-adenine dinucleotide as coenzyme. It was found that the enzyme's activity ratio on the amyl alcohol and ethanol(A/E value) of serum and on the particulate fractions of the liver were different, but the A/E value of the soluble fraction was similar to that of serum. The A/E value of the particulate fractions were higher than that of the soluble fraction. From the results of experimental liver damage in the rat, it seems that estimation of the A/E value of ADH activity in serum is a useful parameter for the diagnosis of active liver injury. Since the A/E values of patients' sera differed from those of the normal subjects, the estimation of the A/E value of serum may give diagnostic information on liver injury, especially in chronic liver injury. PMID:179739

  5. Development of an enzymatic chromatography strip with nicotinamide adenine dinucleotide-tetrazolium coupling reactions for quantitative l-lactate analysis.

    PubMed

    Kan, Shu-Chen; Chang, Wei-Feng; Lan, Min-Chi; Lin, Chia-Chi; Lai, Wei-Shiang; Shieh, Chwen-Jen; Hsiung, Kuang-Pin; Liu, Yung-Chuan

    2015-02-15

    In this study, a dry assay of l-lactate via the enzymatic chromatographic test (ECT) was developed. An l-lactate dehydrogenase plus a nicotinamide adenine dinucleotide (NADH) regeneration reaction were applied simultaneously. Various tetrazolium salts were screened to reveal visible color intensities capable of determining the lactate concentrations in the sample. The optimal analysis conditions were as follows. The diaphorase (0.5 μl, 2(-6)U/μl) was immobilized in the test line of the ECT strip. Nitrotetrazolium blue chloride (5 μl, 12 mM), l-lactate dehydrogenase (1 μl, 0.25U/μl), and NAD(+) (2μl, 1.5×10(-5)M) were added into the mobile phase (100 μl) composed of 0.1% (w/w) Tween 20 in 10mM phosphate buffer (pH 9.0), and the process was left to run for 10 min. This detection had a linear range of 0.039 to 5mM with a detection limit of 0.047 mM. This quantitative analysis process for l-lactate was easy to operate with good stability and was proper for the point-of-care testing applications. PMID:25454507

  6. Molecular beacon based bioassay for highly sensitive and selective detection of nicotinamide adenine dinucleotide and the activity of alanine aminotransferase.

    PubMed

    Tang, Zhiwen; Liu, Pei; Ma, Changbei; Yang, Xiaohai; Wang, Kemin; Tan, Weihong; Lv, Xiaoyuan

    2011-04-01

    We have developed a new approach to detect nicotinamide adenine dinucleotide (NAD(+)) with high specificity and sensitivity using molecular beacons (MBs) and employed it in the investigation of NAD(+) related biological processes, such as calorie restriction and alanine aminotransferase (ALT) activation. The E. coli DNA ligase would catalyze the ligation of two short oligonucleotides that complement with an MB only in the presence of NAD(+), resulting in the opening of the MB and the restoration of fluorescent signal. Thanks to the high sensitivity of the MB probe and the fidelity of E. coli DNA ligase toward its substrates, this approach can detect 0.3 nM NAD(+) with high selectivity against other NAD(+) analogs. This novel assay can also provide a convenient and robust way to analyze NAD(+) in biological samples such as cell lysate. As NAD(+) plays an essential role in many biochemical processes, this method can be used to investigate NAD(+) related life processes. For instance, the effect of calorie restriction on the intracellular NAD(+) level in MCF7 cells has been studied using this new assay. Moreover, this approach was also successfully used to analyze the activity of ALT. Therefore, this novel NAD(+) assay holds wide applicability as an analytical tool in biochemical and biomedical research.

  7. Electrochemical detection of nicotinamide adenine dinucleotide based on molecular beacon-like DNA and E. coli DNA ligase.

    PubMed

    He, Xiaoxiao; Ni, Xiaoqi; Wang, Yonghong; Wang, Kemin; Jian, Lixin

    2011-01-15

    An electrochemical method for nicotinamide adenine dinucleotide (NAD(+)) detection with high sensitivity and selectivity has been developed by using molecular beacon (MB)-like DNA and Escherichia coli DNA ligase. In this method, MB-like DNA labeled with 5'-SH and 3'-biotin was self-assembled onto a gold electrode in its duplex form by means of facile gold-thiol chemistry, which resulted in blockage of electronic transmission. It was eT OFF state. In the presence of NAD(+), E. coli DNA ligase was activated, and the two nucleotide fragments which were complementary to the loop of the MB-like DNA could be ligated by the NAD(+)-dependent E. coli DNA ligase. Hybridization of the ligated DNA with the MB-like DNA induced a large conformational change in this surface-confined DNA structure, which in turn pushed the biotin away from the electrode surface and made the electrons exchange freely with the electrode. Then the generated electrochemical signals can be measured by differential pulse voltammetry (DPV). Under optimized conditions, a linear response to logarithmic concentration of NAD(+) range from 3 nM to 5 μM and a detection limit of 1.8 nM were obtained. Furthermore, the proposed strategy had sufficient selectivity to discriminate NAD(+) from its analogues.

  8. Unique kinetics of nicotinic acid-adenine dinucleotide phosphate (NAADP) binding enhance the sensitivity of NAADP receptors for their ligand.

    PubMed Central

    Patel, S; Churchill, G C; Galione, A

    2000-01-01

    Nicotinic acid-adenine dinucleotide phosphate (NAADP) is a novel and potent Ca(2+)-mobilizing agent in sea urchin eggs and other cell types. Little is known, however, concerning the properties of the putative intracellular NAADP receptor. In the present study we have characterized NAADP binding sites in sea urchin egg homogenates. [(32)P]NAADP bound to a single class of high-affinity sites that were reversibly inhibited by NaCl but insensitive to pH and Ca(2+). Binding of [(32)P]NAADP was lost in preparations that did not mobilize Ca(2+) in response to NAADP, indicating that [(32)P]NAADP probably binds to a receptor mediating Ca(2+) mobilization. Addition of excess unlabelled NAADP, at various times after initiation of [(32)P]NAADP binding, did not result in displacement of bound [(32)P]NAADP. These data show that NAADP becomes irreversibly bound to its receptor immediately upon association. Accordingly, incubation of homogenates with low concentrations of NAADP resulted in maximal labelling of NAADP binding sites. This unique property renders NAADP receptors exquisitely sensitive to their ligand, thereby allowing detection of minute changes in NAADP levels. PMID:11104679

  9. Eliciting the mitochondrial unfolded protein response by nicotinamide adenine dinucleotide repletion reverses fatty liver disease in mice

    PubMed Central

    Gariani, Karim; Menzies, Keir J.; Ryu, Dongryeol; Wegner, Casey J.; Wang, Xu; Ropelle, Eduardo R.; Moullan, Norman; Zhang, Hongbo; Perino, Alessia; Lemos, Vera; Kim, Bohkyung; Park, Young‐Ki; Piersigilli, Alessandra; Pham, Tho X.; Yang, Yue; Ku, Chai Siah; Koo, Sung I.; Fomitchova, Anna; Cantó, Carlos; Schoonjans, Kristina; Sauve, Anthony A.

    2015-01-01

    With no approved pharmacological treatment, nonalcoholic fatty liver disease (NAFLD) is now the most common cause of chronic liver disease in Western countries and its worldwide prevalence continues to increase along with the growing obesity epidemic. Here, we show that a high‐fat high‐sucrose (HFHS) diet, eliciting chronic hepatosteatosis resembling human fatty liver, lowers hepatic nicotinamide adenine dinucleotide (NAD+) levels driving reductions in hepatic mitochondrial content, function, and adenosine triphosphate (ATP) levels, in conjunction with robust increases in hepatic weight, lipid content, and peroxidation in C57BL/6J mice. To assess the effect of NAD+ repletion on the development of steatosis in mice, nicotinamide riboside, a precursor of NAD+ biosynthesis, was added to the HFHS diet, either as a preventive strategy or as a therapeutic intervention. We demonstrate that NR prevents and reverts NAFLD by inducing a sirtuin (SIRT)1‐ and SIRT3‐dependent mitochondrial unfolded protein response, triggering an adaptive mitohormetic pathway to increase hepatic β‐oxidation and mitochondrial complex content and activity. The cell‐autonomous beneficial component of NR treatment was revealed in liver‐specific Sirt1 knockout mice (Sirt1hep−/−), whereas apolipoprotein E‐deficient mice (Apoe −/−) challenged with a high‐fat high‐cholesterol diet affirmed the use of NR in other independent models of NAFLD. Conclusion: Our data warrant the future evaluation of NAD+ boosting strategies to manage the development or progression of NAFLD. (Hepatology 2016;63:1190–1204) PMID:26404765

  10. EPR and potentiometric studies of copper(II) binding to nicotinamide adenine dinucleotide (NAD+) in water solution.

    PubMed

    Hoffmann, Stanisław K; Goslar, Janina; Lijewski, Stefan; Basiński, Kamil; Gąsowska, Anna; Łomozik, Lechosław

    2012-06-01

    Coordination of Cu(II) by nicotinamide adenine dinucleotide (NAD(+)) molecule has been studied in water solutions of various pH by potentiometry and electron paramagnetic resonance (EPR) and electron spin echo (ESE) spectroscopy. Potentiometric results indicate Cu(II) coordination by protonated NAD(+) at low pH and by deprotonated NAD(+) at high pH. At medium pH value (around pH=7) NAD(+) is not able to coordinate Cu(II) ions effectively and mainly the Cu(H(2)O)(6) complexes exist in the studied solution. This has been confirmed by EPR results. Electronic structure of Cu(II)-NAD complex and coordination sites is determined from EPR and ESE measurements in frozen solutions (at 77K and 6K). EPR spectra exclude coordination with nitrogen atoms. Detailed analysis of EPR parameters (g(||)=2.420, g(perpendicular)==2.080, A(||)=-131×10(-4)cm(-1) and A(perpendicular)=8×10(-4)cm(-1)) performed in terms of molecular orbital (MO) theory shows that Cu(II)NAD complex has elongated axial octahedral symmetry with a relatively strong delocalization of unpaired electron density on in-plane and axial ligands. The distortion of octahedron is analyzed using A(||) vs. g(||) diagram for various CuO(x) complexes. Electron spin echo decay modulation excludes the coordination by oxygen atoms of phosphate groups. We postulate a coordination of Cu(II) by two hydroxyl oxygen atoms of two ribose moieties of the NAD molecules and four solvated water molecules both at low and high pH values with larger elongation of the octahedron at higher pH.

  11. Real-time measurements of nicotinamide adenine dinucleotide in live human trabecular meshwork cells: effects of acute oxidative stress.

    PubMed

    Masihzadeh, Omid; Ammar, David A; Lei, Tim C; Gibson, Emily A; Kahook, Malik Y

    2011-09-01

    The trabecular meshwork (TM) region of the eye is exposed to a constant low-level of oxidative insult. The cumulative damage may be the reason behind age-dependent risk for developing primary open angle glaucoma. Chronic and acute effects of hydrogen peroxide (H(2)O(2)) on TM endothelial cells include changes in viability, protein synthesis, and cellular adhesion. However, little if anything is known about the immediate effect of H(2)O(2) on the biochemistry of the TM cells and the initial response to oxidative stress. In this report, we have used two-photon excitation autofluorescence (2PAF) to monitor changes to TM cell nicotinamide adenine dinucleotide (NADPH). 2PAF allows non-destructive, real-time analysis of concentration of intracellular NADPH. Coupled to reduced glutathione, NADPH, is a major component in the anti-oxidant defense of TM cells. Cultured human TM cells were monitored for over 30 min in control and H(2)O(2)-containing solutions. Peroxide caused both a dose- and time-dependent decrease in NADPH signal. NADPH fluorescence in control and in 4 mM H(2)O(2) solutions showed little attenuation of NADPH signal (4% and 9% respectively). TM cell NADPH fluorescence showed a linear decrease with exposure to 20 mM H(2)O(2) (-29%) and 100 mM H(2)O(2) (37%) after a 30 min exposure. Exposure of TM cells to 500 mM H(2)O(2) caused an exponential decrease in NADPH fluorescence to a final attenuation of 46% of starting intensity. Analysis of individual TM cells indicates that cells with higher initial NADPH fluorescence are more refractive to the apparent loss of viability caused by H(2)O(2) than weakly fluorescing TM cells. We conclude that 2PAF of intracellular NADPH is a valuable tool for studying TM cell metabolism in response to oxidative insult. PMID:21354135

  12. ß-nicotinamide adenine dinucleotide is an enteric inhibitory neurotransmitter in human and non-human primate colons

    PubMed Central

    Hwang, Sung Jin; Durnin, Leonie; Dwyer, Laura; Rhee, Poong-Lyul; Ward, Sean M.; Koh, Sang Don; Sanders, Kenton M.; Mutafova-Yambolieva, Violeta N.

    2010-01-01

    Background & Aims An important component of enteric inhibitory neurotransmission is mediated by a purine neurotransmitter, such as adenosine 5’-triphosphate (ATP), binding to P2Y1 receptors and activating small conductance K+ channels. In murine colon ß-nicotinamide adenine dinucleotide (ß-NAD) is released with ATP and mimics the pharmacology of inhibitory neurotransmission better than ATP. Here ß-NAD and ATP were compared as possible inhibitory neurotransmitters in human and monkey colons. Methods A small-volume superfusion assay and HPLC with fluorescence detection were used to evaluate spontaneous and nerve-evoked overflow of ß-NAD, ATP and metabolites. Postjunctional responses to nerve stimulation, ß-NAD and ATP were compared using intracellular membrane potential and force measurements. Effects of ß-NAD on smooth muscle cells (SMCs) were recorded by patch clamp. P2Y receptor transcripts and proteins were assayed by RT-PCR. Results In contrast to ATP, overflow of ß-NAD evoked by electrical field stimulation correlated with stimulation frequency and was diminished by neurotoxins, tetrodotoxin and ω-conotoxin GVIA. Inhibitory junction potentials and responses to exogenous ß-NAD, but not ATP, were blocked by P2Y receptor antagonists suramin, PPADS, MRS2179 and MRS2500. ß-NAD activated non-selective cation currents in SMCs, but failed to activate outward currents. Conclusions ß-NAD meets the criteria for a neurotransmitter better than ATP in human and monkey colons and therefore may contribute to neural regulation of colonic motility. SMCs are unlikely targets for inhibitory purine neurotransmitters because dominant responses of SMCs were activation of net inward, rather than outward, current. PMID:20875415

  13. Morphological features, distribution and compartmental organization of the nicotinamide adenine dinucleotide phosphate reduced-diaphorase interneurons in the human striatum.

    PubMed

    Bernácer, Javier; Prensa, Lucía; Giménez-Amaya, José Manuel

    2005-08-29

    Striatal nicotinamide adenine dinucleotide phosphate reduced-diaphorase (NADPH-d)-positive (+) cells are one of the major classes of striatal interneurons. The present study analyzes their somatodendritic morphology, distribution pattern, and compartmental organization in the caudate nucleus (CN) and putamen (Put) of nine normal human brains. The following striatal territories are examined: 1) the precommissural head of the CN; 2) the postcommissural head of the CN; 3) the body of the CN; 4) the gyrus of the CN; 5) the tail of the CN; 6) the precommissural Put; and 7) the postcommissural Put. Three morphologically distinct types of NADPH-d+ neurons were found in each of these territories. The two most common NADPH-d+ neurons displayed an ovoid or triangular perikaryon from which several thick primary dendrites emerged, although much less numerous, bipolar-shaped NADPH-d+ cells were also observed. The highest density of NADPH-d+ neurons was found in the gyrus of the CN, followed by the body of the CN, tail of the CN, postcommissural head of the CN, postcommissural Put, precommissural head of the CN, and precommissural Put. The matrix was the striatal compartment with the densest NADPH-d+ neuronal population. Some of these cells also occurred in the center and peripheral regions of the striosomes located in the head of the CN and in the Put. In the body and gyrus of the CN, the striosomes were largely devoid of these striatal interneurons. Knowledge of the density and distribution of these interneurons should advance our understanding of the organization of the normal human striatum and help to evaluate the effects of neurodegenerative processes on cell density.

  14. Real-time measurements of nicotinamide adenine dinucleotide in live human trabecular meshwork cells: Effects of acute oxidative stress✩

    PubMed Central

    Masihzadeh, Omid; Ammar, David A.; Lei, Tim C.; Gibson, Emily A.; Kahook, Malik Y.

    2016-01-01

    The trabecular meshwork (TM) region of the eye is exposed to a constant low-level of oxidative insult. The cumulative damage may be the reason behind age-dependent risk for developing primary open angle glaucoma. Chronic and acute effects of hydrogen peroxide (H2O2) on TM endothelial cells include changes in viability, protein synthesis, and cellular adhesion. However, little if anything is known about the immediate effect of H2O2 on the biochemistry of the TM cells and the initial response to oxidative stress. In this report, we have used two-photon excitation autofluorescence (2PAF) to monitor changes to TM cell nicotinamide adenine dinucleotide (NADPH). 2PAF allows non-destructive, real-time analysis of concentration of intracellular NADPH. Coupled to reduced glutathione, NADPH, is a major component in the anti-oxidant defense of TM cells. Cultured human TM cells were monitored for over 30 min in control and H2O2-containing solutions. Peroxide caused both a dose- and time-dependent decrease in NADPH signal. NADPH fluorescence in control and in 4 mM H2O2 solutions showed little attenuation of NADPH signal (4% and 9% respectively). TM cell NADPH fluorescence showed a linear decrease with exposure to 20 mM H2O2 (−29%) and 100 mM H2O2 (37%) after a 30 min exposure. Exposure of TM cells to 500 mM H2O2 caused an exponential decrease in NADPH fluorescence to a final attenuation of 46% of starting intensity. Analysis of individual TM cells indicates that cells with higher initial NADPH fluorescence are more refractive to the apparent loss of viability caused by H2O2 than weakly fluorescing TM cells. We conclude that 2PAF of intracellular NADPH is a valuable tool for studying TM cell metabolism in response to oxidative insult. PMID:21354135

  15. Evidence for the Degradation of Nicotinamide Adenine Dinucleotide Phosphate-Dependent Glutamate Dehydrogenase of Candida utilis During Rapid Enzyme Inactivation

    PubMed Central

    Hemmings, Brian A.

    1978-01-01

    The nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase (NADP-GDH) from the food yeast Candida utilis was found to be rapidly inactivated when cultures were starved of a carbon source. The addition of glutamate or alanine to the starvation medium stimulated the rate of inactivation. Loss of enzyme activity was irreversible since the reappearance of enzyme activity, following the addition of glucose to carbon-starved cultures, was blocked by cycloheximide. A specific rabbit antibody was prepared against the NADP-GDH from C. utilis and used to quantitate the enzyme during inactivation promoted by carbon starvation. The amount of precipitable antigenic material paralleled the rapid decrease of enzyme activity observed after transition of cells from NH4+-glucose to glutamate medium. No additional small-molecular-weight protein was precipitated by the antibody as a result of the inactivation, suggesting that the enzyme is considerably altered during the primary steps of the inactivation process. Analysis by immunoprecipitation of the reappearance of enzyme activity after enzyme inactivation showed that increase of NADP-GDH activity was almost totally due to de novo synthesis, ruling out the possibility that enzyme activity modulation is achieved by reversible covalent modification. Enzyme degradation was also measured during steady-state growth and other changes in nitrogen and carbon status of the culture media. In all instances so far estimated, the enzyme was found to be very stable and not normally subject to high rates of degradation. Therefore, the possibility that inactivation was caused by a change in the ratio of synthesis to degradation can be excluded. Images PMID:24041

  16. A functional role for nicotinic acid adenine dinucleotide phosphate in oxytocin-mediated contraction of uterine smooth muscle from rat.

    PubMed

    Aley, Parvinder K; Noh, Hyun J; Gao, Xin; Tica, Andrei A; Brailoiu, Eugen; Churchill, Grant C

    2010-06-01

    Conventionally, G protein-coupled receptors are thought to increase calcium via inositol 1,4,5-trisphosphate (InsP(3)). More recent evidence shows that an alternative second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), also has a role to play, causing researchers to question established calcium releasing pathways. With the recent development, by our group, of cell-permeant NAADP (NAADP-aceteoxymethyl ester) and a selective NAADP receptor antagonist (Ned-19; 1-(3-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid),the ability to investigate this signaling pathway has improved. Therefore, we investigated a role for NAADP in oxytocin-mediated responses in the rat uterus. Oxytocin- and NAADP-mediated effects were investigated by using contractile measurements of whole uterine strips from rat in organ baths. Responses were correlated to calcium release in cultured rat uterine smooth muscle cells measured by fluorescence microscopy. Inhibition of both oxytocin-induced contraction and calcium release by the traditional NAADP-signaling disrupter bafilomycin and the NAADP receptor antagonist Ned-19 clearly demonstrated a role for NAADP in oxytocin-induced signaling. A cell-permeant form of NAADP was able to produce both uterine contractions and calcium release. This response was unaffected by depletion of sarcoplasmic reticulum stores with thapsigargin, but was abolished by both bafilomycin and Ned-19. Crucially, oxytocin stimulated an increase in NAADP in rat uterine tissue. The present study demonstrates directly that NAADP signaling plays a role in rat uterine contractions. Moreover, investigation of this signaling pathway highlights yet another component of oxytocin-mediated signaling, stressing the need to consider the action of new components as they are discovered, even in signaling pathways that are thought to be well established.

  17. Interaction of reduced nicotinamide adenine dinucleotide with an antifreeze protein from Dendroides canadensis: mechanistic implication of antifreeze activity enhancement

    PubMed Central

    Wen, Xin; Wang, Sen; Amornwittawat, Natapol; Houghton, Eric A.; Sacco, Michael A.

    2016-01-01

    Antifreeze proteins (AFPs) found in many organisms can noncolligatively lower the freezing point of water without altering the melting point. The difference between the depressed freezing point and the melting point, termed thermal hysteresis (TH), is usually a measure of the antifreeze activity of AFPs. Certain low molecular mass molecules and proteins can further enhance the antifreeze activity of AFPs. Interaction between an enhancer and arginine is known to play an important role in enhancing the antifreeze activity of an AFP from the beetle Dendroides canadensis (DAFP-1). Here, we examined the enhancement effects of several prevalent phosphate-containing coenzymes on the antifreeze activity of DAFP-1. β-Nicotinamide adenine dinucleotide (reduced) (NADH) is identified as the most efficient enhancer of DAFP-1, which increases the antifreeze activity of DAFP-1 by around 10 times. Examination of the enhancement abilities of a series of NADH analogs and various molecular fragments of NADH reveals that the modifications of nicotinamide generate a series of highly efficient enhancers, though none as effective as NADH itself, and the whole molecular structure of NADH is necessary for its highly efficient enhancement effect. We also demonstrated a 1:1 binding between DAFP-1 and NADH. The binding was characterized by high-performance liquid chromatography (HPLC) using the gel filtration method of Hummel and Dreyer. The data analysis suggests binding between DAFP-1 and NADH with a dissociation constant in the micromolar range. Interactions between DAFP-1 and NADH are discussed along with molecular mechanisms of enhancer action. PMID:22038809

  18. Facile synthesis of near infrared fluorescent trypsin-stabilized Ag nanoclusters with tunable emission for 1,4-dihydronicotinamide adenine dinucleotide and ethanol sensing.

    PubMed

    Liu, Siyu; Wang, Hui; Cheng, Zhen; Liu, Hongguang

    2015-07-30

    A facile chemical synthetic route was developed to prepare near-infrared fluorescent trypsin-stabilized Ag nanoclusters (Try-Ag NCs). The fluorescence emission wavelength of the produced Try-Ag NCs is tunable by simple adjusting pH value of the synthesis system, and the Try-Ag NCs offer a symmetric fluorescent excitation and emission peak. The fluorescence of Try-Ag NCs remains constant in the presence of various ions and molecules, and it can be effectively quenched by 1,4-dihydronicotinamide adenine dinucleotide (NADH) instead of its oxidized forms nicotinamide adenine dinucleotide (NAD(+)). This property enables the Try-Ag NCs to be a novel analytical platform to monitor biological reaction involved with NADH. In this work, the Try-Ag NCs was also applied to analyze ethanol based on the generation of NADH which was the product of NAD(+) and ethanol in the catalysis of alcohol dehydrogenase. And the proposed platform allowed ethanol to be determined in the range from 10 to 300 μmol/L with 5 μmol/L detection limit.

  19. Changes in phosphorylation of adenosine phosphate and redox state of nicotinamide-adenine dinucleotide (phosphate) in Geobacter sulfurreducens in response to electron acceptor and anode potential variation.

    PubMed

    Rose, Nicholas D; Regan, John M

    2015-12-01

    Geobacter sulfurreducens is one of the dominant bacterial species found in biofilms growing on anodes in bioelectrochemical systems. The intracellular concentrations of reduced and oxidized forms of nicotinamide-adenine dinucleotide (NADH and NAD(+), respectively) and nicotinamide-adenine dinucleotide phosphate (NADPH and NADP(+), respectively) as well as adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) were measured in G. sulfurreducens using fumarate, Fe(III)-citrate, or anodes poised at different potentials (110, 10, -90, and -190 mV (vs. SHE)) as the electron acceptor. The ratios of CNADH/CNAD+ (0.088±0.022) and CNADPH/CNADP+ (0.268±0.098) were similar under all anode potentials tested and with Fe(III)-citrate (reduced extracellularly). Both ratios significantly increased with fumarate as the electron acceptor (0.331±0.094 for NAD and 1.96±0.37 for NADP). The adenylate energy charge (the fraction of phosphorylation in intracellular adenosine phosphates) was maintained near 0.47 under almost all conditions. Anode-growing biofilms demonstrated a significantly higher molar ratio of ATP/ADP relative to suspended cultures grown on fumarate or Fe(III)-citrate. These results provide evidence that the cellular location of reduction and not the redox potential of the electron acceptor controls the intracellular redox potential in G. sulfurreducens and that biofilm growth alters adenylate phosphorylation.

  20. Regulation of Enzymes Involved in the Conversion of Tryptophan to Nicotinamide Adenine Dinucleotide in a Colorless Strain of Xanthomonas pruni1

    PubMed Central

    Brown, Albert T.; Wagner, Conrad

    1970-01-01

    A colorless strain of Xanthomonas pruni was isolated which is capable of converting tryptophan to nicotinamide adenine dinucleotide (NAD). The enzymes responsible for the conversion of tryptophan to quinolinic acid were shown to be present. Nicotinic acid-requiring mutants were isolated, and it was found that the growth of these mutants can be supported by various intermediates on the pathway from tryptophan to NAD. The first three enzymes on this pathway are induced coordinately by l-tryptophan. Gratuitous inducers of these enzymes include d-tryptophan, α-methyl-dl-tryptophan, and 4-methyl-dl-tryptophan; formyl-l-kynurenine and l-kynurenine were not effective as inducers. These data suggest that at least the first three enzymes in the pathway from tryptophan to NAD are under common regulatory control. PMID:4313053

  1. Diabetic complications within the context of aging: Nicotinamide adenine dinucleotide redox, insulin C-peptide, sirtuin 1-liver kinase B1-adenosine monophosphate-activated protein kinase positive feedback and forkhead box O3.

    PubMed

    Ido, Yasuo

    2016-07-01

    Recent research in nutritional control of aging suggests that cytosolic increases in the reduced form of nicotinamide adenine dinucleotide and decreasing nicotinamide adenine dinucleotide metabolism plays a central role in controlling the longevity gene products sirtuin 1 (SIRT1), adenosine monophosphate-activated protein kinase (AMPK) and forkhead box O3 (FOXO3). High nutrition conditions, such as the diabetic milieu, increase the ratio of reduced to oxidized forms of cytosolic nicotinamide adenine dinucleotide through cascades including the polyol pathway. This redox change is associated with insulin resistance and the development of diabetic complications, and might be counteracted by insulin C-peptide. My research and others' suggest that the SIRT1-liver kinase B1-AMPK cascade creates positive feedback through nicotinamide adenine dinucleotide synthesis to help cells cope with metabolic stress. SIRT1 and AMPK can upregulate liver kinase B1 and FOXO3, key factors that help residential stem cells cope with oxidative stress. FOXO3 directly changes epigenetics around transcription start sites, maintaining the health of stem cells. 'Diabetic memory' is likely a result of epigenetic changes caused by high nutritional conditions, which disturb the quiescent state of residential stem cells and impair tissue repair. This could be prevented by restoring SIRT1-AMPK positive feedback through activating FOXO3. PMID:27181414

  2. Diabetic complications within the context of aging: Nicotinamide adenine dinucleotide redox, insulin C-peptide, sirtuin 1-liver kinase B1-adenosine monophosphate-activated protein kinase positive feedback and forkhead box O3.

    PubMed

    Ido, Yasuo

    2016-07-01

    Recent research in nutritional control of aging suggests that cytosolic increases in the reduced form of nicotinamide adenine dinucleotide and decreasing nicotinamide adenine dinucleotide metabolism plays a central role in controlling the longevity gene products sirtuin 1 (SIRT1), adenosine monophosphate-activated protein kinase (AMPK) and forkhead box O3 (FOXO3). High nutrition conditions, such as the diabetic milieu, increase the ratio of reduced to oxidized forms of cytosolic nicotinamide adenine dinucleotide through cascades including the polyol pathway. This redox change is associated with insulin resistance and the development of diabetic complications, and might be counteracted by insulin C-peptide. My research and others' suggest that the SIRT1-liver kinase B1-AMPK cascade creates positive feedback through nicotinamide adenine dinucleotide synthesis to help cells cope with metabolic stress. SIRT1 and AMPK can upregulate liver kinase B1 and FOXO3, key factors that help residential stem cells cope with oxidative stress. FOXO3 directly changes epigenetics around transcription start sites, maintaining the health of stem cells. 'Diabetic memory' is likely a result of epigenetic changes caused by high nutritional conditions, which disturb the quiescent state of residential stem cells and impair tissue repair. This could be prevented by restoring SIRT1-AMPK positive feedback through activating FOXO3.

  3. A label-free fluorescence strategy for selective detection of nicotinamide adenine dinucleotide based on a dumbbell-like probe with low background noise.

    PubMed

    Chen, Xuexu; Lin, Chunshui; Chen, Yiying; Wang, Yiru; Chen, Xi

    2016-03-15

    In this work we developed a novel label-free fluorescence sensing approach for the detection of nicotinamide adenine dinucleotide (NAD(+)) based on a dumbbell-like DNA probe designed for both ligation reaction and digestion reaction with low background noise. SYBR Green I (SG I), a double-helix dye, was chosen as the readout fluorescence signal. In the absence of NAD(+), the ligation reaction did not occur, but the probe was digested to mononucleotides after the addition of exonuclease I (Exo I) and exonuclease I (Exo III), resulting in a weak fluorescence intensity due to the weak interaction between SG I and mononucleotides. In the presence of NAD(+), the DNA probe was ligated by Escherichia coli DNA ligase, blocking the digestion by Exo I and Exo III. As a result, SG I was intercalated into the stem part of the DNA dumbbell probe and fluorescence enhancement was achieved. This method was simple in design, fast to operate, with good sensitivity and selectivity which could discriminate NAD(+) from its analogs. PMID:26454831

  4. Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Endolysosomal Two-pore Channels Modulate Membrane Excitability and Stimulus-Secretion Coupling in Mouse Pancreatic β Cells.

    PubMed

    Arredouani, Abdelilah; Ruas, Margarida; Collins, Stephan C; Parkesh, Raman; Clough, Frederick; Pillinger, Toby; Coltart, George; Rietdorf, Katja; Royle, Andrew; Johnson, Paul; Braun, Matthias; Zhang, Quan; Sones, William; Shimomura, Kenju; Morgan, Anthony J; Lewis, Alexander M; Chuang, Kai-Ting; Tunn, Ruth; Gadea, Joaquin; Teboul, Lydia; Heister, Paula M; Tynan, Patricia W; Bellomo, Elisa A; Rutter, Guy A; Rorsman, Patrik; Churchill, Grant C; Parrington, John; Galione, Antony

    2015-08-28

    Pancreatic β cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca(2+) action potentials due to the activation of voltage-dependent Ca(2+) channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca(2+) release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic β cells. NAADP-regulated Ca(2+) release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca(2+) from the endolysosomal system, resulting in localized Ca(2+) signals. We show here that NAADP-mediated Ca(2+) release from endolysosomal Ca(2+) stores activates inward membrane currents and depolarizes the β cell to the threshold for VDCC activation and thereby contributes to glucose-evoked depolarization of the membrane potential during stimulus-response coupling. Selective pharmacological inhibition of NAADP-evoked Ca(2+) release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca(2+) signals, and insulin secretion. Our findings implicate NAADP-evoked Ca(2+) release from acidic Ca(2+) storage organelles in stimulus-secretion coupling in β cells. PMID:26152717

  5. Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-mediated Calcium Signaling and Arrhythmias in the Heart Evoked by β-Adrenergic Stimulation*♦

    PubMed Central

    Nebel, Merle; Schwoerer, Alexander P.; Warszta, Dominik; Siebrands, Cornelia C.; Limbrock, Ann-Christin; Swarbrick, Joanna M.; Fliegert, Ralf; Weber, Karin; Bruhn, Sören; Hohenegger, Martin; Geisler, Anne; Herich, Lena; Schlegel, Susan; Carrier, Lucie; Eschenhagen, Thomas; Potter, Barry V. L.; Ehmke, Heimo; Guse, Andreas H.

    2013-01-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+-releasing second messenger known to date. Here, we report a new role for NAADP in arrhythmogenic Ca2+ release in cardiac myocytes evoked by β-adrenergic stimulation. Infusion of NAADP into intact cardiac myocytes induced global Ca2+ signals sensitive to inhibitors of both acidic Ca2+ stores and ryanodine receptors and to NAADP antagonist BZ194. Furthermore, in electrically paced cardiac myocytes BZ194 blocked spontaneous diastolic Ca2+ transients caused by high concentrations of the β-adrenergic agonist isoproterenol. Ca2+ transients were recorded both as increases of the free cytosolic Ca2+ concentration and as decreases of the sarcoplasmic luminal Ca2+ concentration. Importantly, NAADP antagonist BZ194 largely ameliorated isoproterenol-induced arrhythmias in awake mice. We provide strong evidence that NAADP-mediated modulation of couplon activity plays a role for triggering spontaneous diastolic Ca2+ transients in isolated cardiac myocytes and arrhythmias in the intact animal. Thus, NAADP signaling appears an attractive novel target for antiarrhythmic therapy. PMID:23564460

  6. Ordered carbohydrate-derived porous carbons immobilized gold nanoparticles as a new electrode material for electrocatalytical oxidation and determination of nicotinamide adenine dinucleotide.

    PubMed

    Hosseini, Hadi; Behbahani, Mohammad; Mahyari, Mojtaba; Kazerooni, Hanif; Bagheri, Akbar; Shaabani, Ahmad

    2014-09-15

    The ordered carbohydrate-derived porous carbons (OC-DPCs) were first functionalized with thiol groups (-SH) and then immobilized with gold nanoparticles (AuNPs). The Au-SH-OC-DPCs were characterized by CHN analysis, transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), and x-ray diffraction (XRD). The Au-SH-OC-DPCs were applied for the fabrication of a new electrochemical sensor. The electrocatalytic capabilities of the new sensor were tested by the oxidation of nicotinamide adenine dinucleotide (NADH) in a 0.1 M Robinson buffer solution (pH 7.0) using cyclic voltammetry (CV), linear sweep voltammetry (LSV), and differential pulse voltammetry (DPV). The Au-SH-OC-DPCs showed a good voltammetric performance in the electrochemical detection of NADH with a low detection limit (1.0 nM), high sensitivity (4.934 μA/μM), and wide linear concentration range (5.0 nM-10 µM).

  7. Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Endolysosomal Two-pore Channels Modulate Membrane Excitability and Stimulus-Secretion Coupling in Mouse Pancreatic β Cells.

    PubMed

    Arredouani, Abdelilah; Ruas, Margarida; Collins, Stephan C; Parkesh, Raman; Clough, Frederick; Pillinger, Toby; Coltart, George; Rietdorf, Katja; Royle, Andrew; Johnson, Paul; Braun, Matthias; Zhang, Quan; Sones, William; Shimomura, Kenju; Morgan, Anthony J; Lewis, Alexander M; Chuang, Kai-Ting; Tunn, Ruth; Gadea, Joaquin; Teboul, Lydia; Heister, Paula M; Tynan, Patricia W; Bellomo, Elisa A; Rutter, Guy A; Rorsman, Patrik; Churchill, Grant C; Parrington, John; Galione, Antony

    2015-08-28

    Pancreatic β cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca(2+) action potentials due to the activation of voltage-dependent Ca(2+) channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca(2+) release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic β cells. NAADP-regulated Ca(2+) release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca(2+) from the endolysosomal system, resulting in localized Ca(2+) signals. We show here that NAADP-mediated Ca(2+) release from endolysosomal Ca(2+) stores activates inward membrane currents and depolarizes the β cell to the threshold for VDCC activation and thereby contributes to glucose-evoked depolarization of the membrane potential during stimulus-response coupling. Selective pharmacological inhibition of NAADP-evoked Ca(2+) release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca(2+) signals, and insulin secretion. Our findings implicate NAADP-evoked Ca(2+) release from acidic Ca(2+) storage organelles in stimulus-secretion coupling in β cells.

  8. Autocrine/paracrine function of nicotinic acid adenine dinucleotide phosphate (NAADP) for glucose homeostasis in pancreatic β-cells and adipocytes.

    PubMed

    Park, Kwang-Hyun; Kim, Byung-Ju; Shawl, Asif Iqbal; Han, Myung-Kwan; Lee, Hon Cheung; Kim, Uh-Hyun

    2013-12-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger for mobilizing Ca(2+) from intracellular stores in various cell types. Extracellular application of NAADP has been shown to elicit intracellular Ca(2+) signals, indicating that it is readily transported into cells. However, little is known about the functional role of this NAADP uptake system. Here, we show that NAADP is effectively transported into selected cell types involved in glucose homeostasis, such as adipocytes and pancreatic β-cells, but not the acinar cells, in a high glucose-dependent manner. NAADP uptake was inhibitable by Ned-19, a NAADP mimic; dipyridamole, a nucleoside inhibitor; or NaN3, a metabolic inhibitor or under Ca(2+)-free conditions. Furthermore, NAADP was found to be released from pancreatic islets upon stimulation by high glucose. Consistently, administration of NAADP to type 2 diabetic mice improved glucose tolerance. We propose that NAADP is functioning as an autocrine/paracrine hormone important in glucose homeostasis. NAADP is thus a potential antidiabetic agent with therapeutic relevance.

  9. Increase of reduced nicotinamide adenine dinucleotide fluorescence lifetime precedes mitochondrial dysfunction in staurosporine-induced apoptosis of HeLa cells

    NASA Astrophysics Data System (ADS)

    Yu, Jia-Sin; Guo, Han-Wen; Wang, Chih-Hao; Wei, Yau-Huei; Wang, Hsing-Wen

    2011-03-01

    In vivo noninvasive detection of apoptosis represents a new tool that may yield a more definite diagnosis, a more accurate prognosis, and help improve therapies for human diseases. The intrinsic fluorescence of reduced nicotinamide adenine dinucleotide (NADH) may be a potential optical biomarker for the apoptosis detection because NADH is involved in the respiration for the mitochondrial membrane potential (ΔΨ) formation and adenosine-5'-triphosphate (ATP) synthesis, and the depletion of ΔΨ and ATP level is the hallmark of apoptosis. We have previously observed the NADH fluorescence lifetime change is associated with staurosporine (STS)-induced mitochondria-mediated apoptosis. However, its relationship with mitochondrial functions such as ΔΨ, ATP, and oxygen consumption rate is not clear. In this study, we investigated this relationship. Our results indicate that the NADH fluorescence lifetime increased when ΔΨ and ATP levels were equal to or higher than their values of controls and decreased before the depletion of ΔΨ and ATP, and the oxygen consumption rate did not change. These findings suggest that the increased NADH fluorescence lifetime in STS-induced cell death occurred before the depletion of ΔΨ and ATP and activation of caspase 3, and was not simply caused by cellular metabolic change. Furthermore, the NADH fluorescence lifetime change is associated with the pace of apoptosis.

  10. Differential regulation of nicotinic acid-adenine dinucleotide phosphate and cADP-ribose production by cAMP and cGMP.

    PubMed Central

    Wilson, H L; Galione, A

    1998-01-01

    The sea urchin egg has been used as a system to study calcium-release mechanisms induced by inositol 1,4,5-trisphosphate (IP3), cADP-ribose (cADPR), and more recently, nicotinic acid-adenine dinucleotide phosphate (NAADP). In order that cADPR and NAADP may be established as endogenous messengers for calcium release, the existence of intracellular enzymes capable of metabolizing these molecules must be demonstrated. In addition, intracellular levels of cADPR and NAADP should be under the control of extracellular stimuli. It has been shown that cGMP stimulates the synthesis of cADPR in the sea urchin egg. The present study shows that the sea urchin egg is capable of synthesizing and degrading NAADP. cADPR and NAADP synthetic activities appear to be separate, with different cellular localizations, pH and temperature optima. We suggest that in the sea urchin egg, cADPR and NAADP production may be differentially regulated by receptor-coupled second messengers, with cADPR production being regulated by cGMP and NAADP production modulated by cAMP. PMID:9560312

  11. Differential effect of pH upon cyclic-ADP-ribose and nicotinate-adenine dinucleotide phosphate-induced Ca2+ release systems.

    PubMed Central

    Chini, E N; Liang, M; Dousa, T P

    1998-01-01

    We investigated the pH dependence and the effects of thimerosal and dithiothreitol (DTT) upon the Ca2+ release induced by cADP-ribose (cADPR) and nicotinate-adenine dinucleotide phosphate (NAADP) in sea urchin egg homogenates. Both Ca2+ release triggered by cADPR and the binding of [3H]cADPR to sea urchin egg homogenates were decreased by alkalization of the assay media from pH 7.2 to 8.9. In contrast, NAADP-triggered Ca2+ release was not influenced by changes in pH. The Ca2+ release induced by cADPR was potentiated by thimerosal and inhibited by DTT, but neither thimerosal nor DTT had any effect upon the Ca2+ release induced by NAADP. We conclude that cADPR-sensitive Ca2+-release mechanisms are dependent on pH of the assay media and are sensitive to thiol group modification. On the other hand, these functional properties are not shared by NAADP-regulated Ca2+ channels. PMID:9794787

  12. A VOLTAMMETRIC FLAVIN MICROELECTRODE FOR USE IN BIOFILMS

    PubMed Central

    Nguyen, Hung Duc; Renslow, Ryan; Babauta, Jerome; Ahmed, Bulbul; Beyenal, Haluk

    2011-01-01

    Biofilms used in bioelectrochemical systems are expected to transfer electrons using electron transfer mediators. One mediator type, flavins, which includes flavin mononucleotide, riboflavin, and flavin adenine dinucleotide, has been found to be endogenously produced by Shewanella oneidensis MR-1. However, the presence and concentration of flavins inside a S. oneidensis MR-1 biofilm have never been reported. The goal of this study was to develop a flavin microelectrode capable of measuring flavins inside a living biofilm and apply it to a biofilm which produces flavins. Because flavins are electrochemically active molecules, the flavin microelectrode was based on detection via square-wave voltammetry. The microelectrode consisted of a carbon working electrode with a 10–30 μm tip diameter, a built-in platinum counter electrode, and a Ag/AgCl reference electrode, all enclosed in a glass outer case. The microelectrode was calibrated between 0.1 μM and 10 μM flavins and showed a linear correlation between flavin concentration and peak currents located at −424 mVAg/AgCl on a square-wave voltammogram. We also developed a model to explain the electrochemical mechanism of flavin detection, and to determine the effective surface area of the microelectrode, the standard reduction potential, and the transfer coefficient. We found that the effective surface area of the microelectrode was close to 100 times the projected surface area. The model predicted a standard reduction potential for RF/RFH2 of −419 mVAg/AgCl at 20 °C and a transfer coefficient of 0.45. Lastly, we measured flavin concentration inside a S. oneidensis MR-1 biofilm grown on a glass surface using oxygen as the electron acceptor. The flavin concentration reached 0.7 μM, increasing near the bottom of the biofilm, where no oxygen was present. This shows the possibility that flavins are produced in the anaerobic zone to act as intermediate electron acceptors in the deeper parts of the biofilm, where

  13. Flavins secreted by roots of iron-deficient Beta vulgaris enable mining of ferric oxide via reductive mechanisms.

    PubMed

    Sisó-Terraza, Patricia; Rios, Juan J; Abadía, Javier; Abadía, Anunciación; Álvarez-Fernández, Ana

    2016-01-01

    Iron (Fe) is abundant in soils but generally poorly soluble. Plants, with the exception of Graminaceae, take up Fe using an Fe(III)-chelate reductase coupled to an Fe(II) transporter. Whether or not nongraminaceous species can convert scarcely soluble Fe(III) forms into soluble Fe forms has deserved little attention so far. We have used Beta vulgaris, one among the many species whose roots secrete flavins upon Fe deficiency, to study whether or not flavins are involved in Fe acquisition. Flavins secreted by Fe-deficient plants were removed from the nutrient solution, and plants were compared with Fe-sufficient plants and Fe-deficient plants without flavin removal. Solubilization of a scarcely soluble Fe(III)-oxide was assessed in the presence or absence of flavins, NADH (nicotinamide adenine dinucleotide, reduced form) or plant roots, and an Fe(II) trapping agent. The removal of flavins from the nutrient solution aggravated the Fe deficiency-induced leaf chlorosis. Flavins were able to dissolve an Fe(III)-oxide in the presence of NADH. The addition of extracellular flavins enabled roots of Fe-deficient plants to reductively dissolve an Fe(III)-oxide. We concluded that root-secretion of flavins improves Fe nutrition in B. vulgaris. Flavins allow B. vulgaris roots to mine Fe from Fe(III)-oxides via reductive mechanisms.

  14. Flavin Electron Shuttles Dominate Extracellular Electron Transfer by Shewanella oneidensis

    PubMed Central

    Kotloski, Nicholas J.; Gralnick, Jeffrey A.

    2013-01-01

    ABSTRACT Shewanella oneidensis strain MR-1 is widely studied for its ability to respire a diverse array of soluble and insoluble electron acceptors. The ability to breathe insoluble substrates is defined as extracellular electron transfer and can occur via direct contact or by electron shuttling in S. oneidensis. To determine the contribution of flavin electron shuttles in extracellular electron transfer, a transposon mutagenesis screen was performed with S. oneidensis to identify mutants unable to secrete flavins. A multidrug and toxin efflux transporter encoded by SO_0702 was identified and renamed bfe (bacterial flavin adenine dinucleotide [FAD] exporter) based on phenotypic characterization. Deletion of bfe resulted in a severe decrease in extracellular flavins, while overexpression of bfe increased the concentration of extracellular flavins. Strains lacking bfe had no defect in reduction of soluble Fe(III), but these strains were deficient in the rate of insoluble Fe(III) oxide reduction, which was alleviated by the addition of exogenous flavins. To test a different insoluble electron acceptor, graphite electrode bioreactors were set up to measure current produced by wild-type S. oneidensis and the Δbfe mutant. With the same concentration of supplemented flavins, the two strains produced similar amounts of current. However, when exogenous flavins were not supplemented to bioreactors, bfe mutant strains produced significantly less current than the wild type. We have demonstrated that flavin electron shuttling accounts for ~75% of extracellular electron transfer to insoluble substrates by S. oneidensis and have identified the first FAD transporter in bacteria. PMID:23322638

  15. Ryanodine receptor type I and nicotinic acid adenine dinucleotide phosphate receptors mediate Ca2+ release from insulin-containing vesicles in living pancreatic beta-cells (MIN6).

    PubMed

    Mitchell, Kathryn J; Lai, F Anthony; Rutter, Guy A

    2003-03-28

    We have demonstrated recently (Mitchell, K. J., Pinton, P., Varadi, A., Tacchetti, C., Ainscow, E. K., Pozzan, T., Rizzuto, R., and Rutter, G. A. (2001) J. Cell Biol. 155, 41-51) that ryanodine receptors (RyR) are present on insulin-containing secretory vesicles. Here we show that pancreatic islets and derived beta-cell lines express type I and II, but not type III, RyRs. Purified by subcellular fractionation and membrane immuno-isolation, dense core secretory vesicles were found to possess a similar level of type I RyR immunoreactivity as Golgi/endoplasmic reticulum (ER) membranes but substantially less RyR II than the latter. Monitored in cells expressing appropriately targeted aequorins, dantrolene, an inhibitor of RyR I channels, elevated free Ca(2+) concentrations in the secretory vesicle compartment from 40.1 +/- 6.7 to 90.4 +/- 14.8 microm (n = 4, p < 0.01), while having no effect on ER Ca(2+) concentrations. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP), a novel Ca(2+)-mobilizing agent, decreased dense core secretory vesicle but not ER free Ca(2+) concentrations in permeabilized MIN6 beta-cells, and flash photolysis of caged NAADP released Ca(2+) from a thapsigargin-insensitive Ca(2+) store in single MIN6 cells. Because dantrolene strongly inhibited glucose-stimulated insulin secretion (from 3.07 +/- 0.51-fold stimulation to no significant glucose effect; n = 3, p < 0.01), we conclude that RyR I-mediated Ca(2+)-induced Ca(2+) release from secretory vesicles, possibly potentiated by NAADP, is essential for the activation of insulin secretion.

  16. Amelioration of nicotinamide adenine dinucleotide phosphate-oxidase mediated stress reduces cell death after blast-induced traumatic brain injury.

    PubMed

    Lucke-Wold, Brandon P; Naser, Zachary J; Logsdon, Aric F; Turner, Ryan C; Smith, Kelly E; Robson, Matthew J; Bailes, Julian E; Lee, John M; Rosen, Charles L; Huber, Jason D

    2015-12-01

    A total of 1.7 million traumatic brain injuries (TBIs) occur each year in the United States, but available pharmacologic options for the treatment of acute neurotrauma are limited. Oxidative stress is an important secondary mechanism of injury that can lead to neuronal apoptosis and subsequent behavioral changes. Using a clinically relevant and validated rodent blast model, we investigated how nicotinamide adenine dinucleotide phosphate oxidase (Nox) expression and associated oxidative stress contribute to cellular apoptosis after single and repeat blast injuries. Nox4 forms a complex with p22phox after injury, forming free radicals at neuronal membranes. Using immunohistochemical-staining methods, we found a visible increase in Nox4 after single blast injury in Sprague Dawley rats. Interestingly, Nox4 was also increased in postmortem human samples obtained from athletes diagnosed with chronic traumatic encephalopathy. Nox4 activity correlated with an increase in superoxide formation. Alpha-lipoic acid, an oxidative stress inhibitor, prevented the development of superoxide acutely and increased antiapoptotic markers B-cell lymphoma 2 (t = 3.079, P < 0.05) and heme oxygenase 1 (t = 8.169, P < 0.001) after single blast. Subacutely, alpha-lipoic acid treatment reduced proapoptotic markers Bax (t = 4.483, P < 0.05), caspase 12 (t = 6.157, P < 0.001), and caspase 3 (t = 4.573, P < 0.01) after repetitive blast, and reduced tau hyperphosphorylation indicated by decreased CP-13 and paired helical filament staining. Alpha-lipoic acid ameliorated impulsive-like behavior 7 days after repetitive blast injury (t = 3.573, P < 0.05) compared with blast exposed animals without treatment. TBI can cause debilitating symptoms and psychiatric disorders. Oxidative stress is an ideal target for neuropharmacologic intervention, and alpha-lipoic acid warrants further investigation as a therapeutic for prevention of chronic neurodegeneration.

  17. β-Nicotinamide adenine dinucleotide acts at prejunctional adenosine A1 receptors to suppress inhibitory musculomotor neurotransmission in guinea pig colon and human jejunum.

    PubMed

    Wang, Guo-Du; Wang, Xi-Yu; Liu, Sumei; Xia, Yun; Zou, Fei; Qu, Meihua; Needleman, Bradley J; Mikami, Dean J; Wood, Jackie D

    2015-06-01

    Intracellular microelectrodes were used to record neurogenic inhibitory junction potentials in the intestinal circular muscle coat. Electrical field stimulation was used to stimulate intramural neurons and evoke contraction of the smooth musculature. Exposure to β-nicotinamide adenine dinucleotide (β-NAD) did not alter smooth muscle membrane potential in guinea pig colon or human jejunum. ATP, ADP, β-NAD, and adenosine, as well as the purinergic P2Y1 receptor antagonists MRS 2179 and MRS 2500 and the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine, each suppressed inhibitory junction potentials in guinea pig and human preparations. β-NAD suppressed contractile force of twitch-like contractions evoked by electrical field stimulation in guinea pig and human preparations. P2Y1 receptor antagonists did not reverse this action. Stimulation of adenosine A1 receptors with 2-chloro-N6-cyclopentyladenosine suppressed the force of twitch contractions evoked by electrical field stimulation in like manner to the action of β-NAD. Blockade of adenosine A1 receptors with 8-cyclopentyl-1,3-dipropylxanthine suppressed the inhibitory action of β-NAD on the force of electrically evoked contractions. The results do not support an inhibitory neurotransmitter role for β-NAD at intestinal neuromuscular junctions. The data suggest that β-NAD is a ligand for the adenosine A1 receptor subtype expressed by neurons in the enteric nervous system. The influence of β-NAD on intestinal motility emerges from adenosine A1 receptor-mediated suppression of neurotransmitter release at inhibitory neuromuscular junctions.

  18. A Novel Nicotinamide Adenine Dinucleotide Correction Method for Mitochondrial Ca(2+) Measurement with FURA-2-FF in Single Permeabilized Ventricular Myocytes of Rat.

    PubMed

    Lee, Jeong Hoon; Ha, Jeong Mi; Leem, Chae Hun

    2015-07-01

    Fura-2 analogs are ratiometric fluoroprobes that are widely used for the quantitative measurement of [Ca(2+)]. However, the dye usage is intrinsically limited, as the dyes require ultraviolet (UV) excitation, which can also generate great interference, mainly from nicotinamide adenine dinucleotide (NADH) autofluorescence. Specifically, this limitation causes serious problems for the quantitative measurement of mitochondrial [Ca(2+)], as no available ratiometric dyes are excited in the visible range. Thus, NADH interference cannot be avoided during quantitative measurement of [Ca(2+)] because the majority of NADH is located in the mitochondria. The emission intensity ratio of two different excitation wavelengths must be constant when the fluorescent dye concentration is the same. In accordance with this principle, we developed a novel online method that corrected NADH and Fura-2-FF interference. We simultaneously measured multiple parameters, including NADH, [Ca(2+)], and pH/mitochondrial membrane potential; Fura-2-FF for mitochondrial [Ca(2+)] and TMRE for Ψm or carboxy-SNARF-1 for pH were used. With this novel method, we found that the resting mitochondrial [Ca(2+)] concentration was 1.03 µM. This 1 µM cytosolic Ca(2+) could theoretically increase to more than 100 mM in mitochondria. However, the mitochondrial [Ca(2+)] increase was limited to ~30 µM in the presence of 1 µM cytosolic Ca(2+). Our method solved the problem of NADH signal contamination during the use of Fura-2 analogs, and therefore the method may be useful when NADH interference is expected.

  19. Effect of telmisartan on the expression of adiponectin receptors and nicotinamide adenine dinucleotide phosphate oxidase in the heart and aorta in type 2 diabetic rats

    PubMed Central

    2012-01-01

    Background Diabetic cardiovascular disease is associated with decreased adiponectin and increased oxidative stress. This study investigated the effect of telmisartan on the expression of adiponectin receptor 2 (adipoR2) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits in the heart and the expression of adiponectin receptor 1 (adipoR1) in aorta in type 2 diabetic rats. Methods Type 2 diabetes was induced by high-fat and high-sugar diet and intraperitoneal injection of a low dose of streptozotocin (STZ). Heart function, adipoR2, p22phox, NOX4, glucose transporter 4(GLUT4), monocyte chemoattractant protein-1(MCP-1) and connective tissue growth factor (CTGF)in the heart, and adipoR1, MCP-1 and nuclear factor kappa B (NF-κB) in aorta were analyzed in controls and diabetic rats treated with or without telmisartan (5mg/kg/d) by gavage for 12 weeks. Results Heart function, plasma and myocardial adiponectin levels, the expression of myocardial adipoR2 and GLUT4 were significantly decreased in diabetic rats (P <0.05). The expression of myocardial p22phox, NOX4, MCP-1, and CTGF was significantly increased in diabetic rats (P <0.05). The expression of adipoR1 was decreased and the expression of MCP-1 and NF-κB was increased in the abdominal aorta in diabetic rats (P <0.05). Telmisartan treatment significantly attenuated these changes in diabetic rats (P <0.05). Conclusions Our results suggest that telmisartan upregulates the expression of myocardial adiponectin, its receptor 2 and GLUT4. Simultaneously, it downregulates the expression of myocardial p22phox, NOX4, MCP-1, and CTGF, contributing so to the improvement of heart function in diabetic rats. Telmisartan also induces a protective role on the vascular system by upregulating the expression of adipoR1 and downregulating the expression of MCP-1 and NF-κB in the abdominal aorta in diabetic rats. PMID:22873349

  20. Fabrication and characterization of Meldola's blue/zinc oxide hybrid electrodes for efficient detection of the reduced form of nicotinamide adenine dinucleotide at low potential.

    PubMed

    Kumar, S Ashok; Chen, Shen-Ming

    2007-05-29

    We report the synthesis and the electrochemical properties of hybrid films made of zinc oxide (ZnO) and Meldola's blue dye (MB) using cyclic voltammetry (CV). MB/ZnO hybrid films were electrochemically deposited onto glassy carbon, gold and indium tin oxide-coated glass (ITO) electrodes at room temperature (25+/-2 degrees C) from the bath solution containing 0.1 M Zn(NO3)2, 0.1 M KNO3 and 1x10(-4) MMB. The surface morphology and deposition kinetics of MB/ZnO hybrid films were studied by means of scanning electron microscopy (SEM), atomic force microscopy (AFM) and electrochemical quartz crystal microbalance (EQCM) techniques, respectively. SEM and AFM images of MB/ZnO hybrid films have revealed that the surfaces are well crystallized, porous and micro structured. MB molecules were immobilized and strongly fixed in a transparent inorganic matrix. MB/ZnO hybrid films modified glassy carbon electrode (MB/ZnO/GC) showed one reversible redox couple centered at formal potential (E0') -0.12 V (pH 6.9). The surface coverage (gamma) of the MB immobilized on ZnO/GC was about 9.86x10(-12) mol cm(-2) and the electron transfer rate constant (ks) was determined to be 38.9 s(-1). The MB/ZnO/GC electrode acted as a sensor and displayed an excellent specific electrocatalytic response to the oxidation of nicotinamide adenine dinucleotide (NADH). The linear response range between 50 and 300 microM NADH concentration at pH 6.9 was observed with a detection limit of 10 microM (S/N=3). The electrode was stable during the time it was used for the full study (about 1 month) without a notable decrease in current. Indeed, dopamine (DA), ascorbic acid (AA), acetaminophen (AP) and uric acid (UA) did not show any interference during the detection of NADH at this modified electrode.

  1. The regulation of respiration of guinea pig taenia coli in high-K medium: the role of nicotinamide-adenine dinucleotide, adenosine diphosphate and Ca++.

    PubMed

    Tsuda, S; Urakawa, N; Saito, Y; Fukami, J

    1975-10-01

    In an attempt to elucidate the regulation mechanism of respiration in the smooth muscle cell, we investigated the roles of nicotinamide-adenine dinucleotide (NAD), adenosine diphosphate (ADP) and Ca++ in the muscle respiration using the tissues and subcellular fractions from guinea pig taenia coli. The tension in the strips of taenia coli increased with a concomitant increase in O2 consumption in high-K medium (40 mM K) containing 2.5 mM Ca. 10(-3) M amytal and 10(-5)M ouabain decreased the high-K induced tension and O2 consumption of the muscle. 10(-4)M 2,4-dinitrophenol (DNP) relieved the decreased respiration induced by ouabain, but not that with amytal. From these data it is suggested that NADH-linked respiration plays an important role in the respiration of the muscle. Ca++ in concentrations ranging from 0.5 to 2.5 mM in the high-K medium resulted in an increase in tension and in O2 concumption progressively. In spectrophotometric observations of subcellular fractions of the taenia coli, ADP increased in absorbance change at 340 m mu. Such occurred in mitochondrial fractions and was initiated by the addition of NADH. Therefore it is deduced that the increase in ADP level of the cytoplasm is primarily due to a contraction triggered by Ca++ thus stimulating respiration. On the other hand, at 0.1 mM of Ca++ concentration, the muscle strip increased O2 consumption without tension development in high-K medium. In the spectrophotometric observations, Ca++ and Sr++ increased the absorbance change in the homogenate and in the mitochondrial fraction. Hence, it seems that one part of the Ca++ entering into the smooth muscle treated with the high-K increased O2 consumption in mitochondia independent of an increase in muscle tension. From these results it is concluded that NADH-linked respiration plays an important role in the smooth muscle respiration in high-K medium and that ADP and Ca++ also play a role in regulating respiration. PMID:176493

  2. Nicotinamide Adenine Dinucleotide Phosphate Oxidase–Mediated Redox Signaling and Vascular Remodeling by 16α-Hydroxyestrone in Human Pulmonary Artery Cells

    PubMed Central

    Hood, Katie Y.; Montezano, Augusto C.; Harvey, Adam P.; Nilsen, Margaret; MacLean, Margaret R.

    2016-01-01

    Estrogen and oxidative stress have been implicated in pulmonary arterial hypertension (PAH). Mechanisms linking these systems are elusive. We hypothesized that estrogen metabolite, 16α-hydroxyestrone (16αOHE1), stimulates nicotinamide adenine dinucleotide phosphate oxidase (Nox)–induced reactive oxygen species (ROS) generation and proliferative responses in human pulmonary artery smooth muscle cells (hPASMCs) and that in PAH aberrant growth signaling promotes vascular remodeling. The pathophysiological significance of estrogen–Nox–dependent processes was studied in female Nox1−/− and Nox4−/− mice with PAH. PASMCs from control subjects (control hPASMCs) and PAH patients (PAH-hPASMCs) were exposed to estrogen and 16αOHE1 in the presence/absence of inhibitors of Nox, cytochrome P450 1B1, and estrogen receptors. Estrogen, through estrogen receptor-α, increased Nox-derived ROS and redox-sensitive growth in hPASMCs, with greater effects in PAH-hPASMCs versus control hPASMCs. Estrogen effects were inhibited by cytochrome P450 1B1 blockade. 16αOHE1 stimulated transient ROS production in hPASMCs, with sustained responses in PAH-hPASMCs. Basal expression of Nox1/Nox4 was potentiated in PAH-hPASMCs. In hPASMCs, 16αOHE1 increased Nox1 expression, stimulated irreversible oxidation of protein tyrosine phosphatases, decreased nuclear factor erythroid–related factor 2 activity and expression of nuclear factor erythroid–related factor 2–regulated antioxidant genes, and promoted proliferation. This was further amplified in PAH-hPASMCs. Nox1−/− but not Nox4−/− mice were protected against PAH and vascular remodeling. Our findings demonstrate that in PAH-hPASMCs, 16αOHE1 stimulates redox-sensitive cell growth primarily through Nox1. Supporting this, in vivo studies exhibited protection against pulmonary hypertension and remodeling in Nox1−/− mice. This study provides new insights through Nox1/ROS and nuclear factor erythroid–related factor 2

  3. Hydrogen-bonding modulation of excited-state properties of flavins in a model of aqueous confined environment.

    PubMed

    Valle, Lorena; Vieyra, Faustino E Morán; Borsarelli, Claudio D

    2012-06-01

    The singlet and triplet excited states properties of lumiflavin (LF), riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in reversed micelles (RM) of sodium docusate (AOT) in n-hexane solutions were evaluated as a function of the water to surfactant molar ratio, w(0) = [H(2)O]/[AOT], by both steady-state and time-resolved absorption and fluorescence spectroscopy. The results indicated that hydrogen-bonding interactions between the isoalloxazine ring of the flavins with the water molecules of the micellar interior play a crucial role on the modulation of the excited state properties of the flavins. Fluorescence dynamic experiments in the RM, allowed the calculation of similar values for both the internal rotational time of the flavins (θ(i)) and the hydrogen-bonding relaxation time (τ(HB)), e.g.≈ 7 and 1.5 ns at w(0) = 1 and 20, respectively. In turn, the triplet state lifetimes of the flavins were also enlarged in RM solutions at low w(0), without modifications of their quantum yields. A hydrogen bonding relaxation model is proposed to explain the singlet excited state properties of the flavins, while the changes of the triplet state decays of the flavins were related with the global composition and strength of the hydrogen bonding network inside of the RM.

  4. Amphiphilic flavins in micelles and Langmuir-Blodgett films

    NASA Astrophysics Data System (ADS)

    Shaffer, Shadonna Patrice

    2002-01-01

    Flavins are important natural coenzymes for a large number of redox enzymes known as flavoenzymes. The oxidation of NADH to NAD is one of many important flavoenzymes because it is a key step in respiration. Surfactants have been known to accelerate or inhibit these biological redox reactions. We used four different flavins and four different surfactants to observe the effect mixing these compounds would have on the oxidation of N-benzyldihydronicotinamide, a synthetic analog of the natural nicotinamide dihydronicotinamide adenine dinucleotide (NADH). The four surfactants used are dodecyltrimethylammonium bromide (DTAB, a cationic surfactant), sodium dodecyl sulfate (SDS, an anionic surfactant), perfluoro-1-octanesulfonicacid tetraethylammonium salt (POAT, a cation fluorinated surfactant), and [3-[[(heptadecafluorooctyl) sulfonyl]amino]propyl]trimethyl-ammonim iodide (17FOSI, an anionic fluorinated surfactant). The four flavins used are N(10)-butyl-7-trimethylflavin, N(10)-dodecylflavin-7-carboxylic acid, N(10)-butylflavin-7-carboxylic acid, and N(10)-octylflavin-7-carboxylic acid. The kinetics study demonstrated mostly an inhibitory effect on the catalytic oxidation reactions. The second part of the dissertation dealt with monolayers and Langmuir-Blodgett (LB) films. We used N(10)-hexadecyl-isoalloxazine-7-carboxylic acid (C16Fl) as our representative flavin and used two amino acid surfactants N-stearoyl-L-tyrosine methyl ester (STME) and N stearoyl-L-serine methyl ester (SSME) to form mixed monolayers. The monolayer studies provided information about how close in proximity the flavin's ring and the flavin's hydrocarbons are to the liquid surface. For the LB films, the transfer of the monolayers to the indium tin oxide (ITO) glass slides with the mixtures of the amino acids occurred with high transfer ratios, the ratio for STME being higher than the ratio for SSME. The electron transfer rates for LB films of flavin derivatives are slower than those reported for

  5. C4a-hydroperoxyflavin formation in N-hydroxylating flavin monooxygenases is mediated by the 2'-OH of the nicotinamide ribose of NADP⁺.

    PubMed

    Robinson, Reeder; Badieyan, Somayesadat; Sobrado, Pablo

    2013-12-23

    Flavin-dependent monooxygenases must stabilize a C4a-hydroperoxyflavin intermediate to hydroxylate their respective substrates. Formation and decay of the C4a-hydroperoxyflavin were monitored under rapid reaction kinetic conditions in SidA, an N-hydroxylating monooxygenase involved in siderophore biosynthesis. Solvent kinetic isotope effect studies of flavin oxidation indicate that both hydrogen peroxide elimination and water elimination occur via abstraction of hydrogen from the N5 of the flavin. Kinetic isotope effect and density functional theory results are consistent with the transfer of a proton from the 2'-OH of the nicotinamide ribose of nicotinamide adenine dinucleotide phosphate (NADP⁺) to the C4a-peroxyflavin to form the C4a-hydroperoxyflavin. This represents a novel role for NADP⁺ in the reaction of flavin-dependent enzymes.

  6. Flavins contained in yeast extract are exploited for anodic electron transfer by Lactococcus lactis.

    PubMed

    Masuda, Masaki; Freguia, Stefano; Wang, Yung-Fu; Tsujimura, Seiya; Kano, Kenji

    2010-06-01

    Cyclic voltammograms of yeast extract-containing medium exhibit a clear redox peak around -0.4V vs. Ag|AgCl. Fermentative bacterium Lactococcus lactis was hereby shown to exploit this redox compound for extracellular electron transfer towards a graphite anode using glucose as an electron donor. High performance liquid chromatography revealed that this may be a flavin-type compound. The ability of L. lactis to exploit exogenous flavins for anodic glucose oxidation was confirmed by tests where flavin-type compounds were supplied to the bacterium in well defined media. Based on its mid-point potential, riboflavin can be regarded as a near-optimal mediator for microbially catalyzed anodic electron transfer. Riboflavin derivative flavin mononucleotide (FMN) was also exploited by L. lactis as a redox shuttle, unlike flavin adenine dinucleotide (FAD), possibly due to the absence of a specific transporter for the latter. The use of yeast extract in microbial fuel cell media is herein discouraged based on the related unwanted artificial addition of redox mediators which may distort experimental results.

  7. Distinguishing two groups of flavin reductases by analyzing the protonation state of an active site carboxylic acid.

    PubMed

    Dumit, Verónica I; Cortez, Néstor; Matthias Ullmann, G

    2011-07-01

    Flavin-containing reductases are involved in a wide variety of physiological reactions such as photosynthesis, nitric oxide synthesis, and detoxification of foreign compounds, including therapeutic drugs. Ferredoxin-NADP(H)-reductase (FNR) is the prototypical enzyme of this family. The fold of this protein is highly conserved and occurs as one domain of several multidomain enzymes such as the members of the diflavin reductase family. The enzymes of this family have emerged as fusion of a FNR and a flavodoxin. Although the active sites of these enzymes are very similar, different enzymes function in opposite directions, that is, some reduce oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)) and some oxidize reduced nicotinamide adenine dinucleotide phosphate (NADPH). In this work, we analyze the protonation behavior of titratable residues of these enzymes through electrostatic calculations. We find that a highly conserved carboxylic acid in the active site shows a different titration behavior in different flavin reductases. This residue is deprotonated in flavin reductases present in plastids, but protonated in bacterial counterparts and in diflavin reductases. The protonation state of the carboxylic acid may also influence substrate binding. The physiological substrate for plastidic enzymes is NADP(+), but it is NADPH for the other mentioned reductases. In this article, we discuss the relevance of the environment of this residue for its protonation and its importance in catalysis. Our results allow to reinterpret and explain experimental data. PMID:21538544

  8. Bacterial flavin-containing monooxygenase is trimethylamine monooxygenase

    PubMed Central

    Chen, Yin; Patel, Nisha A.; Crombie, Andrew; Scrivens, James H.; Murrell, J. Colin

    2011-01-01

    Flavin-containing monooxygenases (FMOs) are one of the most important monooxygenase systems in Eukaryotes and have many important physiological functions. FMOs have also been found in bacteria; however, their physiological function is not known. Here, we report the identification and characterization of trimethylamine (TMA) monooxygenase, termed Tmm, from Methylocella silvestris, using a combination of proteomic, biochemical, and genetic approaches. This bacterial FMO contains the FMO sequence motif (FXGXXXHXXXF/Y) and typical flavin adenine dinucleotide and nicotinamide adenine dinucleotide phosphate-binding domains. The enzyme was highly expressed in TMA-grown M. silvestris and absent during growth on methanol. The gene, tmm, was expressed in Escherichia coli, and the purified recombinant protein had high Tmm activity. Mutagenesis of this gene abolished the ability of M. silvestris to grow on TMA as a sole carbon and energy source. Close homologs of tmm occur in many Alphaproteobacteria, in particular Rhodobacteraceae (marine Roseobacter clade, MRC) and the marine SAR11 clade (Pelagibacter ubique). We show that the ability of MRC to use TMA as a sole carbon and/or nitrogen source is directly linked to the presence of tmm in the genomes, and purified Tmm of MRC and SAR11 from recombinant E. coli showed Tmm activities. The tmm gene is highly abundant in the metagenomes of the Global Ocean Sampling expedition, and we estimate that 20% of the bacteria in the surface ocean contain tmm. Taken together, our results suggest that Tmm, a bacterial FMO, plays an important yet overlooked role in the global carbon and nitrogen cycles. PMID:22006322

  9. Protective effects of Guanxin Shutong capsule drug-containing serum on tumor necrosis factor-α-induced endothelial dysfunction through nicotinamide adenine dinucleotide phosphate oxidase and the nitric oxide pathway

    PubMed Central

    CAO, YANJUN; LIU, FENG; HUANG, ZHUANGZHUANG; ZHANG, YANMIN

    2014-01-01

    The Chinese medicinal formula Guanxin Shutong capsule (GXSTC) has been used for almost 10 years as a clinical treatment for chest pain, depression, palpitation and cardiovascular diseases. The aim of this study was to investigate the effects of GXSTC drug-containing serum on tumor necrosis factor-α (TNF-α)-stimulated endothelial cells. Cell viability was measured by MTT assay, and nitric oxide (NO) levels and NO synthase (NOS) activity were measured as standards of endothelial dysfunction. Malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity were evaluated using commercial kits. In addition, the protein expression of endothelial NOS (eNOS), AKT and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits was examined to evaluate the effect of GXSTC drug-containing serum on ECV304 cells. GXSTC significantly reversed the decrease in NO production induced by TNF-α (5 ng/ml) in ECV304 cells. The expression of NADPH oxidase subunits was increased by TNF-α treatment, but markedly inhibited by treatment with GXSTC in TNF-α-stimulated cells. In summary, GXSTC increased the production of NO in ECV304 cells and exerted a protective effect on ECV304 cells stimulated with TNF-α by upregulating the mRNA and protein expression of eNOS. This was accompanied by increased SOD activity and reduced MDA levels. These results suggested that GXSTC protects the endothelium via the NO pathway and exhibits antioxidant effects. PMID:25120637

  10. Molecular characterization of Fasciola hepatica and phylogenetic analysis based on mitochondrial (nicotiamide adenine dinucleotide dehydrogenase subunit I and cytochrome oxidase subunit I) genes from the North-East of Iran

    PubMed Central

    Reaghi, Saber; Haghighi, Ali; Harandi, Majid Fasihi; Spotin, Adel; Arzamani, Kourosh; Rouhani, Soheila

    2016-01-01

    Aim: Fascioliasis is one of the most zoonotic diseases with global extension. As the epidemiological distribution of Fasciola may lead to various genetic patterns of the parasite, the aim of this study is to identify Fasciola hepatica based on spermatogenesis, and phylogenetic analysis using mitochondrial (nicotiamide adenine dinucleotide dehydrogenase subunit I [ND1] and cytochrome oxidase subunit I) gene marker. Materials and Methods: In this study, 90 F. hepatica collected from 30 cattle at slaughterhouse located in three different geographical locations in the North-East of Iran were evaluated based on spermatogenetic ability and internal transcribed spacer 1 gene restriction fragment length polymorphism pattern. Genetic diversity and phylogenetic relationship using mtDNA gene marker for the isolates from the North-East of Iran, and other countries were then analyzed. Results: Partial sequences of mtDNA showed eight haplotypes in both genes. The phylogenic analysis using neighbor joining as well as maximum likelihood methods showed similar topologies of trees. Pairwise fixation index between different F. hepatica populations calculated from the nucleotide data set of ND1 gene are statistically significant and show the genetic difference. Conclusion: F. hepatica found in this region of Iran has different genetic structures through the other Fasciola populations in the world. PMID:27733809

  11. Sinorhizobium meliloti flavin secretion and bacteria-host interaction: role of the bifunctional RibBA protein.

    PubMed

    Yurgel, Svetlana N; Rice, Jennifer; Domreis, Elizabeth; Lynch, Joseph; Sa, Na; Qamar, Zeeshan; Rajamani, Sathish; Gao, Mengsheng; Roje, Sanja; Bauer, Wolfgang D

    2014-05-01

    Sinorhizobium meliloti, the nitrogen-fixing bacterial symbiont of Medicago spp. and other legumes, secretes a considerable amount of riboflavin. This precursor of the cofactors flavin mononucleotide and flavin adenine dinucleotide is a bioactive molecule that has a beneficial effect on plant growth. The ribBA gene of S. meliloti codes for a putative bifunctional enzyme with dihydroxybutanone phosphate synthase and guanosine triphosphate (GTP) cyclohydrolase II activities, catalyzing the initial steps of the riboflavin biosynthesis pathway. We show here that an in-frame deletion of ribBA does not cause riboflavin auxotrophy or affect the ability of S. meliloti to establish an effective symbiosis with the host plant but does affect the ability of the bacteria to secrete flavins, colonize host-plant roots, and compete for nodulation. A strain missing the RibBA protein retains considerable GTP cyclohydrolase II activity. Based on these results, we hypothesize that S. meliloti has two partly interchangeable modules for biosynthesis of riboflavin, one fulfilling the internal need for flavins in bacterial metabolism and the other producing riboflavin for secretion. Our data also indicate that bacteria-derived flavins play a role in communication between rhizobia and the legume host and that the RibBA protein is important in this communication process even though it is not essential for riboflavin biosynthesis and symbiosis.

  12. An essential role for UshA in processing of extracellular flavin electron shuttles by Shewanella oneidensis.

    PubMed

    Covington, Elizabeth D; Gelbmann, Christopher B; Kotloski, Nicholas J; Gralnick, Jeffrey A

    2010-10-01

    The facultative anaerobe Shewanella oneidensis can reduce a number of insoluble extracellular metals. Direct adsorption of cells to the metal surface is not necessary, and it has been shown that S. oneidensis releases low concentrations flavins, including riboflavin and flavin mononucleotide (FMN), into the surrounding medium to act as extracellular electron shuttles. However, the mechanism of flavin release by Shewanella remains unknown. We have conducted a transposon mutagenesis screen to identify mutants deficient in extracellular flavin accumulation. Mutations in ushA, encoding a predicted 5'-nucleotidase, resulted in accumulation of flavin adenine dinucleotide (FAD) in culture supernatants, with a corresponding decrease in FMN and riboflavin. Cellular extracts of S. oneidensis convert FAD to FMN, whereas extracts of ushA mutants do not, and fractionation experiments show that UshA activity is periplasmic. We hypothesize that S. oneidensis secretes FAD into the periplasmic space, where it is hydrolysed by UshA to FMN and adenosine monophosphate (AMP). FMN diffuses through outer membrane porins where it accelerates extracellular electron transfer, and AMP is dephosphorylated by UshA and reassimilated by the cell. We predict that transport of FAD into the periplasm also satisfies the cofactor requirement of the unusual periplasmic fumarate reductase found in Shewanella.

  13. Chloramphenicol Biosynthesis: The Structure of CmlS, a Flavin-Dependent Halogenase Shwing a Covalent Flavin-Aspartate Bond

    SciTech Connect

    Podzelinska, K.; Latimer, R; Bhattacharya, A; Vining, L; Zechel, D; Jia, Z

    2010-01-01

    Chloramphenicol is a halogenated natural product bearing an unusual dichloroacetyl moiety that is critical for its antibiotic activity. The operon for chloramphenicol biosynthesis in Streptomyces venezuelae encodes the chloramphenicol halogenase CmlS, which belongs to the large and diverse family of flavin-dependent halogenases (FDH's). CmlS was previously shown to be essential for the formation of the dichloroacetyl group. Here we report the X-ray crystal structure of CmlS determined at 2.2 {angstrom} resolution, revealing a flavin monooxygenase domain shared by all FDHs, but also a unique 'winged-helix' C-terminal domain that creates a T-shaped tunnel leading to the halogenation active site. Intriguingly, the C-terminal tail of this domain blocks access to the halogenation active site, suggesting a structurally dynamic role during catalysis. The halogenation active site is notably nonpolar and shares nearly identical residues with Chondromyces crocatus tyrosyl halogenase (CndH), including the conserved Lys (K71) that forms the reactive chloramine intermediate. The exception is Y350, which could be used to stabilize enolate formation during substrate halogenation. The strictly conserved residue E44, located near the isoalloxazine ring of the bound flavin adenine dinucleotide (FAD) cofactor, is optimally positioned to function as a remote general acid, through a water-mediated proton relay, which could accelerate the reaction of the chloramine intermediate during substrate halogenation, or the oxidation of chloride by the FAD(C4{alpha})-OOH intermediate. Strikingly, the 8{alpha} carbon of the FAD cofactor is observed to be covalently attached to D277 of CmlS, a residue that is highly conserved in the FDH family. In addition to representing a new type of flavin modification, this has intriguing implications for the mechanism of FDHs. Based on the crystal structure and in analogy to known halogenases, we propose a reaction mechanism for CmlS.

  14. Identification of a flavin-containing S-oxygenating monooxygenase involved in alliin biosynthesis in garlic.

    PubMed

    Yoshimoto, Naoko; Onuma, Misato; Mizuno, Shinya; Sugino, Yuka; Nakabayashi, Ryo; Imai, Shinsuke; Tsuneyoshi, Tadamitsu; Sumi, Shin-ichiro; Saito, Kazuki

    2015-09-01

    S-Alk(en)yl-l-cysteine sulfoxides are cysteine-derived secondary metabolites highly accumulated in the genus Allium. Despite pharmaceutical importance, the enzymes that contribute to the biosynthesis of S-alk-(en)yl-l-cysteine sulfoxides in Allium plants remain largely unknown. Here, we report the identification of a flavin-containing monooxygenase, AsFMO1, in garlic (Allium sativum), which is responsible for the S-oxygenation reaction in the biosynthesis of S-allyl-l-cysteine sulfoxide (alliin). Recombinant AsFMO1 protein catalyzed the stereoselective S-oxygenation of S-allyl-l-cysteine to nearly exclusively yield (RC SS )-S-allylcysteine sulfoxide, which has identical stereochemistry to the major natural form of alliin in garlic. The S-oxygenation reaction catalyzed by AsFMO1 was dependent on the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and flavin adenine dinucleotide (FAD), consistent with other known flavin-containing monooxygenases. AsFMO1 preferred S-allyl-l-cysteine to γ-glutamyl-S-allyl-l-cysteine as the S-oxygenation substrate, suggesting that in garlic, the S-oxygenation of alliin biosynthetic intermediates primarily occurs after deglutamylation. The transient expression of green fluorescent protein (GFP) fusion proteins indicated that AsFMO1 is localized in the cytosol. AsFMO1 mRNA was accumulated in storage leaves of pre-emergent nearly sprouting bulbs, and in various tissues of sprouted bulbs with green foliage leaves. Taken together, our results suggest that AsFMO1 functions as an S-allyl-l-cysteine S-oxygenase, and contributes to the production of alliin both through the conversion of stored γ-glutamyl-S-allyl-l-cysteine to alliin in storage leaves during sprouting and through the de novo biosynthesis of alliin in green foliage leaves.

  15. Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers†

    PubMed Central

    Abbas, Charles A.; Sibirny, Andriy A.

    2011-01-01

    Summary: Riboflavin [7,8-dimethyl-10-(1′-d-ribityl)isoalloxazine, vitamin B2] is an obligatory component of human and animal diets, as it serves as the precursor of flavin coenzymes, flavin mononucleotide, and flavin adenine dinucleotide, which are involved in oxidative metabolism and other processes. Commercially produced riboflavin is used in agriculture, medicine, and the food industry. Riboflavin synthesis starts from GTP and ribulose-5-phosphate and proceeds through pyrimidine and pteridine intermediates. Flavin nucleotides are synthesized in two consecutive reactions from riboflavin. Some microorganisms and all animal cells are capable of riboflavin uptake, whereas many microorganisms have distinct systems for riboflavin excretion to the medium. Regulation of riboflavin synthesis in bacteria occurs by repression at the transcriptional level by flavin mononucleotide, which binds to nascent noncoding mRNA and blocks further transcription (named the riboswitch). In flavinogenic molds, riboflavin overproduction starts at the stationary phase and is accompanied by derepression of enzymes involved in riboflavin synthesis, sporulation, and mycelial lysis. In flavinogenic yeasts, transcriptional repression of riboflavin synthesis is exerted by iron ions and not by flavins. The putative transcription factor encoded by SEF1 is somehow involved in this regulation. Most commercial riboflavin is currently produced or was produced earlier by microbial synthesis using special selected strains of Bacillus subtilis, Ashbya gossypii, and Candida famata. Whereas earlier RF overproducers were isolated by classical selection, current producers of riboflavin and flavin nucleotides have been developed using modern approaches of metabolic engineering that involve overexpression of structural and regulatory genes of the RF biosynthetic pathway as well as genes involved in the overproduction of the purine precursor of riboflavin, GTP. PMID:21646432

  16. Genetic control of biosynthesis and transport of riboflavin and flavin nucleotides and construction of robust biotechnological producers.

    PubMed

    Abbas, Charles A; Sibirny, Andriy A

    2011-06-01

    Riboflavin [7,8-dimethyl-10-(1'-d-ribityl)isoalloxazine, vitamin B₂] is an obligatory component of human and animal diets, as it serves as the precursor of flavin coenzymes, flavin mononucleotide, and flavin adenine dinucleotide, which are involved in oxidative metabolism and other processes. Commercially produced riboflavin is used in agriculture, medicine, and the food industry. Riboflavin synthesis starts from GTP and ribulose-5-phosphate and proceeds through pyrimidine and pteridine intermediates. Flavin nucleotides are synthesized in two consecutive reactions from riboflavin. Some microorganisms and all animal cells are capable of riboflavin uptake, whereas many microorganisms have distinct systems for riboflavin excretion to the medium. Regulation of riboflavin synthesis in bacteria occurs by repression at the transcriptional level by flavin mononucleotide, which binds to nascent noncoding mRNA and blocks further transcription (named the riboswitch). In flavinogenic molds, riboflavin overproduction starts at the stationary phase and is accompanied by derepression of enzymes involved in riboflavin synthesis, sporulation, and mycelial lysis. In flavinogenic yeasts, transcriptional repression of riboflavin synthesis is exerted by iron ions and not by flavins. The putative transcription factor encoded by SEF1 is somehow involved in this regulation. Most commercial riboflavin is currently produced or was produced earlier by microbial synthesis using special selected strains of Bacillus subtilis, Ashbya gossypii, and Candida famata. Whereas earlier RF overproducers were isolated by classical selection, current producers of riboflavin and flavin nucleotides have been developed using modern approaches of metabolic engineering that involve overexpression of structural and regulatory genes of the RF biosynthetic pathway as well as genes involved in the overproduction of the purine precursor of riboflavin, GTP. PMID:21646432

  17. Nicotinamide Adenine Dinucleotide Based Therapeutics, Update.

    PubMed

    Pankiewicz, K W; Petrelli, R; Singh, R; Felczak, K

    2015-01-01

    About 500 NAD (P)-dependent enzymes in the cell use NAD (P) as a cofactor or a substrate. This family of broadly diversified enzymes is crucial for maintaining homeostasis of all living organisms. The NAD binding domain of these enzymes is conserved and it was believed that NAD mimics would not be of therapeutic value due to lack of selectivity. Consequently, only mycophenolic acid which selectively binds at the cofactor pocket of NAD-dependent IMP-dehydrogenase (IMPDH) has been approved as an immunosuppressant. Recently, it became clear that the NAD (P)-binding domain was structurally much more diversified than anticipated and numerous highly potent and selective inhibitors of NAD (P) dependent enzymes have been reported. It is likely, that as in the case of protein kinases inhibitors, inhibitors of NAD (P)-dependent enzymes would find soon their way to the clinic. In this review, recent developments of selective inhibitors of NAD-dependent human IMPDH, as well as inhibitors of IMPDHs from parasites, and from bacterial sources are reported. Therapies against Cryptosporidium parvum and the development of new antibiotics that are on the horizon will be discussed. New inhibitors of bacterial NAD-ligases, NAD-kinases, NMN-adenylyl transferases, as well as phosphoribosyl transferases are also described. Although none of these compounds has yet to be approved, the progress in revealing and understanding crucial factors that might allow for designing more potent and efficient drug candidates is enormous and highly encouraging. PMID:26295463

  18. A regulatory role of NAD redox status on flavin cofactor homeostasis in S. cerevisiae mitochondria.

    PubMed

    Giancaspero, Teresa Anna; Locato, Vittoria; Barile, Maria

    2013-01-01

    Flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD) are two redox cofactors of pivotal importance for mitochondrial functionality and cellular redox balance. Despite their relevance, the mechanism by which intramitochondrial NAD(H) and FAD levels are maintained remains quite unclear in Saccharomyces cerevisiae. We investigated here the ability of isolated mitochondria to degrade externally added FAD and NAD (in both its reduced and oxidized forms). A set of kinetic experiments demonstrated that mitochondrial FAD and NAD(H) destroying enzymes are different from each other and from the already characterized NUDIX hydrolases. We studied here, in some detail, FAD pyrophosphatase (EC 3.6.1.18), which is inhibited by NAD(+) and NADH according to a noncompetitive inhibition, with Ki values that differ from each other by an order of magnitude. These findings, together with the ability of mitochondrial FAD pyrophosphatase to metabolize endogenous FAD, presumably deriving from mitochondrial holoflavoproteins destined to degradation, allow for proposing a novel possible role of mitochondrial NAD redox status in regulating FAD homeostasis and/or flavoprotein degradation in S. cerevisiae. PMID:24078860

  19. A Regulatory Role of NAD Redox Status on Flavin Cofactor Homeostasis in S. cerevisiae Mitochondria

    PubMed Central

    Giancaspero, Teresa Anna; Barile, Maria

    2013-01-01

    Flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD) are two redox cofactors of pivotal importance for mitochondrial functionality and cellular redox balance. Despite their relevance, the mechanism by which intramitochondrial NAD(H) and FAD levels are maintained remains quite unclear in Saccharomyces cerevisiae. We investigated here the ability of isolated mitochondria to degrade externally added FAD and NAD (in both its reduced and oxidized forms). A set of kinetic experiments demonstrated that mitochondrial FAD and NAD(H) destroying enzymes are different from each other and from the already characterized NUDIX hydrolases. We studied here, in some detail, FAD pyrophosphatase (EC 3.6.1.18), which is inhibited by NAD+ and NADH according to a noncompetitive inhibition, with Ki values that differ from each other by an order of magnitude. These findings, together with the ability of mitochondrial FAD pyrophosphatase to metabolize endogenous FAD, presumably deriving from mitochondrial holoflavoproteins destined to degradation, allow for proposing a novel possible role of mitochondrial NAD redox status in regulating FAD homeostasis and/or flavoprotein degradation in S. cerevisiae. PMID:24078860

  20. Role of inorganic phosphate during electron transfer in the redox system NADH+flavin

    NASA Astrophysics Data System (ADS)

    Lozinova, T. A.; Brzhevskaya, O. N.; Kisin, A. V.; Nosova, V. M.; Rykov, S. V.; Tychinskaya, L. Yu.

    2006-05-01

    We have used 31P NMR and EPR to study the effect of inorganic phosphate (Pi) on the process of electron transfer in oxidation of reduced nicotinamide adenine dinucleotide (NADH) by flavin in aqueous solutions. Observation of a significant (up to 40%) drop in the integrated intensity of the 31P NMR signal for the inorganic phosphate over the course of the reaction is interpreted as a manifestation of the effect of chemically induced polarization of the phosphorus nuclei during singlet-triplet conversion in charge-transfer complexes [NADH…Pi], and is evidence for a possible catalytic role for Pi in processes involving transfer of an electron (hydrogen atom) from NADH to appropriate acceptors. The results presented support the possibility of direct involvement of Pi in a chain of one-electron conversions in chemical systems.

  1. Replacement of Tyr50 stacked on the si-face of the isoalloxazine ring of the flavin adenine dinucleotide prosthetic group modulates Bacillus subtilis ferredoxin-NADP(+) oxidoreductase activity toward NADPH.

    PubMed

    Seo, Daisuke; Naito, Hiroshi; Nishimura, Erika; Sakurai, Takeshi

    2015-08-01

    Ferredoxin-NAD(P)(+) oxidoreductases ([EC 1.18.1.2], [EC 1.18.1.3], FNRs) from green sulfur bacteria, purple non-sulfur bacteria and most of Firmicutes, such as Bacillus subtilis (BsFNR) are homo-dimeric flavoproteins homologous to bacterial NADPH-thioredoxin reductase. These FNRs contain two unique aromatic residues stacked on the si- and re-face of the isoalloxazine ring moiety of the FAD prosthetic group whose configurations are often found among other types of flavoproteins including plant-type FNR and flavodoxin, but not in bacterial NADPH-thioredoxin reductase. To investigate the role of the si-face Tyr50 residue in BsFNR, we replaced Tyr50 with Gly, Ser, and Trp and examined its spectroscopic properties and enzymatic activities in the presence of NADPH and ferredoxin (Fd) from B. subtilis (BsFd). The replacement of Tyr50 to Gly (Y50G), Ser (Y50S), and Trp (Y50W) in BsFNR resulted in a blue shift of the FAD transition bands. The Y50G and Y50S mutations enhanced the FAD fluorescence emission, whereas those of the wild type and Y50W mutant were quenched. All three mutants decreased thermal stabilities compared to wild type. Using a diaphorase assay, the k cat values for the Y50G and Y50S mutants in the presence of NADPH and ferricyanide were decreased to less than 5 % of the wild type activity. The Y50W mutant retained approximately 20 % reactivity in the diaphorase assay and BsFd-dependent cytochrome c reduction assay relative to wild type. The present results suggest that Tyr50 modulates the electronic properties and positioning of the prosthetic group.

  2. Flavin-Induced Oligomerization in Escherichia coli Adaptive Response Protein AidB

    SciTech Connect

    Hamill, Michael J.; Jost, Marco; Wong, Cintyu; Elliott, Sean J.; Drennan, Catherine L.

    2011-11-21

    The process known as 'adaptive response' allows Escherichia coli to respond to small doses of DNA-methylating agents by upregulating the expression of four proteins. While the role of three of these proteins in mitigating DNA damage is well understood, the function of AidB is less clear. Although AidB is a flavoprotein, no catalytic role has been established for the bound cofactor. Here we investigate the possibility that flavin plays a structural role in the assembly of the AidB tetramer. We report the generation and biophysical characterization of deflavinated AidB and of an AidB mutant that has greatly reduced affinity for flavin adenine dinucleotide (FAD). Using fluorescence quenching and analytical ultracentrifugation, we find that apo AidB has a high affinity for FAD, as indicated by an apparent dissociation constant of 402.1 {+-} 35.1 nM, and that binding of substoichiometric amounts of FAD triggers a transition in the AidB oligomeric state. In particular, deflavinated AidB is dimeric, whereas the addition of FAD yields a tetramer. We further investigate the dimerization and tetramerization interfaces of AidB by determining a 2.8 {angstrom} resolution crystal structure in space group P3{sub 2} that contains three intact tetramers in the asymmetric unit. Taken together, our findings provide strong evidence that FAD plays a structural role in the formation of tetrameric AidB.

  3. Flavin-Induced Oligomerization in Escherichia coli Adaptive Response Protein AidB

    PubMed Central

    2011-01-01

    The process known as “adaptive response” allows Escherichia coli to respond to small doses of DNA-methylating agents by upregulating the expression of four proteins. While the role of three of these proteins in mitigating DNA damage is well understood, the function of AidB is less clear. Although AidB is a flavoprotein, no catalytic role has been established for the bound cofactor. Here we investigate the possibility that flavin plays a structural role in the assembly of the AidB tetramer. We report the generation and biophysical characterization of deflavinated AidB and of an AidB mutant that has greatly reduced affinity for flavin adenine dinucleotide (FAD). Using fluorescence quenching and analytical ultracentrifugation, we find that apo AidB has a high affinity for FAD, as indicated by an apparent dissociation constant of 402.1 ± 35.1 nM, and that binding of substoichiometric amounts of FAD triggers a transition in the AidB oligomeric state. In particular, deflavinated AidB is dimeric, whereas the addition of FAD yields a tetramer. We further investigate the dimerization and tetramerization interfaces of AidB by determining a 2.8 Å resolution crystal structure in space group P32 that contains three intact tetramers in the asymmetric unit. Taken together, our findings provide strong evidence that FAD plays a structural role in the formation of tetrameric AidB. PMID:22004173

  4. Chopping and Changing: the Evolution of the Flavin-dependent Monooxygenases.

    PubMed

    Mascotti, Maria Laura; Juri Ayub, Maximiliano; Furnham, Nicholas; Thornton, Janet M; Laskowski, Roman A

    2016-07-31

    Flavin-dependent monooxygenases play a variety of key physiological roles and are also very powerful biotechnological tools. These enzymes have been classified into eight different classes (A-H) based on their sequences and biochemical features. By combining structural and sequence analysis, and phylogenetic inference, we have explored the evolutionary history of classes A, B, E, F, and G and demonstrate that their multidomain architectures reflect their phylogenetic relationships, suggesting that the main evolutionary steps in their divergence are likely to have arisen from the recruitment of different domains. Additionally, the functional divergence within in each class appears to have been the result of other mechanisms such as a complex set of single-point mutations. Our results reinforce the idea that a main constraint on the evolution of cofactor-dependent enzymes is the functional binding of the cofactor. Additionally, a remarkable feature of this family is that the sequence of the key flavin adenine dinucleotide-binding domain is split into at least two parts in all classes studied here. We propose a complex set of evolutionary events that gave rise to the origin of the different classes within this family. PMID:27423402

  5. Unprecedented head-to-head right-handed cross-links between the antitumor bis(mu-N,N'-di-p-tolylformamidinate) dirhodium(II,II) core and the dinucleotide d(ApA) with the adenine bases in the rare imino form.

    PubMed

    Chifotides, Helen T; Dunbar, Kim R

    2007-10-17

    Reactions of the anticancer active compound cis-[Rh2(DTolF)2(CH3CN)6](BF4)2 with 9-ethyladenine (9-EtAdeH) or the dinucleotide d(ApA) proceed with bridging adenine bases in the rare imino form (A*), spanning the Rh-Rh bond at equatorial positions via N7/N6. The inflection points for the pH-dependent H2 and H8 NMR resonance curves of cis-[Rh2(DTolF)2(9-EtAdeH)2](BF4)2 correspond to N1H deprotonation of the metal-stabilized rare imino tautomer, which takes place at pKa approximately 7.5 in CD3CN-d3, a considerably reduced value as compared to that of the imino form of 9-EtAdeH. Similarly, coordination of the metal atoms to the N7/N6 adenine sites in Rh2(DTolF)2{d(ApA)} induces formation of the rare imino tautomer of the bases with a concomitant substantial decrease in the basicity of the N1H sites (pKa approximately 7.0 in CD3CN-d3), as compared to the imino form of the free dinucleotide. The presence of the adenine bases in the rare imino form, due to bidentate metalation of the N6/N7 sites, is further corroborated by DQF-COSY H2/N1H and ROE N1H/N6H cross-peaks in the 2D NMR spectra of Rh2(DTolF)2{d(ApA)} in CD3CN-d3 at -38 degrees C. Due to the N7/N6 bridging mode of the adenine bases in Rh2(DTolF)2{d(ApA)}, only the anti orientation of the imino tautomer is possible. The imino form A* of adenine in DNA may result in AT-->CG transversions or AT-->GC transitions, which can eventually lead to lethal mutations. The HH arrangement of the bases in Rh2(DTolF)2{d(ApA)} is indicated by the H8/H8 NOE cross-peaks in the 2D ROESY NMR spectrum, whereas the formamidinate bridging groups dictate the presence of one right-handed conformer HH1R in solution. Complete characterization of Rh2(DTolF)2{d(ApA)} by 2D NMR spectroscopy and molecular modeling supports the presence of the HH1R conformer, anti orientation of both sugar residues about the glycosyl bonds, and N-type conformation for the 5'-A base.

  6. Oxygen uptake after electron transfer from amines, amino acids and ascorbic acid to triplet flavins in air-saturated aqueous solution.

    PubMed

    Görner, Helmut

    2007-05-25

    The photolysis of lumichrome, riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) was studied in air-saturated aqueous solution at room temperature in the presence of appropriate electron donors: ascorbic acid, aromatic amino acids or amines, e.g. ethylenediaminetetraacetate (EDTA). The overall reaction is conversion of oxygen via the hydroperoxyl/superoxide radical into hydrogen peroxide. The quantum yield of oxygen uptake increases with the donor concentration, e.g. up to 0.3 for riboflavin, FMN or FAD in the presence of EDTA or ascorbic acid (0.3-10mM). The formation of H(2)O(2) is initiated by quenching of the acceptor triplet state by the electron donor and subsequent reaction of the semiquinone radical with oxygen. Specific properties of flavins are discussed including the radicals involved and the pH and concentration dependences. The quantum yield of photodegradation is low under air, but substantial under argon, where the major product absorbing in the visible spectral range is the corresponding hydroquinone. PMID:17395476

  7. The structure of a Xanthomonas general stress protein involved in citrus canker reveals its flavin-binding property.

    PubMed

    Hilario, Eduardo; Li, Yang; Niks, Dimitri; Fan, Li

    2012-07-01

    Xanthomonas citri pv. citri (Xac) causes citrus canker and affects citrus agriculture worldwide. Functional genetic analysis has indicated that a putative general stress protein (XacGSP) encoded by the Xac2369 gene is involved in the bacterial infection. In this report, the crystal structure of XacGSP was determined to 2.5 Å resolution. There are four XacGSP molecules in the crystal asymmetric unit. Each XacGSP monomer folds into a six-stranded antiparallel β-barrel flanked by five α-helices. A C-terminal extension protrudes from the sixth β-strand of the β-barrel and pairs with its counterpart from another monomer to form a bridge between the two subunits of an XacGSP dimer. Two XacGSP dimers cross over each other to form a tetramer; the β-barrels from one dimer contact the β-barrels of the other, while the two bridges are distant from each other and do not make contacts. The three-dimensional structure of the XacGSP monomer is very similar to those of pyridoxine 5-phosphate oxidases, a group of enzymes that use flavin mononucleotide (FMN) as a cofactor. Consistent with this, purified XacGSP protein binds to both FMN and flavin adenine dinucleotide (FAD), suggesting that XacGSP may help the bacteria to react against the oxidative stress induced by the defense mechanisms of the plant. PMID:22751670

  8. Evidence for posttranslational protein flavinylation in the syphilis spirochete Treponema pallidum: Structural and biochemical insights from the catalytic core of a periplasmic flavin-trafficking protein

    DOE PAGES

    Deka, Ranjit K.; Brautigam, Chad A.; Liu, Wei Z.; Tomchick, Diana R.; Norgard, Michael V.

    2015-05-05

    The syphilis spirochete Treponema pallidum is an important human pathogen but a highly enigmatic bacterium that cannot be cultivated in vitro. T. pallidum lacks many biosynthetic pathways and therefore has evolved the capability to exploit host-derived metabolites via its periplasmic lipoprotein repertoire. We recently reported a flavin-trafficking protein in T. pallidum (Ftp_Tp; TP0796) as the first bacterial metal-dependent flavin adenine dinucleotide (FAD) pyrophosphatase that hydrolyzes FAD into AMP and flavin mononucleotide (FMN) in the spirochete’s periplasm. However, orthologs of Ftp_Tp from other bacteria appear to lack this hydrolytic activity; rather, they bind and flavinylate subunits of a cytoplasmic membrane redoxmore » system (Nqr/Rnf). To further explore this dichotomy, biochemical analyses, protein crystallography, and structure-based mutagenesis were used to show that a single amino acid change (N55Y) in Ftp_Tp converts it from an Mg²⁺-dependent FAD pyrophosphatase to an FAD-binding protein. We also demonstrated that Ftp_Tp has a second enzymatic activity (Mg²⁺-FMN transferase); it flavinylates protein(s) covalently with FMN on a threonine side chain of an appropriate sequence motif using FAD as the substrate. Moreover, mutation of a metal-binding residue (D284A) eliminates Ftp_Tp’s dual activities, thereby underscoring the role of Mg²⁺ in the enzyme-catalyzed reactions. The posttranslational flavinylation activity that can target a periplasmic lipoprotein (TP0171) has not previously been described. The observed activities reveal the catalytic flexibility of a treponemal protein to perform multiple functions. Together, these findings imply mechanisms by which a dynamic pool of flavin cofactor is maintained and how flavoproteins are generated by Ftp_Tp locally in the T. pallidum periplasm.« less

  9. Evidence for Posttranslational Protein Flavinylation in the Syphilis Spirochete Treponema pallidum: Structural and Biochemical Insights from the Catalytic Core of a Periplasmic Flavin-Trafficking Protein

    PubMed Central

    Deka, Ranjit K.; Brautigam, Chad A.; Liu, Wei Z.

    2015-01-01

    ABSTRACT The syphilis spirochete Treponema pallidum is an important human pathogen but a highly enigmatic bacterium that cannot be cultivated in vitro. T. pallidum lacks many biosynthetic pathways and therefore has evolved the capability to exploit host-derived metabolites via its periplasmic lipoprotein repertoire. We recently reported a flavin-trafficking protein in T. pallidum (Ftp_Tp; TP0796) as the first bacterial metal-dependent flavin adenine dinucleotide (FAD) pyrophosphatase that hydrolyzes FAD into AMP and flavin mononucleotide (FMN) in the spirochete’s periplasm. However, orthologs of Ftp_Tp from other bacteria appear to lack this hydrolytic activity; rather, they bind and flavinylate subunits of a cytoplasmic membrane redox system (Nqr/Rnf). To further explore this dichotomy, biochemical analyses, protein crystallography, and structure-based mutagenesis were used to show that a single amino acid change (N55Y) in Ftp_Tp converts it from an Mg2+-dependent FAD pyrophosphatase to an FAD-binding protein. We also demonstrated that Ftp_Tp has a second enzymatic activity (Mg2+-FMN transferase); it flavinylates protein(s) covalently with FMN on a threonine side chain of an appropriate sequence motif using FAD as the substrate. Moreover, mutation of a metal-binding residue (D284A) eliminates Ftp_Tp’s dual activities, thereby underscoring the role of Mg2+ in the enzyme-catalyzed reactions. The posttranslational flavinylation activity that can target a periplasmic lipoprotein (TP0171) has not previously been described. The observed activities reveal the catalytic flexibility of a treponemal protein to perform multiple functions. Together, these findings imply mechanisms by which a dynamic pool of flavin cofactor is maintained and how flavoproteins are generated by Ftp_Tp locally in the T. pallidum periplasm. PMID:25944861

  10. Role of aromatic stacking interactions in the modulation of the two-electron reduction potentials of flavin and substrate/product in Megasphaera elsdenii short-chain acyl-coenzyme A dehydrogenase.

    PubMed

    Pellett, J D; Becker, D F; Saenger, A K; Fuchs, J A; Stankovich, M T

    2001-06-26

    The effects of aromatic stacking interactions on the stabilization of reduced flavin adenine dinucleotide (FAD) and substrate/product have been investigated in short-chain acyl-coenzyme A dehydrogenase (SCAD) from Megasphaera elsdenii. Mutations were made at the aromatic residues Phe160 and Tyr366, which flank either face of the noncovalently bound flavin cofactor. The electrochemical properties of the mutants were then measured in the presence and absence of a butyryl-CoA/crotonyl-CoA mixture. Results from these redox studies suggest that the phenylalanine and tyrosine both engage in favorable pi-sigma interactions with the isoalloxazine ring of the flavin to help stabilize formation of the anionic flavin hydroquinone. Disruption of these interactions by replacing either residue with a leucine (F160L and Y366L) causes the midpoint potential for the oxidized/hydroquinone couple (E(ox/hq)) to shift negative by 44-54 mV. The E(ox/hq) value was also found to decrease when aromatic residues containing electron-donating heteroatoms were introduced at the 160 position. Potential shifts of -32 and -43 mV for the F160Y and F160W mutants, respectively, are attributed to increased pi-pi repulsive interactions between the ring systems. This study also provides evidence for thermodynamic regulation of the substrate/product couple in the active site of SCAD. Binding to the wild-type enzyme caused the midpoint potential for the butyryl-CoA/crotonyl-CoA couple (E(BCoA/CCoA)) to shift 14 mV negative, stabilizing the oxidized product. Formation of product was found to be even more favorable in complexes with the F160Y and F160W mutants, suggesting that the electrostatic environment around the flavin plays a role in substrate/product activation.

  11. How pH Modulates the Reactivity and Selectivity of a Siderophore-Associated Flavin Monooxygenase

    PubMed Central

    2015-01-01

    Flavin-containing monooxygenases (FMOs) catalyze the oxygenation of diverse organic molecules using O2, NADPH, and the flavin adenine dinucleotide (FAD) cofactor. The fungal FMO SidA initiates peptidic siderophore biosynthesis via the highly selective hydroxylation of l-ornithine, while the related amino acid l-lysine is a potent effector of reaction uncoupling to generate H2O2. We hypothesized that protonation states could critically influence both substrate-selective hydroxylation and H2O2 release, and therefore undertook a study of SidA’s pH-dependent reaction kinetics. Consistent with other FMOs that stabilize a C4a-OO(H) intermediate, SidA’s reductive half reaction is pH independent. The rate constant for the formation of the reactive C4a-OO(H) intermediate from reduced SidA and O2 is likewise independent of pH. However, the rate constants for C4a-OO(H) reactions, either to eliminate H2O2 or to hydroxylate l-Orn, were strongly pH-dependent and influenced by the nature of the bound amino acid. Solvent kinetic isotope effects of 6.6 ± 0.3 and 1.9 ± 0.2 were measured for the C4a-OOH/H2O2 conversion in the presence and absence of l-Lys, respectively. A model is proposed in which l-Lys accelerates H2O2 release via an acid–base mechanism and where side-chain position determines whether H2O2 or the hydroxylation product is observed. PMID:24490904

  12. The reduced flavin-dependent monooxygenase SfnG converts dimethylsulfone to methanesulfinate.

    PubMed

    Wicht, Denyce K

    2016-08-15

    The biochemical pathway through which sulfur may be assimilated from dimethylsulfide (DMS) is proposed to proceed via oxidation of DMS to dimethylsulfoxide (DMSO) and subsequent conversion of DMSO to dimethylsulfone (DMSO2). Analogous chemical oxidation processes involving biogenic DMS in the atmosphere result in the deposition of DMSO2 into the terrestrial environment. Elucidating the enzymatic pathways that involve DMSO2 contribute to our understanding of the global sulfur cycle. Dimethylsulfone monooxygenase SfnG and flavin mononucleotide (FMN) reductase MsuE from the genome of the aerobic soil bacterium Pseudomonas fluorescens Pf0-1 were produced in Escherichia coli, purified, and biochemically characterized. The enzyme MsuE functions as a reduced nicotinamide adenine dinucleotide (NADH)-dependent FMN reductase with apparent steady state kinetic parameters of Km = 69 μM and kcat/Km = 9 min(-1) μM (-1) using NADH as the variable substrate, and Km = 8 μM and kcat/Km = 105 min(-1) μM (-1) using FMN as the variable substrate. The enzyme SfnG functions as a flavoprotein monooxygenase and converts DMSO2 to methanesulfinate in the presence of FMN, NADH, and MsuE, as evidenced by (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy. The results suggest that methanesulfinate is a biochemical intermediate in sulfur assimilation. PMID:27392454

  13. Is Nicotinamide Adenine Dinucleotide Phosphate an Obligatory Intermediate in Photosynthesis?

    PubMed Central

    Ben-Amotz, Ami; Avron, Mordhay

    1972-01-01

    The site of action of the inhibitors disalicylidenepropanediamine and pyrophosphate was more closely defined as acting on ferredoxin. Three inhibitors which act on the electron transport path between ferredoxin and NADP: disalicylidenepropanediamine, pyrophosphate, and phosphoadenosinediphosphate ribose, had no effect on photosynthesis in cell free preparations of Dunaliela parva at concentrations which completely inhibited the enzymic activity on which each inhibitor acts. The addition of disalicylidenepropanediamine to dark-grown Euglena gracilis cells prevented the light-induced formation of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, but not of photosynthesis, chlorophyll synthesis, or NAD-dependent glyceraldehyde-3-phosphate dehydrogenase. The above results are interpreted as indicating that, at least under some conditions, a reduced product of photosystem I preceding ferredoxin in the electron transport path can serve as the reductant of CO2 in photosynthesis. PMID:16657933

  14. Binding of nicotinamide–adenine dinucleotides to diphtheria toxin

    PubMed Central

    Montanaro, L.; Sperti, Simonetta

    1967-01-01

    1. Changes in protein fluorescence have been utilized in determining the stoicheiometry and dissociation constants of the complexes of diphtheria toxin with NADH2, NAD, NADPH2 and NADP. 2. The binding stoicheiometry is 2moles of NADH2 and 1mole of NADPH2/mole of diphtheria toxin. The binding sites for NADH2 appear to be equivalent and independent. 3. The toxin shows a higher affinity for the reduced than for the oxidized forms of the nucleotides. 4. Dissociation constants at 0·01I, pH7 and 25° are 0·7×10−6m for NADH2 and 0·45×10−6m for NADPH2. Dissociation constants increase with increasing ionic strength, indicating that the binding is mainly electrostatic. 5. Bound NADH2 and NADPH2 may be activated to fluoresce by the transfer of energy from the excited aromatic amino acids of the toxin. Activation and emission spectra of bound and free nucleotides are compared. 6. Since NAD and NADH2 are cofactors specifically required for the inhibition of protein synthesis by diphtheria toxin, the possible role of toxin–nucleotide complexes is discussed in this regard. PMID:4384596

  15. Evidence for posttranslational protein flavinylation in the syphilis spirochete Treponema pallidum: Structural and biochemical insights from the catalytic core of a periplasmic flavin-trafficking protein

    SciTech Connect

    Deka, Ranjit K.; Brautigam, Chad A.; Liu, Wei Z.; Tomchick, Diana R.; Norgard, Michael V.

    2015-05-05

    The syphilis spirochete Treponema pallidum is an important human pathogen but a highly enigmatic bacterium that cannot be cultivated in vitro. T. pallidum lacks many biosynthetic pathways and therefore has evolved the capability to exploit host-derived metabolites via its periplasmic lipoprotein repertoire. We recently reported a flavin-trafficking protein in T. pallidum (Ftp_Tp; TP0796) as the first bacterial metal-dependent flavin adenine dinucleotide (FAD) pyrophosphatase that hydrolyzes FAD into AMP and flavin mononucleotide (FMN) in the spirochete’s periplasm. However, orthologs of Ftp_Tp from other bacteria appear to lack this hydrolytic activity; rather, they bind and flavinylate subunits of a cytoplasmic membrane redox system (Nqr/Rnf). To further explore this dichotomy, biochemical analyses, protein crystallography, and structure-based mutagenesis were used to show that a single amino acid change (N55Y) in Ftp_Tp converts it from an Mg²⁺-dependent FAD pyrophosphatase to an FAD-binding protein. We also demonstrated that Ftp_Tp has a second enzymatic activity (Mg²⁺-FMN transferase); it flavinylates protein(s) covalently with FMN on a threonine side chain of an appropriate sequence motif using FAD as the substrate. Moreover, mutation of a metal-binding residue (D284A) eliminates Ftp_Tp’s dual activities, thereby underscoring the role of Mg²⁺ in the enzyme-catalyzed reactions. The posttranslational flavinylation activity that can target a periplasmic lipoprotein (TP0171) has not previously been described. The observed activities reveal the catalytic flexibility of a treponemal protein to perform multiple functions. Together, these findings imply mechanisms by which a dynamic pool of flavin cofactor is maintained and how flavoproteins are generated by Ftp_Tp locally in the T. pallidum periplasm.

  16. Fluorescence of the Flavin group in choline oxidase. Insights and analytical applications for the determination of choline and betaine aldehyde.

    PubMed

    Ortega, E; de Marcos, S; Sanz-Vicente, I; Ubide, C; Ostra, M; Vidal, M; Galbán, J

    2016-01-15

    Choline oxidase (ChOx) is a flavoenzyme catalysing the oxidation of choline (Ch) to betaine aldehyde (BA) and glycine betaine (GB). In this paper a fundamental study of the intrinsic fluorescence properties of ChOx due to Flavin Adenine Dinucleotide (FAD) is presented and some analytical applications are studied in detail. Firstly, an unusual alteration in the excitation spectra, in comparison with the absorption spectra, has been observed as a function of the pH. This is ascribed to a change of polarity in the excited state. Secondly, the evolution of the fluorescence spectra during the reaction seems to indicate that the reaction takes place in two consecutive, but partially overlapped, steps and each of them follows a different mechanism. Thirdly, the chemical system can be used to determine the Ch concentration in the range from 5×10(-6)M to 5×10(-5)M (univariate and multivariate calibration) in the presence of BA as interference, and the joint Ch+BA concentration in the range 5×10(-6)-5×10(-4)M (multivariate calibration) with mean errors under 10%; a semiquantitative determination of the BA concentration can be deduced by difference. Finally, Ch has been successfully determined in an infant milk sample.

  17. What is adenine doing in photolyase?

    PubMed

    Acocella, Angela; Jones, Garth A; Zerbetto, Francesco

    2010-03-25

    The short answer to the title question is that it acts as an electrostatic bouncer that shoves the charge flow from flavin toward the DNA lesion that photolyase repairs. This explanation is provided by an explicit time-dependent quantum mechanical approach, which is used to investigate the electron transfer process that triggers the repair mechanism. The transfer occurs from the flavin photolyase cofactor to the cyclobutane ring of DNA, previously formed by light-induced cycloaddition of adjacent pyrimidine bases. The electron wave function dynamics accurately accounts for the previously proposed mechanism of transfer via the terminal methyl group of the flavin moiety present in the catalytic electron-donor cofactor, FADH(-), which also contains adenine. This latter moiety, which has often been assumed to be present mainly for structural reasons, instantaneously modifies the interaction between acceptor and donor by a variation of the electrostatic interactions so that the presence of its local atomic charges is necessary to trigger the transfer. In principle, knowledge of the details of the electron transfer dynamics and of the important role of polarization effects can be exploited to improve the efficiency of the repair mechanism in artificial systems.

  18. A study of solvent effects on the phosphorescence properties of flavins.

    PubMed Central

    Brunk, G R; Martin, K A; Nishimura, A M

    1976-01-01

    A combination of zero field triplet state techniques are used to study the excited electronic states of a series of flavin and flavin related molecules both in single crystals and glass matrices. Particular attention is given to the effects of solvent interaction on the triplet state properties of the flavin molecules. The total phosphorescence decay rate constants at 1.4 degrees K are reported for the flavin molecules in polar and nonpolar solvents. The rate constants are then correlated to the degree of solvent interaction. Results indicate possible complex formation between the isoalloxazine and adenine groups in FAD. Finally, the results and possible interpretation on the study of a flavoenzyme, L-amino acid oxidase are presented. PMID:990392

  19. Aspartate 120 of Escherichia coli methylenetetrahydrofolate reductase: evidence for major roles in folate binding and catalysis and a minor role in flavin reactivity.

    PubMed

    Trimmer, Elizabeth E; Ballou, David P; Galloway, Lara J; Scannell, Sara A; Brinker, Danielle R; Casas, Katie R

    2005-05-10

    Escherichia coli methylenetetrahydrofolate reductase (MTHFR) catalyzes the NADH-linked reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate) using flavin adenine dinucleotide (FAD) as cofactor. MTHFR is unusual among flavin oxidoreductases because it contains a conserved, negatively rather than positively charged amino acid (aspartate 120) near the N1-C2=O position of the flavin. At this location, Asp 120 is expected to influence the redox properties of the enzyme-bound FAD. Modeling of the CH(3)-H(4)folate product into the enzyme active site suggests that Asp 120 may also play crucial roles in folate binding and catalysis. We have replaced Asp 120 with Asn, Ser, Ala, Val, and Lys and have characterized the mutant enzymes. Consistent with a loss of negative charge near the flavin, the midpoint potentials of the mutants increased from 17 to 30 mV. A small kinetic effect on the NADH reductive half-reaction was also observed as the mutants exhibited a 1.2-1.5-fold faster reduction rate than the wild-type enzyme. Catalytic efficiency (k(cat)/K(m)) in the CH(2)-H(4)folate oxidative half-reaction was decreased significantly (up to 70000-fold) and in a manner generally consistent with the negative charge density of position 120, supporting a major role for Asp 120 in electrostatic stabilization of the putative 5-iminium cation intermediate during catalysis. Asp 120 is also intimately involved in folate binding as increases in the apparent K(d) of up to 15-fold were obtained for the mutants. Examining the E(red) + CH(2)-H(4)folate reaction at 4 degrees C, we obtained, for the first time, evidence for the rapid formation of a reduced enzyme-folate complex with wild-type MTHFR. The more active Asp120Ala mutant, but not the severely impaired Asp120Lys mutant, demonstrated the species, suggesting a connection between the extent of complex formation and catalytic efficiency. PMID:15865426

  20. Search for interstellar adenine

    NASA Astrophysics Data System (ADS)

    Chakrabarti, Sandip K.; Majumdar, Liton; Das, Ankan; Chakrabarti, Sonali

    2015-05-01

    It is long debated if pre-biotic molecules are indeed present in the interstellar medium. Despite substantial works pointing to their existence, pre-biotic molecules are yet to be discovered with a complete confidence. In this paper, our main aim is to study the chemical evolution of interstellar adenine under various circumstances. We prepare a large gas-grain chemical network by considering various pathways for the formation of adenine. Majumdar et al. (New Astron. 20:15, 2013) proposed that in the absence of adenine detection, one could try to trace two precursors of adenine, namely, HCCN and NH2CN. Recently Merz et al. (J. Phys. Chem. A 118:3637-3644, 2014), proposed another route for the formation of adenine in interstellar condition. They proposed two more precursor molecules. But it was not verified by any accurate gas-grain chemical model. Neither was it known if the production rate would be high or low. Our paper fills this important gap. We include this new pathways to find that the contribution through this pathways for the formation of Adenine is the most dominant one in the context of interstellar medium. We propose that observers may look for the two precursors (C3NH and HNCNH) in the interstellar media which are equally important for predicting abundances of adenine. We perform quantum chemical calculations to find out spectral properties of adenine and its two new precursor molecules in infrared, ultraviolet and sub-millimeter region. Our present study would be useful for predicting abundance of adenine.

  1. Camphor Pathway Redux: Functional Recombinant Expression of 2,5- and 3,6-Diketocamphane Monooxygenases of Pseudomonas putida ATCC 17453 with Their Cognate Flavin Reductase Catalyzing Baeyer-Villiger Reactions

    PubMed Central

    Iwaki, Hiroaki; Grosse, Stephan; Bergeron, Hélène; Leisch, Hannes; Morley, Krista; Hasegawa, Yoshie

    2013-01-01

    Whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-DKCMO and 3,6-DKCMO, respectively) in the initial catabolic steps of (+) and (−) isomeric forms of camphor (CAM) metabolism in Pseudomonas putida ATCC 17453 are relatively well characterized, the actual identity of the flavin reductase (Fred) component that provides the reduced flavin to the oxygenases has hitherto been ill defined. In this study, a 37-kDa Fred was purified from a camphor-induced culture of P. putida ATCC 17453 and this facilitated cloning and characterization of the requisite protein. The active Fred is a homodimer with a subunit molecular weight of 18,000 that uses NADH as an electron donor (Km = 32 μM), and it catalyzes the reduction of flavin mononucleotide (FMN) (Km = 3.6 μM; kcat = 283 s−1) in preference to flavin adenine dinucleotide (FAD) (Km = 19 μM; kcat = 128 s−1). Sequence determination of ∼40 kb of the CAM degradation plasmid revealed the locations of two isofunctional 2,5-DKCMO genes (camE25–1 for 2,5-DKCMO-1 and camE25–2 for 2,5-DKCMO-2) as well as that of a 3,6-DKCMO-encoding gene (camE36). In addition, by pulsed-field gel electrophoresis, the CAM plasmid was established to be linear and ∼533 kb in length. To enable functional assessment of the two-component monooxygenase system in Baeyer-Villiger oxidations, recombinant plasmids expressing Fred in tandem with the respective 2,5-DKCMO- and 3,6-DKCMO-encoding genes in Escherichia coli were constructed. Comparative substrate profiling of the isofunctional 2,5-DCKMOs did not yield obvious differences in Baeyer-Villiger biooxidations, but they are distinct from 3,6-DKCMO in the stereoselective oxygenations with various mono- and bicyclic ketone substrates. PMID:23524667

  2. Flavin reduction activates Drosophila cryptochrome

    PubMed Central

    Vaidya, Anand T.; Top, Deniz; Manahan, Craig C.; Tokuda, Joshua M.; Zhang, Sheng; Pollack, Lois; Young, Michael W.; Crane, Brian R.

    2013-01-01

    Entrainment of circadian rhythms in higher organisms relies on light-sensing proteins that communicate to cellular oscillators composed of delayed transcriptional feedback loops. The principal photoreceptor of the fly circadian clock, Drosophila cryptochrome (dCRY), contains a C-terminal tail (CTT) helix that binds beside a FAD cofactor and is essential for light signaling. Light reduces the dCRY FAD to an anionic semiquinone (ASQ) radical and increases CTT proteolytic susceptibility but does not lead to CTT chemical modification. Additional changes in proteolytic sensitivity and small-angle X-ray scattering define a conformational response of the protein to light that centers at the CTT but also involves regions remote from the flavin center. Reduction of the flavin is kinetically coupled to CTT rearrangement. Chemical reduction to either the ASQ or the fully reduced hydroquinone state produces the same conformational response as does light. The oscillator protein Timeless (TIM) contains a sequence similar to the CTT; the corresponding peptide binds dCRY in light and protects the flavin from oxidation. However, TIM mutants therein still undergo dCRY-mediated degradation. Thus, photoreduction to the ASQ releases the dCRY CTT and promotes binding to at least one region of TIM. Flavin reduction by either light or cellular reductants may be a general mechanism of CRY activation. PMID:24297896

  3. Evolution probabilities and phylogenetic distance of dinucleotides.

    PubMed

    Michel, Christian J

    2007-11-21

    We develop here an analytical evolution model based on a dinucleotide mutation matrix 16 x 16 with six substitution parameters associated with the three types of substitutions in the two dinucleotide sites. It generalizes the previous models based on the nucleotide mutation matrices 4 x 4. It determines at some time t the exact occurrence probabilities of dinucleotides mutating randomly according to these six substitution parameters. Furthermore, several properties and two applications of this model allow to derive 16 evolutionary analytical solutions of dinucleotides and also a dinucleotide phylogenetic distance. Finally, based on this mathematical model, the SED (Stochastic Evolution of Dinucleotides) web server has been developed for deriving evolutionary analytical solutions of dinucleotides.

  4. Photodissociation of dinucleotide ions in a storage ring

    NASA Astrophysics Data System (ADS)

    Worm, Esben S.; Andersen, Inge Hald; Andersen, Jens Ulrik; Holm, Anne I. S.; Hvelplund, Preben; Kadhane, Umesh; Nielsen, Steen Brøndsted; Poully, Jean-Christophe; Støchkel, Kristian

    2007-04-01

    The decay of protonated DNA dinucleotides, dA2+ , dG2+ , dT2+ , dC2+ and deprotonated ones, dA2- and dT2- , after 260nm photon absorption was measured in an electrostatic ion storage ring (A denotes adenine, G guanine, T thymine, and C cytosine). Fragmentation on the microsecond time scale was observed and assigned to statistical dissociation. Good fits to the decay spectra were obtained with a model based on microcanonical rate constants of the Arrhenius type with activation energies and preexponential factors for the dissociation that agree well with literature values. In accordance with results from other groups, dT2+ was found to have the longest lifetime among the cations. The importance of decay processes faster than the microsecond time scale is elucidated by a comparison between the total ion beam depletion and that due to the observed statistical decay. We find that such processes play a major role for all of the dinucleotides, being more than 25 times more probable than the microsecond statistical dissociation for dA2+ , dG2+ , and dC2+ , about 10 times for dT2+ , and between 2 and 6 times for dA2- and dT2- . For the cations, we ascribe these processes to nonstatistical dissociation prior to randomization of the excitation energy among all degrees of freedom whereas direct photoelectron detachment may play a role for the anions. Thus, our data indicate that the propensity for nonstatistical dissociation increases upon nucleobase protonation. Consistent with this trend, the propensity is less for dT2+ than for the other dinucleotide cations because the phosphoric acid group competes with thymine for the proton.

  5. Structure and general properties of flavins.

    PubMed

    Edwards, Ana Maria

    2014-01-01

    Flavins are a family of yellow-colored compounds with the basic structure of 7,8-dimethyl-10-alkylisoalloxazine. Riboflavin, commonly known as vitamin B2, is an essential component of living organisms and is the precursor of all biologically important flavins. In this chapter, the redox properties of flavins are described, with special emphasis in their ability to participate in both one-electron and two-electron transfer processes; hence, flavins are indispensable mediators between two-electron and one-electron processes in biological systems. The photophysical and photochemical properties of flavins are also discussed. All oxidized flavins exhibit strong absorption in the ultraviolet and visible regions and an intense yellow-green fluorescence (in their neutral oxidized form). Flavins are thermostable compounds; however, they are photosensitive. In the absence of an external reductant, the isoalloxazine ring system undergoes intramolecular photoreduction. Some flavins are efficient photosensitizers; they can induce photomodifications of compounds that are not directly modified by visible light.

  6. Flavin Redox Switching of Protein Functions

    PubMed Central

    Zhu, Weidong; Moxley, Michael A.

    2011-01-01

    Abstract Flavin cofactors impart remarkable catalytic diversity to enzymes, enabling them to participate in a broad array of biological processes. The properties of flavins also provide proteins with a versatile redox sensor that can be utilized for converting physiological signals such as cellular metabolism, light, and redox status into a unique functional output. The control of protein functions by the flavin redox state is important for transcriptional regulation, cell signaling pathways, and environmental adaptation. A significant number of proteins that have flavin redox switches are found in the Per-Arnt-Sim (PAS) domain family and include flavoproteins that act as photosensors and respond to changes in cellular redox conditions. Biochemical and structural studies of PAS domain flavoproteins have revealed key insights into how flavin redox changes are propagated to the surface of the protein and translated into a new functional output such as the binding of a target protein in a signaling pathway. Mechanistic details of proteins unrelated to the PAS domain are also emerging and provide novel examples of how the flavin redox state governs protein–membrane interactions in response to appropriate stimuli. Analysis of different flavin switch proteins reveals shared mechanistic themes for the regulation of protein structure and function by flavins. Antioxid. Redox Signal. 14, 1079–1091. PMID:21028987

  7. Whence flavins? Redox-active ribonucleotides link metabolism and genome repair to the RNA world.

    PubMed

    Nguyen, Khiem Van; Burrows, Cynthia J

    2012-12-18

    Present-day organisms are under constant environmental stress that damages bases in DNA, leading to mutations. Without DNA repair processes to correct these errors, such damage would be catastrophic. Organisms in all kingdoms have repair processes ranging from direct reversal to base excision and nucleotide excision repair, and the recently characterized giant viruses also include these mechanisms. At what point in the evolution of genomes did active repair mechanisms become critical? In particular, how did early RNA genomes protect themselves from UV photodamage that would have hampered nonenzymatic replication and led to a mutation rate too high to pass on accurate sequence information from one generation to the next? Photolyase is a widespread and phylogenetically ancient enzyme that utilizes longer wavelength light to cleave thymine dimers in DNA produced via photodamage. The protein serves as a binding scaffold but does not contribute to the catalytic chemistry; the action of the dinucleotide cofactor FADH(2) breaks the chemical bonds. This small bit of RNA, hailed as a "fossil of the RNA World," contains the flavin heterocycle, whose redox activity has been harnessed for myriad functions of life from metabolism to DNA repair. In present-day biochemistry, flavin biosynthesis begins with guanosine and proceeds through seven steps catalyzed by protein-based enzymes. This leads to the question of how flavins originally evolved. Did the RNA world include ancestral RNA bases with greater redox activity than G, A, C, and U that were capable of photorepair of uracil dimers? Could those ancestral bases have chemically evolved to the current flavin structure? Or did flavins already exist from prebiotic chemical synthesis? And were they then co-opted as catalysts for repair sometime after metabolism was established? In this Account, we analyze simple derivatives of guanosine and other bases that show two prerequisites for flavin-like photolyase activity: a significantly

  8. Spectroscopic and kinetic properties of a recombinant form of the flavin domain of spinach NADH: nitrate reductase.

    PubMed

    Quinn, G B; Trimboli, A J; Prosser, I M; Barber, M J

    1996-03-01

    The C-terminal 268 residues of the spinach assimilatory NADH:nitrate reductase amino acid sequence that correspond to the flavin-containing domain of the enzyme have been selectively amplified and expressed as a recombinant protein in Escherichia coli. The recombinant protein, which was produced in both soluble and insoluble forms, was purified to homogeneity using a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP-agarose and FPLC gel filtration. The purified domain exhibited a molecular weight of approximately 30 kDa, estimated by polyacrylamide gel electrophoresis, and a molecular mass of 30,169 for the apoprotein determined by mass spectrometry, which also confirmed the presence of FAD. The UV/visible spectrum was typical of a flavoprotein, with maxima at 272, 386, and 461 nm in the oxidized form while CD spectroscopy yielded both positive and negative maxima at 313 and 382 nm and 461 and 484 nm, respectively. The purified domain showed immunological cross-reactivity with anti-spinach nitrate reductase polyclonal antibodies while both N-terminal and internal amino acid sequencing of isolated peptides confirmed the fidelity of the domain's primary sequence. The protein retained NADH-ferricyanide reductase activity (Vmax=84 micromol NADH consumer/min/nmol FAD) with Km's of 17 and 34 microM for NADH and ferricyanide, respectively, with a pH optimum of approximately 6.5 A variety of NADH-analogs could also function as electron donors, though with decreased efficiency, the most effective being reduced nicotinamide hypoxanthine dinucleotide (V(max) = 35 micromol NHDH consumer/min/nmol FAD) and Km = 22 microM). NAD+ was demonstrated to be a competitive inhibitor (Ki = 1.9 mM) while analysis of inhibition by a variety of NAD+-analogs indicated the most efficient inhibitor to be ADP (Ki = 0.2 mM), with analogs devoid of either the phosphate, ribose, or adenine moieties proving to be markedly less-efficient inhibitors. The isolated domain

  9. Vertical Ionization Energies of Adenine and 9-Methyl Adenine

    NASA Astrophysics Data System (ADS)

    Dolgounitcheva, O.; Zakrzewski, V. G.; Ortiz, J. V.

    2009-07-01

    Vertical ionization energies of 9-H adenine and 9-methyl adenine have been calculated with the following, ab initio, electron propagator methods: the outer valence Green's function (OVGF), partial third-order theory (P3), and the third-order algebraic diagrammatic construction, or ADC(3). Basis set effects have been systematically examined. All methods predict near degeneracy in the π2-n1 and π3-n2 pairs of cationic, adenine final states and larger splittings of the corresponding, cationic states of 9-methyl adenine. P3 results for adenine predict the following order of the first six final states: π1, n1, π2, n2, π3, n3. Coupled-cluster calculations on the first three cationic states of adenine confirm these predictions. OVGF and ADC(3) calculations reverse the order of the second and third states and of the fourth and fifth states. All results confirm previous interpretations of experiments in which the second and third spectral bands correspond to the aforementioned pairs of final states and disagree with a recent reassignment based on time-resolved photoelectron spectra. Lower ionization energies and larger splittings in the methylated molecule are interpreted in terms of phase relationships in the Dyson orbitals. ADC(3) results confirm the qualitative validity of the one-electron approximation for the first six final states of both molecules and disclose its inadequacies for higher ionization energies.

  10. Inactivation of yeast alcohol dehydrogenase by alkylperoxyl radicals. Characteristics and influence of nicotinamide-adenine dinucleotides.

    PubMed

    Videla, L A; Salim-Hanna, M; Lissi, E A

    1992-10-01

    The study of the interaction of alkylperoxyl radicals generated by the aerobic thermolysis of 2,2'-azobis(2-amidinopropane) (AAP) with yeast alcohol dehydrogenase (YADH) revealed a high reactivity of the enzyme, with an average of about 20 radicals per added YADH tetramer being needed to elicit its total inactivation. NAD+ enhanced YADH inactivation at NAD+/YADH molar ratios from 0.25 to 1, decreasing the rate of the process when added in excess to the enzyme concentration. At NADH/YADH molar ratios greater than 1, NADH exhibited a protective effect characterized by a poorly defined induction time and lower inactivation rates, which progressively increased during the reaction period. These changes occurred concomitantly with the oxidation of NADH into NAD+, which might counteract the protective effect of NADH. Under similar conditions, NADP+ did not modify AAP-induced YADH inactivation, while NADPH exhibited a modest protection at NADPH/YADH molar ratios greater than 1. It is concluded that YADH inactivation by alkylperoxyl radicals is strongly dependent on the redox state of the NADH-NAD+ couple, as the rates of the process at different time intervals inversely correlate with the respective NADH/NAD+ ratios.

  11. The responses of isolated plant mitochondria to external nicotinamide adenine dinucleotide.

    PubMed

    Soole, K L; Dry, I B; Wiskich, J T

    1986-06-01

    The effects of added NAD on substrate oxidation by turnip (Brassica rapa L.) and beetroot (Beta vulgaris L.) mitochondria were investigated. State 3 malate and 2-oxoglutarate oxidation rates with turnip mitochondria were stimulated 25 to 40% by external NAD. Following NAD-depletion this stimulation by NAD was increased to 70 to 80%. With purified beetroot mitochondria, state 3 malate and 2-oxoglutarate oxidation rates were only marginally increased (10-15%) by the addition of NAD but after NAD-depletion treatments this stimulation increased to 55%. The effect of added NAD on oxidation rates could be reduced by preloading mitochondria with NAD in the presence of succinate. Oxidation rates were found to be most sensitive to the addition of external NAD when rotenone was present. The uptake of external NAD into beetroot mitochondria appeared to be composed of both an active and a diffusive component. The active component displayed saturation kinetics with an approximate K(m) of 0.105 +/- 0.046 millimolar. These results provide further evidence, reported previously with potato mitochondria, that NAD can move across the inner membrane of plant mitochondria. They are particularly significant with respect to beetroot mitochondria which in contrast to other plant mitochondria, have not demonstrated any response to added NAD.

  12. The activity of liver alcohol dehydrogenase with nicotinamide–adenine dinucleotide phosphate as coenzyme

    PubMed Central

    Dalziel, K.; Dickinson, F. M.

    1965-01-01

    1. The separation of nucleotide impurities from commercial NADP preparations by chromatography is described. All the preparations studied contained 0·1–0·2% of NAD. 2. The activity of pure crystalline liver alcohol dehydrogenase with NADP as coenzyme has been confirmed. Initial-rate data are reported for the reaction at pH 6·0 and 7·0 with ethanol and acetaldehyde as substrates. With NADP and NADPH2 of high purity, the maximal specific rates were similar to those obtained with NAD and NADH2, but the Michaelis constants for the former coenzymes were much greater than those for the latter. 3. The oxidation of ethanol by NADP is greatly inhibited by NADH2, and this accounts for low values of certain initial-rate parameters obtained with commercial NADP preparations containing NAD. The kinetics of the inhibition are consistent with competitive inhibition in a compulsory-order mechanism. 4. Initial-rate data with NAD and NADPH2 do not conform to the requirements of the mechanism proposed by Theorell & Chance (1951), in contrast with results previously obtained with NAD and NADH2. The possibility that the deviations are due to competing nucleotide impurity in the oxidized coenzyme cannot be excluded. The data show that the enzyme reacts more slowly with, and has a smaller affinity for, NADP and NADPH2 than NAD and NADH2. 5. Phosphate behaves as a competitive inhibitor towards NADP. PMID:14340079

  13. Ruling out pyridine dinucleotides as true TRPM2 channel activators reveals novel direct agonist ADP-ribose-2′-phosphate

    PubMed Central

    Tóth, Balázs; Iordanov, Iordan

    2015-01-01

    Transient receptor potential melastatin 2 (TRPM2), a Ca2+-permeable cation channel implicated in postischemic neuronal cell death, leukocyte activation, and insulin secretion, is activated by intracellular ADP ribose (ADPR). In addition, the pyridine dinucleotides nicotinamide-adenine-dinucleotide (NAD), nicotinic acid–adenine-dinucleotide (NAAD), and NAAD-2′-phosphate (NAADP) have been shown to activate TRPM2, or to enhance its activation by ADPR, when dialyzed into cells. The precise subset of nucleotides that act directly on the TRPM2 protein, however, is unknown. Here, we use a heterologously expressed, affinity-purified–specific ADPR hydrolase to purify commercial preparations of pyridine dinucleotides from substantial contaminations by ADPR or ADPR-2′-phosphate (ADPRP). Direct application of purified NAD, NAAD, or NAADP to the cytosolic face of TRPM2 channels in inside-out patches demonstrated that none of them stimulates gating, or affects channel activation by ADPR, indicating that none of these dinucleotides directly binds to TRPM2. Instead, our experiments identify for the first time ADPRP as a true direct TRPM2 agonist of potential biological interest. PMID:25918360

  14. Protein Modification by Adenine Propenal

    PubMed Central

    2015-01-01

    Base propenals are products of the reaction of DNA with oxidants such as peroxynitrite and bleomycin. The most reactive base propenal, adenine propenal, is mutagenic in Escherichia coli and reacts with DNA to form covalent adducts; however, the reaction of adenine propenal with protein has not yet been investigated. A survey of the reaction of adenine propenal with amino acids revealed that lysine and cysteine form adducts, whereas histidine and arginine do not. Nε-Oxopropenyllysine, a lysine–lysine cross-link, and S-oxopropenyl cysteine are the major products. Comprehensive profiling of the reaction of adenine propenal with human serum albumin and the DNA repair protein, XPA, revealed that the only stable adduct is Nε-oxopropenyllysine. The most reactive sites for modification in human albumin are K190 and K351. Three sites of modification of XPA are in the DNA-binding domain, and two sites are subject to regulatory acetylation. Modification by adenine propenal dramatically reduces XPA’s ability to bind to a DNA substrate. PMID:25211669

  15. Flavin-Dependent Enzymes in Cancer Prevention

    PubMed Central

    Wojcieszyńska, Danuta; Hupert-Kocurek, Katarzyna; Guzik, Urszula

    2012-01-01

    Statistical studies have demonstrated that various agents may reduce the risk of cancer’s development. One of them is activity of flavin-dependent enzymes such as flavin-containing monooxygenase (FMO)GS-OX1, FAD-dependent 5,10-methylenetetrahydrofolate reductase and flavin-dependent monoamine oxidase. In the last decade, many papers concerning their structure, reaction mechanism and role in the cancer prevention were published. In our work, we provide a more in-depth analysis of flavin-dependent enzymes and their contribution to the cancer prevention. We present the actual knowledge about the glucosinolate synthesized by flavin-containing monooxygenase (FMO)GS-OX1 and its role in cancer prevention, discuss the influence of mutations in FAD-dependent 5,10-methylenetetrahydrofolate reductase on the cancer risk, and describe FAD as an important cofactor for the demethylation of histons. We also present our views on the role of riboflavin supplements in the prevention against cancer. PMID:23222680

  16. Interactions between Flavins and Quadruplex Nucleic Acids.

    PubMed

    Merkle, Tobias; Sinn, Malte; Hartig, Jörg S

    2015-11-01

    Quadruplex nucleic acids are widespread in genomes. They influence processes such as transcription, translation, replication, recombination, and the regulation of gene expression. Several synthetic ligands have been demonstrated to target quadruplex nucleic acids. However, only very few metabolites have been reported to interact with quadruplexes. In principle, an intracellular metabolite that selectively binds to four-stranded sequences could modulate quadruplex formation, stability, and thus functions in a riboswitch (or deoxyriboswitch) manner. Here we report quadruplex interactions with flavin derivatives such as FMN and FAD. The affinities were highest with parallel quadruplexes, with low (14-20 μm) dissociation constants. Taking into account combined intracellular flavin concentrations of 243 μm in E. coli, the observed interactions in principle open up the possibility of flavin levels affecting gene expression and other processes by modulating quadruplex formation.

  17. Quantitative bioluminescent detection of bacteria

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Picciolo, G. L.

    1976-01-01

    Phosphoflavins in sample are measured using photobacterial luciferase assay technique for flavin mononucleotide (FMN). Boiling perchloric acid is used to rupture cells to free bound flavin and to hydrolyze flavin adenine dinucleotide to FMN. Base-stabilized water solution of sodium borohydride is used as reactant.

  18. Quantum-chemical study of interactions of trans-resveratrol with guanine-thymine dinucleotide and DNA-nucleobases.

    PubMed

    Mikulski, Damian; Szeląg, Małgorzata; Molski, Marcin

    2011-12-01

    Trans-resveratrol, a natural phytoalexin present in red wine and grapes, has gained considerable attention because of its antiproliferative, chemopreventive and proapoptotic activity against human cancer cells. The accurate quantum-chemical computations based on the density functional theory (DFT) and ab initio second-order Møller-Plesset perturbation method (MP2) have been performed for the first time to study interactions of trans-resveratrol with guanine-thymine dinucleotide and DNA-derived nitrogenous bases: adenine, guanine, cytosine and thymine in vacuum and water medium. This compound is found to show high affinity to nitrogenous bases and guanine-thymine dinucleotide. The electrostatic interactions from intermolecular hydrogen bonding increase the stability of complexes studied. In particular, significantly strong hydrogen bonds between 4'-H atom of trans-resveratrol and imidazole nitrogen as well as carbonyl oxygen atoms of nucleobases studied stabilize these systems. The stabilization energies computed reveal that the negatively charged trans-resveratrol-dinucleotide complex is more energetically stable in water medium than in vacuum. MP2 method gives more reliable and significantly high values of stabilization energy of trans-resveratrol-dinucleotide, trans-resveratrol-guanine and trans-resveratrol-thymine complexes than B3LYP exchange-correlation functional because it takes into account London dispersion energy. According to the results, in the presence of trans-resveratrol the 3'-5' phosphodiester bond in dinucleotide can be cleaved and the proton from 4'-OH group of trans-resveratrol migrates to the 3'-O atom of dinucleotide. It is concluded that trans-resveratrol is able to break the DNA strand. Hence, the findings obtained help understand antiproliferative and anticancer properties of this polyphenol.

  19. Automated genotyping of dinucleotide repeat markers

    SciTech Connect

    Perlin, M.W.; Hoffman, E.P. |

    1994-09-01

    The dinucleotide repeats (i.e., microsatellites) such as CA-repeats are a highly polymorphic, highly abundant class of PCR-amplifiable markers that have greatly streamlined genetic mapping experimentation. It is expected that over 30,000 such markers (including tri- and tetranucleotide repeats) will be characterized for routine use in the next few years. Since only size determination, and not sequencing, is required to determine alleles, in principle, dinucleotide repeat genotyping is easily performed on electrophoretic gels, and can be automated using DNA sequencers. Unfortunately, PCR stuttering with these markers generates not one band for each allele, but a pattern of bands. Since closely spaced alleles must be disambiguated by human scoring, this poses a key obstacle to full automation. We have developed methods that overcome this obstacle. Our model is that the observed data is generated by arithmetic superposition (i.e., convolution) of multiple allele patterns. By quantitatively measuring the size of each component band, and exploiting the unique stutter pattern associated with each marker, closely spaced alleles can be deconvolved; this unambiguously reconstructs the {open_quotes}true{close_quotes} allele bands, with stutter artifact removed. We used this approach in a system for automated diagnosis of (X-linked) Duchenne muscular dystrophy; four multiplexed CA-repeats within the dystrophin gene were assayed on a DNA sequencer. Our method accurately detected small variations in gel migration that shifted the allele size estimate. In 167 nonmutated alleles, 89% (149/167) showed no size variation, 9% (15/167) showed 1 bp variation, and 2% (3/167) showed 2 bp variation. We are currently developing a library of dinucleotide repeat patterns; together with our deconvolution methods, this library will enable fully automated genotyping of dinucleotide repeats from sizing data.

  20. Synthesis and Characterization of Naphthalenediimide-Functionalized Flavin Derivatives

    PubMed Central

    Zainalabdeen, Nada; Fitzpatrick, Brian; Kareem, Mohanad Mousa; Nandwana, Vikas; Cooke, Graeme; Rotello, Vincent M.

    2013-01-01

    Two acceptor–acceptor dyads have been synthesized featuring a flavin moiety and a naphthalenediimide (NDI) unit. The NDI unit is linked to the flavin through a short spacer group via either the N(3) or N(10) positions of the flavin. We have investigated the UV-Vis and redox properties of these multi-electron accepting systems which indicate that these materials display the collective properties of their component systems. Fluorescence spectroscopy measurements have revealed that their emission properties are dominated by the flavin unit. PMID:23552831

  1. Synthesis and characterization of naphthalenediimide-functionalized flavin derivatives.

    PubMed

    Zainalabdeen, Nada; Fitzpatrick, Brian; Kareem, Mohanad Mousa; Nandwana, Vikas; Cooke, Graeme; Rotello, Vincent M

    2013-01-01

    Two acceptor-acceptor dyads have been synthesized featuring a flavin moiety and a naphthalenediimide (NDI) unit. The NDI unit is linked to the flavin through a short spacer group via either the N(3) or N(10) positions of the flavin. We have investigated the UV-Vis and redox properties of these multi-electron accepting systems which indicate that these materials display the collective properties of their component systems. Fluorescence spectroscopy measurements have revealed that their emission properties are dominated by the flavin unit. PMID:23552831

  2. Vertical Singlet Excitations on Adenine Dimer: A Time Dependent Density Functional Study

    NASA Astrophysics Data System (ADS)

    Crespo-Hernández, Carlos E.; Marai, Christopher N. J.

    2007-12-01

    The condense phase, excited state dynamics of the adenylyl(3'→5')adenine (ApA) dinucleotide has been previously studied using transient absorption spectroscopy with femtosecond time resolution (Crespo-Hernández et al. Chem. Rev. 104, 1977-2019 (2004)). An ultrafast and a long-lived component were observed with time constants of <1 ps and 60±16 ps, respectively. Comparison of the time constants measured for the dinucleotide with that for the adenine nucleotide suggested that the fast component observed in ApA could be assigned to monomer dynamics. The long-lived component observed in ApA was assigned to an excimer state that originates from a fraction of base stacked conformations present at the time of excitation. In this contribution, supermolecule calculations using the time dependent implementation of density functional theory is used to provide more insights on the origin of the initial Franck-Condon excitations. Monomer-like, localized excitations are observed for conformations having negligible base stacking interactions, whereas delocalized excitations are predicted for conformations with significant vertical base-base overlap.

  3. Quantitive determination of flavin nucleotide using the bacterial bioluminescent reaction

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Picciolo, G. L.

    1969-01-01

    Photometric method based on the use of bacterial luminiscent reaction quantitatively detects the presence of flavin compounds in all forms of life. Aqueous cellular dispersion of a biological sample with an aqueous perchloric acid ruptures the cells and frees the flavin coenzymes from their proteins.

  4. Solution conformation of 2-aminopurine (2-AP) dinucleotide determined by ultraviolet 2D fluorescence spectroscopy (UV-2D FS)

    PubMed Central

    Widom, Julia R.; Johnson, Neil P.; von Hippel, Peter H.; Marcus, Andrew H.

    2013-01-01

    We have observed the conformation-dependent electronic coupling between the monomeric subunits of a dinucleotide of 2-aminopurine (2-AP), a fluorescent analog of the nucleic acid base adenine. This was accomplished by extending two-dimensional fluorescence spectroscopy (2D FS) – a fluorescence-detected variation of 2D electronic spectroscopy – to excite molecular transitions in the ultraviolet (UV) regime. A collinear sequence of four ultrafast laser pulses centered at 323 nm was used to resonantly excite the coupled transitions of 2-AP dinucleotide. The phases of the optical pulses were continuously swept at kilohertz frequencies, and the ensuing nonlinear fluorescence was phase-synchronously detected at 370 nm. Upon optimization of a point-dipole coupling model to our data, we found that in aqueous buffer the 2-AP dinucleotide adopts an average conformation in which the purine bases are non-helically stacked (center-to-center distance R12 = 3.5 Å ± 0.5 Å, twist angle θ12 = 5° ± 5°), which differs from the conformation of such adjacent bases in duplex DNA. These experiments establish UV-2D FS as a method for examining the local conformations of an adjacent pair of fluorescent nucleotides substituted into specific DNA or RNA constructs, which will serve as a powerful probe to interpret, in structural terms, biologically significant local conformational changes within the nucleic acid framework of protein-nucleic acid complexes. PMID:24223491

  5. Bound anionic states of adenine

    SciTech Connect

    Haranczyk, Maciej; Gutowski, Maciej S; Li, Xiang; Bowen, Kit H

    2007-03-20

    Anionic states of nucleic acid bases are involved in DNA damage by low-energy electrons and in charge transfer through DNA. Previous gas phase studies of free, unsolvated nucleic acid base parent anions probed only dipole-bound states, which are not present in condensed phase environments, but did not observe valence anionic states, which for purine bases, are thought to be adiabatically unbound. Contrary to this expectation, we have demonstrated that some thus far ignored tautomers of adenine, which result from enamine-imine transformations, support valence anionic states with electron vertical detachment energies as large as 2.2 eV, and at least one of these anionic tautomers is adiabatically bound. Moreover, we predict that the new anionic tautomers should also dominate in solutions and should be characterized by larger values of electron vertical detachment energy than the canonical valence anion. All of the new-found anionic tautomers might be formed in the course of dissociative electron attachment followed by a hydrogen atom attachment to a carbon atom, and they might affect the structure and properties of DNA and RNA exposed to low-energy electrons. The discovery of these valence anionic states of adenine was facilitated by the development of: (i) a new experimental method for preparing parent anions of nucleic acid bases for photoelectron experiments, and (ii) a new combinatorial/ quantum chemical approach for identification of the most stable tautomers of organic molecules. The computational portion of this work was supported by the: (i) Polish State Committee for Scientific Research (KBN) Grants: DS/8000-4-0140-7 (M.G.) and N204 127 31/2963 (M.H.), (ii) European Social Funds (EFS) ZPORR/2.22/II/2.6/ARP/U/2/05 (M.H.), and (iii) US DOE Office of Biological and Environmental Research, Low Dose Radiation Research Program (M.G.). M.H. holds the Foundation for Polish Science (FNP) award for young scientists. The calculations were performed at the Academic

  6. The role of nicotinamide–adenine dinucleotide phosphate-dependent malate dehydrogenase and isocitrate dehydrogenase in the supply of reduced nicotinamide–adenine dinucleotide phosphate for steroidogenesis in the superovulated rat ovary

    PubMed Central

    Flint, A. P. F.; Denton, R. M.

    1970-01-01

    1. Superovulated rat ovary was found to contain high activities of NADP–malate dehydrogenase and NADP–isocitrate dehydrogenase. The activity of each enzyme was approximately four times that of glucose 6-phosphate dehydrogenase and equalled or exceeded the activities reported to be present in other mammalian tissues. Fractionation of a whole tissue homogenate of superovulated rat ovary indicated that both enzymes were exclusively cytoplasmic. The tissue was also found to contain pyruvate carboxylase (exclusively mitochondrial), NAD–malate dehydrogenase and aspartate aminotransferase (both mitochondrial and cytoplasmic) and ATP–citrate lyase (exclusively cytoplasmic). 2. The kinetic properties of glucose 6-phosphate dehydrogenase, NADP–malate dehydrogenase and NADP–isocitrate dehydrogenase were determined and compared with the whole-tissue concentrations of their substrates and NADPH; NADPH is a competitive inhibitor of all three enzymes. The concentrations of glucose 6-phosphate, malate and isocitrate in incubated tissue slices were raised at least tenfold by the addition of glucose to the incubation medium, from the values below to values above the respective Km values of the dehydrogenases. Glucose doubled the tissue concentration of NADPH. 3. Steroidogenesis from acetate is stimulated by glucose in slices of superovulated rat ovary incubated in vitro. It was found that this stimulatory effect of glucose can be mimicked by malate, isocitrate, lactate and pyruvate. 4. It is concluded that NADP–malate dehydrogenase or NADP–isocitrate dehydrogenase or both may play an important role in the formation of NADPH in the superovulated rat ovary. It is suggested that the stimulatory effect of glucose on steroidogenesis from acetate results from an increased rate of NADPH formation through one or both dehydrogenases, brought about by the increases in the concentrations of malate, isocitrate or both. Possible pathways involving the two enzymes are discussed. PMID:4393612

  7. DiProDB: a database for dinucleotide properties.

    PubMed

    Friedel, Maik; Nikolajewa, Swetlana; Sühnel, Jürgen; Wilhelm, Thomas

    2009-01-01

    DiProDB (http://diprodb.fli-leibniz.de) is a database of conformational and thermodynamic dinucleotide properties. It includes datasets both for DNA and RNA, as well as for single and double strands. The data have been shown to be important for understanding different aspects of nucleic acid structure and function, and they can also be used for encoding nucleic acid sequences. The database is intended to facilitate further applications of dinucleotide properties. A number of property datasets is highly correlated. Therefore, the database comes with a correlation analysis facility. Authors having determined new sets of dinucleotide property values are invited to submit these data to DiProDB.

  8. Formation of the imidazolides of dinucleotides under potentially prebiotic conditions

    NASA Technical Reports Server (NTRS)

    Sleeper, H. L.; Lohrmann, R.; Orgel, L. E.

    1978-01-01

    Imidazolides of dinucleotides such as ImpApA can be formed from the corresponding dinucleotides in a two-stage process, which gives up to 15% yields under potentially prebiotic conditions. First a solution of the dinucleotide and sodium trimetaphosphate is dried out at constant temperature and humidity. This produces polyphosphates such as p(n)ApA in excellent yield (greater than or equal to 80%). The products are dissolved in water, imidazole is added, and the solution is dried out again. This yields the 5'-phosphorimidazolides.

  9. An investigation of the nicotinamide-adenine dinucleotide-induced 'tightening' of the structure of glyceraldehyde 3-phosphate dehydrogenase.

    PubMed Central

    Osborne, H H; Hollaway, M R

    1976-01-01

    An investigation was made of the effect of NAD+ analogues on subunit interactions in yeast and rabbit muscle glyceraldehyde 3-phosphate dehydrogenases by using the subunit exchange (hybridization) method described previously [e.g. see Osborne & Hollaway (1975) Biochem. J. 151, 37-45]. The ligands ATP, ITP, ADP, AMP, cyclic AMP and ADP-ribose like NADH, all caused an apparent weakening of intramolecular subunit interactions, whereas NAD+ caused an apparent increase in the stability of the tetrameric enzyme molecules. A mixture of NMN and AMP, although it did not simulate completely the NAD+-induced 'tightening' of the enzyme structure, did result in a more than 20-fold decrease in the rate of subunit exchange compared with that in the presence of AMP alone. These results show that occupancy of the NMN subsite of the enzyme NAD+-binding site is insufficient in itself to give the marked tightening of the enzyme structure induced by NAD+. The 'tightening' effect is specific in that it seems to require a phosphodiester link between NMN and ADP-ribose. These effects are discussed in terms of the detailed X-ray structure of the lobster holoenzyme [Buehner et al. (1974) J. Mol. Biol. 90, 25-49]. Images PLATE 1 PLATE 2 PMID:183744

  10. The synthesis of nicotinamide–adenine dinucleotide and poly(adenosine diphosphate ribose) in various classes of rat liver nuclei

    PubMed Central

    Haines, M. E.; Johnston, I. R.; Mathias, A. P.; Ridge, D.

    1969-01-01

    1. The activities of NMN adenylyltransferase and an enzyme that synthesizes poly (ADP-ribose) from NAD were investigated in the various classes of rat liver nuclei fractionated by zonal centrifugation. 2. The highest specific activities of these two nuclear enzymes occur in different classes of nuclei. In very young and in mature rats it was shown that a correlation exists between DNA synthesis and NMN adenylyltransferase activity, but in rats of intermediate age this correlation is less evident. The highest activities of the enzyme that catalyses formation of poly (ADP-ribose) are in the nuclei involved in the synthesis of RNA. 3. The significance of these results in relation to NAD metabolism is discussed. PMID:4311824

  11. Evaluation of equilibrium constants for the interaction of lactate dehydrogenase isoenzymes with reduced nicotinamide-adenine dinucleotide by affinity chromatography.

    PubMed Central

    Brinkworth, R I; Masters, C J; Winzor, D J

    1975-01-01

    Rabbit muscle lactate dehydrogenase was subjected to frontal affinity chromatography on Sepharose-oxamate in the presence of various concentrations of NADH and sodium phosphate buffer (0.05 M, pH 6.8) containing 0.5 M-NaCl. Quantitative interpretation of the results yields an intrinsic association constant of 9.0 x 10 (4)M-1 for the interaction of enzyme with NADH at 5 degrees C, a value that is confirmed by equilibrium-binding measurements. In a second series of experiments, zonal affinity chromatography of a mouse tissue extract under the same conditions was used to evaluate assoication constants of the order 2 x 10(5)M-1, 3 x 10(5)M-1, 4 x 10(5)M-1, 7 x 10(5)M-1 and 2 x 10(6)M-1 for the interaction of NADH with the M4, M3H, M2H2, MH3 and H4 isoenzymes respectively of lactate dehydrogenase. PMID:175784

  12. Nicotinamide Adenine Dinucleotide Phosphate Oxidase (NOX) in Experimental Liver Fibrosis: GKT137831 as a Novel Potential Therapeutic Agent

    PubMed Central

    Aoyama, Tomonori; Paik, Yong-Han; Watanabe, Sumio; Laleu, Benoît; Gaggini, Francesca; Fioraso-Cartier, Laetitia; Molango, Sophie; Heitz, Freddy; Merlot, Cédric; Szyndralewiez, Cédric; Page, Patrick; Brenner, David A.

    2012-01-01

    Background & Aims NADPH oxidase (NOX) generates reactive oxygen species (ROS) in hepatic stellate cells (HSCs) during liver fibrosis. In response to fibrogenic agonists, such as angiotensin II (Ang II), the NOX1 components form an active complex including Rac1. Superoxide dismutase 1 (SOD1) interacts with the NOX-Rac1 complex to stimulate NOX activity. NOX4 is also induced in activated HSCs/myofibroblast by increased gene expression. Here, we investigate the role of an enhanced activity SOD1 G37R mutation (SODmu) and the effects of GKT137831, a dual NOX1/4 inhibitor, on HSCs and liver fibrosis. Methods To induce liver fibrosis, wild-type (WT) and SOD1mu mice were treated with carbon tetrachloride (CCl4) or bile duct ligation (BDL). Then, to address the role of NOX-SOD1-mediated ROS production in HSC activation and liver fibrosis, mice were treated with a NOX1/4 inhibitor. Fibrosis and ROS generation was assessed by histology and measurement of TBARS and NOX related genes. Primary cultured HSCs isolated from WT, SODmu, and NOX1 knock-out (KO) mice were assessed for ROS production, Rac1 activity, and NOX gene expression. Results Liver fibrosis was increased in SOD1mu mice, and ROS production and Rac1 activity were increased in SOD1mu HSCs. The NOX1/4 inhibitor GKT137831 attenuated liver fibrosis and ROS production in both SOD1mu and WT mice as well as mRNA expression of fibrotic and NOX genes. Treatment with GKT137831 suppressed ROS production and NOX and fibrotic gene expression, but not Rac1 activity, in SOD1mut and WT HSCs. Both Ang II and TGFb upregulated NOX4, but AngII required NOX1. Conclusions SOD1mu induces excessive NOX1 activation through Rac1 in HSCs, causing enhanced NOX4 upregulation, ROS generation, and liver fibrosis. Treatment targeting NOX1/4 may be a new therapy for liver fibrosis. PMID:22806357

  13. Enzymatic production by tissue extracts of a metabolite of nicotinamide adenine dinucleotide with calcium-releasing ability

    SciTech Connect

    Tich, N.R.

    1989-01-01

    This research investigated the occurrence and characterization of the metabolite in mammalian tissues. In all mammalian tissues tested, including rabbit liver, heart, spleen, kidney, and brain, the factor to convert NAD into its active metabolite was present. The conversion exhibited many characteristics of an enzymatic process such as temperature sensitivity, concentration dependence and protease sensitivity. Production of the NAD metabolite occurred within a time frame of 15-45 minutes at 37{degree}C, depending upon the particular preparation. The metabolite was isolated using high performance liquid chromatography from all mammalian tissues. This purified metabolite was then tested for its effectiveness in releasing intracellular calcium in an intact cell by microinjecting it into unfertilized sea urchin eggs. These eggs undergo a massive morphological change upon fertilization which is dependent upon the release of calcium from inside the cell. Upon injection of the NAD metabolite into unfertilized eggs, this same morphological change was observed showing indirectly that the metabolite released intracellular calcium from an intact, viable cell. In addition, radioactive studies using {sup 45}Ca{sup 2+} loaded into permeabilized hepatocytes, indicated in preliminary studies that the NAD metabolite could also release calcium from intracellular stores of mammalian cells.

  14. Syntheses of nicotinamide riboside and derivatives: effective agents for increasing nicotinamide adenine dinucleotide concentrations in mammalian cells.

    PubMed

    Yang, Tianle; Chan, Noel Yan-Ki; Sauve, Anthony A

    2007-12-27

    A new two-step methodology achieves stereoselective synthesis of beta-nicotinamide riboside and a series of related amide, ester, and acid nucleosides. Compounds were prepared through a triacetylated-nicotinate ester nucleoside, via coupling of either ethylnicotinate or phenylnicotinate with 1,2,3,5-tetra-O-acetyl-beta-D-ribofuranose. Nicotinamide riboside, nicotinic acid riboside, O-ethylnicotinate riboside, O-methylnicotinate riboside, and several N-alkyl derivatives increased NAD+ concentrations from 1.2-2.7-fold in several mammalian cell lines. These findings establish bioavailability and potent effects of these nucleosides in stimulating the increase of NAD+ concentrations in mammalian cells. PMID:18052316

  15. Accumulation of DNA damage and reduced levels of nicotine adenine dinucleotide in the brains of Atm-deficient mice.

    PubMed

    Stern, Nora; Hochman, Ayala; Zemach, Naty; Weizman, Nir; Hammel, Ilan; Shiloh, Yosef; Rotman, Galit; Barzilai, Ari

    2002-01-01

    Ataxia-telangiectasia (A-T) is a human genetic disorder caused by mutational inactivation of the ATM gene. A-T patients display a pleiotropic phenotype, in which a major neurological feature is progressive ataxia due to degeneration of cerebellar Purkinje and granule neurons. Disruption of the mouse Atm locus creates a murine model of A-T that exhibits most of the clinical and cellular features of the human disease, but the neurological phenotype is barely expressed. We present evidence for the accumulation of DNA strand breaks in the brains of Atm(-/-), supporting the notion that ATM plays a major role in maintaining genomic stability. We also show a perturbation of the steady state levels of pyridine nucleotides. There is a significant decrease in both the reduced and the oxidized forms of NAD and in the total levels of NADP(T) and NADP(+) in the brains of Atm(-/-) mice. The changes in NAD(T), NADH, NAD(+), NADP(T), and NADP(+) were progressive and observed primarily in the cerebellum of 4-month-old Atm(-/-) mice. Higher rates of mitochondrial respiration were also recorded in 4-month-old Atm(-/-) cerebella. Taken together, our findings support the hypothesis that absence of functional ATM results in continuous stress, which may be an important cause of the degeneration of cerebellar neurons in A-T. PMID:11679583

  16. The ascorbic acid-dependent oxidation of reduced nicotinamide–adenine dinucleotide by ciliary and retinal microsomes

    PubMed Central

    Heath, H.; Fiddick, Rosemary

    1965-01-01

    1. The presence of an ascorbic acid-dependent NADH oxidation in ocular tissues has been established. Subcellular fractionation revealed that the enzyme is localized in the microsomes. The distribution of the enzyme in some ocular tissues has been determined; microsomes from the ciliary processes and the retina have comparable activities, which are much higher than those from the cornea or lens. 2. NADPH cannot replace NADH, and cysteine, reduced glutathione, ergothioneine and dehydroascorbic acid cannot be substituted for ascorbic acid in the reaction. The rate of NADH oxidation was greatly increased in the presence of cucumber ascorbate oxidase, and the enzyme appears to be NADH–monodehydroascorbate transhydrogenase. 3. Cytochrome b5 is present in retinal microsomes. 4. The enzyme is inhibited by p-chloromercuribenzoate and iodoacetate, but not by cyanide, Amytal or malonate. 5. High concentrations of chloroquine cause a partial inhibition of the reaction, probably owing to interaction of this compound with the enzyme thiol groups. Low concentrations of Diamox, comparable with those attained in tissues during therapy with this drug, bring about partial inhibition of the reaction. Eserine, cortisone, hydrocortisone, 11-deoxycorticosterone and dexamethasone have no effect on the rate of oxidation. 6. The possible role of ascorbic acid and NADH–monodehydroascorbate transhydrogenase in the formation of aqueous humour and secretory mechanisms is discussed. PMID:14345883

  17. The purification and properties of the respiratory-chain reduced nicotinamide–adenine dinucleotide dehydrogenase of Torulopsis utilis

    PubMed Central

    Tottmar, S. O. C.; Ragan, C. I.

    1971-01-01

    1. An NADH–ferricyanide reductase activity has been isolated from the respiratory chain of Torulopsis utilis by using detergents. The isolated enzyme contains non-haem iron, acid-labile sulphide and FMN in the molar proportions 27.5:28.4:1. The preparation is free of FAD and largely free of cytochrome. 2. The enzyme catalyses ferricyanide reduction by NADPH at about 1% of the rate with NADH, and reacts poorly with acceptors other than ferricyanide. The rates of reduction of some acceptors are, as percentages of the rate with ferricyanide: menadione, 0.35%; lipoate, 0.01%; cytochrome c, 0.065%; dichlorophenolindophenol, 0.35%; ubiquinone-1, 0.08%. 3. Several properties of submitochondrial particles of T. utilis (non-haem iron, acid-labile sulphide, FMN and an NADH-reducible electron-paramagnetic-resonance signal) were found to co-purify with the NADH–ferricyanide reductase activity. Thus about 70% of the FMN and, within the limits of accuracy of the experiments, 100% of the non-haem iron and acid-labile sulphide of submitochondrial particles derived from T. utilis cells grown under conditions of glycerol limitation (but relatively low iron availability) can be attributed to the NADH–ferricyanide reductase. 4. It was also shown that the component of submitochondrial particles specifically bleached at 460nm by NADH [species 1 of Ragan & Garland (1971)] co-purifies with the NADH–ferricyanide reductase. 5. This successful purification of an NADH dehydrogenase from T. utilis forms a starting point for investigating the molecular properties of phenotypically modified mitochondrial NADH oxidation pathways that lack energy conservation between NADH and the cytochromes. PMID:4399788

  18. A biomimetic redox flow battery based on flavin mononucleotide

    PubMed Central

    Orita, Akihiro; Verde, Michael G.; Sakai, Masanori; Meng, Ying Shirley

    2016-01-01

    The versatility in design of redox flow batteries makes them apt to efficiently store energy in large-scale applications at low cost. The discovery of inexpensive organic electroactive materials for use in aqueous flow battery electrolytes is highly attractive, but is thus far limited. Here we report on a flow battery using an aqueous electrolyte based on the sodium salt of flavin mononucleotide. Flavins are highly versatile electroactive molecules, which catalyse a multitude of redox reactions in biological systems. We use nicotinamide (vitamin B3) as a hydrotropic agent to enhance the water solubility of flavin mononucleotide. A redox flow battery using flavin mononucleotide negative and ferrocyanide positive electrolytes in strong base shows stable cycling performance, with over 99% capacity retention over the course of 100 cycles. We hypothesize that this is enabled due to the oxidized and reduced forms of FMN-Na being stabilized by resonance structures. PMID:27767026

  19. Complexation of cyclodextrins with flavines in aqueous solutions

    NASA Astrophysics Data System (ADS)

    Terekhova, I. V.

    2014-01-01

    Data on the binding mode and thermodynamics of complex formation for various cyclodextrins (CDs) with flavines are summarized. It is shown that the governing factors of complexation are the size, degree of hydration, and hydrophobicity of the guest molecule. It is found that the presence of small hydrophobic substituents in a flavine's structure increases their affinity toward cyclodextrin cavities, raising the stability of a complex. In contrast, the presence of bulky and polar side groups in a flavine's structure prevents its inclusion in a macrocyclic cavity and weakens complexation. The size of a CD cavity plays a minor role in the interaction between CDs and flavines, since the inclusion of a guest molecule is only partial.

  20. Metal enhanced fluorescence of flavin mononucleotide using new plasmonic platform

    NASA Astrophysics Data System (ADS)

    Synak, Anna; Grobelna, Beata; Raut, Sangram; Bojarski, Piotr; Gryczyński, Ignacy; Karczewski, Jakub; Shtoyko, Tanya

    2016-09-01

    New plasmonic platform was successfully obtained to investigate the increase of fluorescence intensity of a fluorophore in the presence of silver nanoparticles. A flavin mononucleotide, was selected by us as a fluorophore for this study as a very important biological compound playing a key role in many biochemical process. Plasmonic platforms were characterized by means of luminescence spectroscopy. Flavin mononucleotide deposited on plasmonic platform exhibits dramatic emission enhancements in presence of silver nanoparticles deposited on gold mirror.

  1. Photophysical deactivation pathways in adenine oligonucleotides.

    PubMed

    Spata, Vincent A; Matsika, Spiridoula

    2015-12-14

    In this work we study deactivation processes in adenine oligomers after absorption of UV radiation using Quantum Mechanics combined with Molecular Mechanics (QM/MM). Correlated electronic structure methods appropriate for describing the excited states are used to describe a π-stacked dimer of adenine bases incorporated into (dA)20(dT)20. The results of these calculations reveal three different types of excited state minima which play a role in deactivation processes. Within this set of minima there are minima where the excited state is localized on one adenine (monomer-like) as well as minima where the excited state is delocalized on two adenines, forming different types of excimers and bonded excimers of varying but inter-related character. The proximity of their energies reveals that the minima can decay into one another along a flat potential energy surface dependent on the interbase separation. Additionally, analysis of the emissive energies and other physical properties, including theoretical anisotropy calculations, and comparison with fluorescence experiments, provides evidence that excimers play an important role in long-lived signals in adenine oligonucleotides while the subpicosecond decay is attributed to monomer-like minima. The necessity for a close approach of the nucleobases reveals that the deactivation mechanism is tied to macro-molecular motion. PMID:26536353

  2. The catalase activity of diiron adenine deaminase.

    PubMed

    Kamat, Siddhesh S; Holmes-Hampton, Gregory P; Bagaria, Ashima; Kumaran, Desigan; Tichy, Shane E; Gheyi, Tarun; Zheng, Xiaojing; Bain, Kevin; Groshong, Chris; Emtage, Spencer; Sauder, J Michael; Burley, Stephen K; Swaminathan, Subramanyam; Lindahl, Paul A; Raushel, Frank M

    2011-12-01

    Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn(2+) before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO(4). Inductively coupled plasma mass spectrometry and Mössbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe(II) /Fe(II) ]-ADE catalyzed the conversion of H(2)O(2) to O(2) and H(2)O. The values of k(cat) and k(cat)/K(m) for the catalase activity are 200 s(-1) and 2.4 × 10(4) M(-1) s(-1), respectively. [Fe(II)/Fe(II)]-ADE underwent more than 100 turnovers with H(2)O(2) before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g(ave) = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H(2)O(2) by [Fe(II)/Fe(II)]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS. PMID:21998098

  3. Adenine auxotrophy--be aware: some effects of adenine auxotrophy in Saccharomyces cerevisiae strain W303-1A.

    PubMed

    Kokina, Agnese; Kibilds, Juris; Liepins, Janis

    2014-08-01

    Adenine auxotrophy is a commonly used genetic marker in haploid yeast strains. Strain W303-1A, which carries the ade2-1 mutation, is widely used in physiological and genetic research. Yeast extract-based rich medium contains a low level of adenine, so that adenine is often depleted before glucose. This could affect the cell physiology of adenine auxotrophs grown in rich medium. The aim of our study was to assess the effects of adenine auxotrophy on cell morphology and stress physiology. Our results show that adenine depletion halts cell division, but that culture optical density continues to increase due to cell swelling. Accumulation of trehalose and a coincident 10-fold increase in desiccation stress tolerance is observed in adenine auxotrophs after adenine depletion, when compared to prototrophs. Under adenine starvation, long-term survival of W303-1A is lower than during carbon starvation, but higher than during leucine starvation. We observed drastic adenine-dependent changes in cell stress physiology, suggesting that results may be biased when adenine auxotrophs are grown in rich media without adenine supplementation.

  4. Excited flavin and pterin coenzyme molecules in evolution.

    PubMed

    Kritsky, M S; Telegina, T A; Vechtomova, Y L; Kolesnikov, M P; Lyudnikova, T A; Golub, O A

    2010-10-01

    Excited flavin and pterin molecules are active in intermolecular energy transfer and in photocatalysis of redox reactions resulting in conservation of free energy. Flavin-containing pigments produced in models of the prebiotic environment are capable of converting photon energy into the energy of phosphoanhydride bonds of ATP. However, during evolution photochemical reactions involving excited FMN or FAD molecules failed to become participants of bioenergy transfer systems, but they appear in enzymes responsible for repair of UV-damaged DNA (DNA photolyases) and also in receptors of blue and UV-A light regulating vital functions of organisms. The families of these photoproteins (DNA-photolyases and cryptochromes, LOV-domain- and BLUF-domain-containing proteins) are different in the structure and in mechanisms of the photoprocesses. The excited flavin molecules are involved in photochemical processes in reaction centers of these photoproteins. In DNA photolyases and cryptochromes the excitation energy on the reaction center flavin is supplied from an antenna molecule that is bound with the same polypeptide. The role of antenna is played by MTHF or by 8-HDF in some DNA photolyases, i.e. also by molecules with known coenzyme functions in biocatalysis. Differences in the structure of chromophore-binding domains suggest an independent origin of the photoprotein families. The analysis of structure and properties of coenzyme molecules reveals some specific features that were significant in evolution for their being selected as chromophores in these proteins.

  5. Cofactor Trapping, a New Method To Produce Flavin Mononucleotide ▿

    PubMed Central

    Krauss, Ulrich; Svensson, Vera; Wirtz, Astrid; Knieps-Grünhagen, Esther; Jaeger, Karl-Erich

    2011-01-01

    We have purified flavin mononucleotide (FMN) from a flavoprotein-overexpressing Escherichia coli strain by cofactor trapping. This approach uses an overexpressed flavoprotein to trap FMN, which is thus removed from the cascade regulating FMN production in E. coli. This, in turn, allows the isolation of highly pure FMN. PMID:21131527

  6. Covalent immobilization of a flavoprotein monooxygenase via its flavin cofactor.

    PubMed

    Krzek, Marzena; van Beek, Hugo L; Permentier, Hjalmar P; Bischoff, Rainer; Fraaije, Marco W

    2016-01-01

    A generic approach for flavoenzyme immobilization was developed in which the flavin cofactor is used for anchoring enzymes onto the carrier. It exploits the tight binding of flavin cofactors to their target apo proteins. The method was tested for phenylacetone monooxygenase (PAMO) which is a well-studied and industrially interesting biocatalyst. Also a fusion protein was tested: PAMO fused to phosphite dehydrogenase (PTDH-PAMO). The employed flavin cofactor derivative, N6-(6-carboxyhexyl)-FAD succinimidylester (FAD*), was covalently anchored to agarose beads and served for apo enzyme immobilization by their reconstitution into holo enzymes. The thus immobilized enzymes retained their activity and remained active after several rounds of catalysis. For both tested enzymes, the generated agarose beads contained 3 U per g of dry resin. Notably, FAD-immobilized PAMO was found to be more thermostable (40% activity after 1 h at 60 °C) when compared to PAMO in solution (no activity detected after 1 h at 60 °C). The FAD-decorated agarose material could be easily recycled allowing multiple rounds of immobilization. This method allows an efficient and selective immobilization of flavoproteins via the FAD flavin cofactor onto a recyclable carrier.

  7. Why flavins are not competitors of chlorophyll in the evolution of biological converters of solar energy.

    PubMed

    Kritsky, Mikhail S; Telegina, Taisiya A; Vechtomova, Yulia L; Buglak, Andrey A

    2012-12-27

    Excited flavin molecules can photocatalyze reactions, leading to the accumulation of free energy in the products, and the data accumulated through biochemical experiments and by modeling prebiological processes suggest that flavins were available in the earliest stages of evolution. Furthermore, model experiments have shown that abiogenic flavin conjugated with a polyamino acid matrix, a pigment that photocatalyzes the phosphorylation of ADP to form ATP, could have been present in the prebiotic environment. Indeed, excited flavin molecules play key roles in many photoenzymes and regulatory photoreceptors, and the substantial structural differences between photoreceptor families indicate that evolution has repeatedly used flavins as chromophores for photoreceptor proteins. Some of these photoreceptors are equipped with a light-harvesting antenna, which transfers excitation energy to chemically reactive flavins in the reaction center. The sum of the available data suggests that evolution could have led to the formation of a flavin-based biological converter to convert light energy into energy in the form of ATP.

  8. The catalase activity of diiron adenine deaminase

    SciTech Connect

    Kamat S. S.; Swaminathan S.; Holmes-Hampton, G. P.; Bagaria, A.; Kumaran, D.; Tichy, S. E.; Gheyi, T.; Zheng, X.; Bain, K.; Groshong, C.; Emtage, S.; Sauder, J. M.; Burley, S. K.; Lindahl, P. A.; Raushel, F. M.

    2011-12-01

    Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn{sup 2+} before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO{sub 4}. Inductively coupled plasma mass spectrometry and Moessbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe{sup II}/Fe{sup II}]-ADE catalyzed the conversion of H{sub 2}O{sub 2} to O{sub 2} and H{sub 2}O. The values of k{sub cat} and k{sub cat}/K{sub m} for the catalase activity are 200 s{sup -1} and 2.4 x 10{sup 4} M{sup -1} s{sup -1}, respectively. [Fe{sup II}/Fe{sup II}]-ADE underwent more than 100 turnovers with H{sub 2}O{sub 2} before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g{sub ave} = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H{sub 2}O{sub 2} by [Fe{sup II}/Fe{sup II}]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS.

  9. The Nature's Clever Trick for Making Cyclic Dinucleotide.

    PubMed

    Fang, Pengfei; Guo, Min

    2015-05-01

    Ever since their initial discovery few years ago, cyclic dinucleotides (cDNs), their biosynthesis, and their biological function have been in focus of intense research efforts. In this issue, Kato et al. (2015) present strong evidence that the key enzyme in cDN biosynthesis, DncV, is poised on a tipping point such that, given a nudge, the enzyme, can link the nucleotides into a distinct cyclic loop, leading to a specific innate immune response.

  10. Background correction using dinucleotide affinities improves the performance of GCRMA

    PubMed Central

    Gharaibeh, Raad Z; Fodor, Anthony A; Gibas, Cynthia J

    2008-01-01

    Background High-density short oligonucleotide microarrays are a primary research tool for assessing global gene expression. Background noise on microarrays comprises a significant portion of the measured raw data, which can have serious implications for the interpretation of the generated data if not estimated correctly. Results We introduce an approach to calculate probe affinity based on sequence composition, incorporating nearest-neighbor (NN) information. Our model uses position-specific dinucleotide information, instead of the original single nucleotide approach, and adds up to 10% to the total variance explained (R2) when compared to the previously published model. We demonstrate that correcting for background noise using this approach enhances the performance of the GCRMA preprocessing algorithm when applied to control datasets, especially for detecting low intensity targets. Conclusion Modifying the previously published position-dependent affinity model to incorporate dinucleotide information significantly improves the performance of the model. The dinucleotide affinity model enhances the detection of differentially expressed genes when implemented as a background correction procedure in GeneChip preprocessing algorithms. This is conceptually consistent with physical models of binding affinity, which depend on the nearest-neighbor stacking interactions in addition to base-pairing. PMID:18947404

  11. Cyclic Dinucleotide-Controlled Regulatory Pathways in Streptomyces Species

    PubMed Central

    2015-01-01

    The cyclic dinucleotides cyclic 3′,5′-diguanylate (c-di-GMP) and cyclic 3′,5′-diadenylate (c-di-AMP) have emerged as key components of bacterial signal transduction networks. These closely related second messengers follow the classical general principles of nucleotide signaling by integrating diverse signals into regulatory pathways that control cellular responses to changing environments. They impact distinct cellular processes, with c-di-GMP having an established role in promoting bacterial adhesion and inhibiting motility and c-di-AMP being involved in cell wall metabolism, potassium homeostasis, and DNA repair. The involvement of c-dinucleotides in the physiology of the filamentous, nonmotile streptomycetes remained obscure until recent discoveries showed that c-di-GMP controls the activity of the developmental master regulator BldD and that c-di-AMP determines the level of the resuscitation-promoting factor A(RpfA) cell wall-remodelling enzyme. Here, I summarize our current knowledge of c-dinucleotide signaling in Streptomyces species and highlight the important roles of c-di-GMP and c-di-AMP in the biology of these antibiotic-producing, multicellular bacteria. PMID:26216850

  12. Association of bone mineral density with a dinucleotide repeat polymorphism at the calcitonin (CT) locus.

    PubMed

    Miyao, M; Hosoi, T; Emi, M; Nakajima, T; Inoue, S; Hoshino, S; Shiraki, M; Orimo, H; Ouchi, Y

    2000-01-01

    Calcitonin (CT), a calcium-regulating hormone, lowers the calcium level in serum by inhibiting bone resorption. Because CT may play a role in the pathogenesis of osteoporosis, genetic variations in or adjacent to the CT gene may be associated with variations in bone mineral density (BMD). The present study examined the correlation between a dinucleotide (cytosine-adenine; CA) repeat polymorphism at the CT locus and BMD in 311 Japanese postmenopausal women (mean age, 64.1 years). Seven alleles were present in this population; each allele contained 10, 11, 16, 17, 18, 19, or 20 CA repeats. Thus, we designated the respective genotypes A10, A11, A16, A17, A18, A19, and A20. The A10 and A17 alleles were the predominant alleles in the population studied. Z scores (a parameter representing deviation from the age-specific weight-adjusted average BMD) were compared between individuals that possessed one or two alleles of each genotype and those that did not possess the allele. Subjects who possessed one or two A10 alleles had lower BMD Z scores than those who did not (lumbar 2-4 BMD Z score; -0.148 +/- 1.23 vs 0.182 +/- 1.54; P = 0.04). No significant relationships were observed between allelic status and background data or biochemical parameters. The significant association observed between BMD and genetic variations at the CT locus implies that polymorphism at this locus may be a useful marker for the genetic study of osteoporosis. PMID:11185743

  13. Methods for detection of methyl-CpG dinucleotides

    SciTech Connect

    Dunn, John J

    2013-11-26

    The invention provides methods for enriching methyl-CpG sequences from a DNA sample. The method makes use of conversion of cytosine residues to uracil under conditions in which methyl-cytosine residues are preserved. Additional methods of the invention enable to preservation of the context of me-CpG dinucleotides. The invention also provides a recombinant, full length and substantially pure McrA protein (rMcrA) for binding and isolation of DNA fragments containing the sequence 5'-C.sup.MeCpGG-3'. Methods for making and using the rMcrA protein, and derivatives thereof are provided.

  14. Methods for detection of methyl-CpG dinucleotides

    SciTech Connect

    Dunn, John J.

    2013-01-29

    The invention provides methods for enriching methyl-CpG sequences from a DNA sample. The method makes use of conversion of cytosine residues to uracil under conditions in which methyl-cytosine residues are preserved. Additional methods of the invention enable to preservation of the context of me-CpG dinucleotides. The invention also provides a recombinant, full length and substantially pure McrA protein (rMcrA) for binding and isolation of DNA fragments containing the sequence 5'-C.sup.MeCpGG-3'. Methods for making and using the rMcrA protein, and derivatives thereof are provided.

  15. Methods for detection of methyl-CpG dinucleotides

    SciTech Connect

    Dunn, John J.

    2012-09-11

    The invention provides methods for enriching methyl-CpG sequences from a DNA sample. The method makes use of conversion of cytosine residues to uracil under conditions in which methyl-cytosine residues are preserved. Additional methods of the invention enable to preservation of the context of me-CpG dinucleotides. The invention also provides a recombinant, full length and substantially pure McrA protein (rMcrA) for binding and isolation of DNA fragments containing the sequence 5'-C.sup.MeCpGG-3'. Methods for making and using the rMcrA protein, and derivatives thereof are provided.

  16. The flavin inhibitor diphenyleneiodonium renders Trichomonas vaginalis resistant to metronidazole, inhibits thioredoxin reductase and flavin reductase, and shuts off hydrogenosomal enzymatic pathways.

    PubMed

    Leitsch, David; Kolarich, Daniel; Duchêne, Michael

    2010-05-01

    Infections with the microaerophilic protozoan parasite Trichomonas vaginalis are commonly treated with metronidazole, a 5-nitroimidazole drug. Metronidazole is selectively toxic to microaerophiles and anaerobes because reduction at the drug's nitro group, which is a precondition for toxicity, occurs only quantitatively in these organisms. In our previous work we identified the flavin enzyme thioredoxin reductase as an electron donor to 5-nitroimidazole drugs in T. vaginalis and observed that highly metronidazole-resistant cell lines lack thioredoxin reductase and flavin reductase activities. In this study we added the flavin inhibitor diphenyleneiodonium (DPI) to T. vaginalis cultures in order to test our hypothesis that metronidazole reduction is catalyzed by flavin enzymes, e.g. thioredoxin reductase, and intracellular free flavins. Indeed, within hours, DPI rendered T. vaginalis insensitive to metronidazole concentrations as high as 1mM and prevented the formation of metronidazole adducts with proteins. Thioredoxin reductase activity was absent from DPI-treated cells and flavin reductase activity was sharply decreased. In addition, DPI-treated cells also upregulated the expression of antioxidant enzymes, i.e. thioredoxin peroxidases and superoxide dismutases, and displayed a fundamentally altered metabolism caused by inactivation of pyruvate:ferredoxin oxidoreductase (PFOR) and concomitant upregulation of lactate dehydrogenase (LDH) activity. Thus, the disruption of the cellular flavin metabolism by DPI mediated metabolic steps which are similar to that of cells with metronidazole resistance induced in vitro. Finally, we present direct evidence that the increased expression of antioxidant enzymes is dispensable for acquiring resistance to metronidazole. PMID:20093143

  17. Adenine nucleotide transporters in organelles: novel genes and functions.

    PubMed

    Traba, Javier; Satrústegui, Jorgina; del Arco, Araceli

    2011-04-01

    In eukaryotes, cellular energy in the form of ATP is produced in the cytosol via glycolysis or in the mitochondria via oxidative phosphorylation and, in photosynthetic organisms, in the chloroplast via photophosphorylation. Transport of adenine nucleotides among cell compartments is essential and is performed mainly by members of the mitochondrial carrier family, among which the ADP/ATP carriers are the best known. This work reviews the carriers that transport adenine nucleotides into the organelles of eukaryotic cells together with their possible functions. We focus on novel mechanisms of adenine nucleotide transport, including mitochondrial carriers found in organelles such as peroxisomes, plastids, or endoplasmic reticulum and also mitochondrial carriers found in the mitochondrial remnants of many eukaryotic parasites of interest. The extensive repertoire of adenine nucleotide carriers highlights an amazing variety of new possible functions of adenine nucleotide transport across eukaryotic organelles.

  18. Radiation and thermal stabilities of adenine nucleotides.

    PubMed

    Demidov, V V; Potaman, V N; Solyanina, I P; Trofimov, V I

    1995-03-01

    We have investigated in detail radiation and thermal stabilities and transformations of adenosine mono- and triphosphates in liquid and frozen solid aqueous solutions within a wide range of absorbed radiation dose (up to 75 kGy) and temperature (up to 160 degrees C). Dephosphorylation is the main pathway of high temperature hydrolysis of adenine nucleotides. Basic thermodynamic and kinetic parameters of this process have been determined. Radiolysis of investigated compounds at room temperature results in scission of N-glycosidic bond with a radiation yield about of 1 mol/100 eV. Solution freezing significantly enhances radiation stability of nucleotides as well as other biomolecules. This circumstance is essential in the discussion of panspermia concepts.

  19. Dynamic determination of the functional state in photolyase and the implication for cryptochrome.

    PubMed

    Liu, Zheyun; Zhang, Meng; Guo, Xunmin; Tan, Chuang; Li, Jiang; Wang, Lijuan; Sancar, Aziz; Zhong, Dongping

    2013-08-01

    The flavin adenine dinucleotide cofactor has an unusual bent configuration in photolyase and cryptochrome, and such a folded structure may have a functional role in initial photochemistry. Using femtosecond spectroscopy, we report here our systematic characterization of cyclic intramolecular electron transfer (ET) dynamics between the flavin and adenine moieties of flavin adenine dinucleotide in four redox forms of the oxidized, neutral, and anionic semiquinone, and anionic hydroquinone states. By comparing wild-type and mutant enzymes, we have determined that the excited neutral oxidized and semiquinone states absorb an electron from the adenine moiety in 19 and 135 ps, whereas the excited anionic semiquinone and hydroquinone states donate an electron to the adenine moiety in 12 ps and 2 ns, respectively. All back ET dynamics occur ultrafast within 100 ps. These four ET dynamics dictate that only the anionic hydroquinone flavin can be the functional state in photolyase due to the slower ET dynamics (2 ns) with the adenine moiety and a faster ET dynamics (250 ps) with the substrate, whereas the intervening adenine moiety mediates electron tunneling for repair of damaged DNA. Assuming ET as the universal mechanism for photolyase and cryptochrome, these results imply anionic flavin as the more attractive form of the cofactor in the active state in cryptochrome to induce charge relocation to cause an electrostatic variation in the active site and then lead to a local conformation change to initiate signaling.

  20. CpG dinucleotide frequencies reveal the role of host methylation capabilities in parvovirus evolution.

    PubMed

    Upadhyay, Mohita; Samal, Jasmine; Kandpal, Manish; Vasaikar, Suhas; Biswas, Banhi; Gomes, James; Vivekanandan, Perumal

    2013-12-01

    Parvoviruses are rapidly evolving viruses that infect a wide range of hosts, including vertebrates and invertebrates. Extensive methylation of the parvovirus genome has been recently demonstrated. A global pattern of methylation of CpG dinucleotides is seen in vertebrate genomes, compared to "fractional" methylation patterns in invertebrate genomes. It remains unknown if the loss of CpG dinucleotides occurs in all viruses of a given DNA virus family that infect host species spanning across vertebrates and invertebrates. We investigated the link between the extent of CpG dinucleotide depletion among autonomous parvoviruses and the evolutionary lineage of the infected host. We demonstrate major differences in the relative abundance of CpG dinucleotides among autonomous parvoviruses which share similar genome organization and common ancestry, depending on the infected host species. Parvoviruses infecting vertebrate hosts had significantly lower relative abundance of CpG dinucleotides than parvoviruses infecting invertebrate hosts. The strong correlation of CpG dinucleotide depletion with the gain in TpG/CpA dinucleotides and the loss of TpA dinucleotides among parvoviruses suggests a major role for CpG methylation in the evolution of parvoviruses. Our data present evidence that links the relative abundance of CpG dinucleotides in parvoviruses to the methylation capabilities of the infected host. In sum, our findings support a novel perspective of host-driven evolution among autonomous parvoviruses.

  1. Hepatitis C virus RNA: dinucleotide frequencies and cleavage by RNase L.

    PubMed

    Washenberger, Christopher L; Han, Jian-Qiu; Kechris, Katherina J; Jha, Babal Kant; Silverman, Robert H; Barton, David J

    2007-12-01

    Ribonuclease L (RNase L) is an antiviral endoribonuclease that cleaves hepatitis C virus (HCV) RNA at single-stranded UA and UU dinucleotides throughout the open reading frame (ORF). To determine whether RNase L exerts evolutionary pressure on HCV we examined the frequencies of UA and UU dinucleotides in 162 RNA sequences from the Los Alamos National Labs HCV Database (http://hcv.lanl.gov). Considering the base composition of the HCV ORFs, both UA and UU dinucleotides were less frequent than predicted in each of 162 HCV RNAs. UA dinucleotides were significantly less frequent than predicted at each of the three codon positions while UU dinucleotides were less frequent than predicted predominantly at the wobble position of codons. UA and UU dinucleotides were among the least abundant dinucleotides in HCV RNA ORFs. Furthermore, HCV genotype 1 RNAs have a lower frequency of UA and UU dinucleotides than genotype 2 and 3 RNAs, perhaps contributing to increased resistance of HCV genotype 1 infections to interferon therapy. In vitro, RNase L cleaved both HCV genotype 1 and 2 RNAs efficiently. Thus, RNase L can cleave HCV RNAs efficiently and variably reduced frequencies of UA and UU dinucleotides in HCV RNA ORFs are consistent with the selective pressure of RNase L.

  2. An Arabidopsis FAD pyrophosphohydrolase, AtNUDX23, is involved in flavin homeostasis.

    PubMed

    Maruta, Takanori; Yoshimoto, Tadashi; Ito, Daisuke; Ogawa, Takahisa; Tamoi, Masahiro; Yoshimura, Kazuya; Shigeoka, Shigeru

    2012-06-01

    Although flavins, riboflavin (RF), FMN and FAD, are essential for primary and secondary metabolism in plants, the metabolic regulation of flavins is still largely unknown. Recently, we found that an Arabidopsis Nudix hydrolase, AtNUDX23, has FAD pyrophosphohydrolase activity and is distributed in plastids. Levels of RF and FAD but not FMN in Arabidopsis leaves significantly increased under continuous light and decreased in the dark. The transcript levels of AtNUDX23 as well as genes involved in flavin metabolism (AtFADS, AtRibF1, AtRibF2, AtFMN/FHy, LS and AtRibA) significantly increased under continuous light. The pyrophosphohydrolase activity toward FAD was enhanced in AtNUDX23-overexpressing (OX-NUDX23) plants and reduced in AtNUDX23-suppressed (KD-nudx23) plants, compared with the control plants. Interestingly intracellular levels of RF, FMN and FAD significantly decreased in not only OX-NUDX23 but also KD-nudx23 plants. The transcript levels of the flavin metabolic genes also decreased in both plants. Similarly, the increase in intracellular levels on treatment with flavins caused a reduction in the transcript levels of genes involved in flavin metabolism. These results suggest that negative feedback regulation of the metabolism of flavins through the hydrolysis of FAD by AtNUDX23 in plastids is involved in flavin homeostasis in plant cells.

  3. Ultrafast 2D-IR spectroelectrochemistry of flavin mononucleotide

    NASA Astrophysics Data System (ADS)

    El Khoury, Youssef; Van Wilderen, Luuk J. G. W.; Bredenbeck, Jens

    2015-06-01

    We demonstrate the coupling of ultrafast two-dimensional infrared (2D-IR) spectroscopy to electrochemistry in solution and apply it to flavin mononucleotide, an important cofactor of redox proteins. For this purpose, we designed a spectroelectrochemical cell optimized for 2D-IR measurements in reflection and measured the time-dependent 2D-IR spectra of the oxidized and reduced forms of flavin mononucleotide. The data show anharmonic coupling and vibrational energy transfer between different vibrational modes in the two redox species. Such information is inaccessible with redox-controlled steady-state FTIR spectroscopy. The wide range of applications offered by 2D-IR spectroscopy, such as sub-picosecond structure determination, IR band assignment via energy transfer, disentangling reaction mixtures through band connectivity in the 2D spectra, and the measurement of solvation dynamics and chemical exchange can now be explored under controlled redox potential. The development of this technique furthermore opens new horizons for studying the dynamics of redox proteins.

  4. Ultrafast 2D-IR spectroelectrochemistry of flavin mononucleotide.

    PubMed

    El Khoury, Youssef; Van Wilderen, Luuk J G W; Bredenbeck, Jens

    2015-06-01

    We demonstrate the coupling of ultrafast two-dimensional infrared (2D-IR) spectroscopy to electrochemistry in solution and apply it to flavin mononucleotide, an important cofactor of redox proteins. For this purpose, we designed a spectroelectrochemical cell optimized for 2D-IR measurements in reflection and measured the time-dependent 2D-IR spectra of the oxidized and reduced forms of flavin mononucleotide. The data show anharmonic coupling and vibrational energy transfer between different vibrational modes in the two redox species. Such information is inaccessible with redox-controlled steady-state FTIR spectroscopy. The wide range of applications offered by 2D-IR spectroscopy, such as sub-picosecond structure determination, IR band assignment via energy transfer, disentangling reaction mixtures through band connectivity in the 2D spectra, and the measurement of solvation dynamics and chemical exchange can now be explored under controlled redox potential. The development of this technique furthermore opens new horizons for studying the dynamics of redox proteins.

  5. Ultraviolet Photostability of Adenine on Gold and Silicon Surfaces

    NASA Astrophysics Data System (ADS)

    Mateo-Martí, Eva; Pradier, Claire-Marie; Martín-Gago, Jose-Angel

    2009-08-01

    The adenine molecule is a DNA nucleobase, an essential component of genetic material. Because of the important role of DNA nucleobases in terrestrial biochemistry, we have studied the molecular adsorption, orientation, and chemical binding of adenine on metallic and semiconducting surfaces, such as gold and silicon, respectively, and their stability toward ultraviolet radiation by X-ray photoelectron spectroscopy (XPS) and reflection absorption infrared spectroscopy (RAIRS) techniques. We have exposed the adenine surface system to UV radiation (200-400 nm) under a high-vacuum environment (10-7 mbar) to study the photostability and photochemistry of adenine on different surfaces. After 10 or 24 hours of exposure under interplanetary space conditions, UV radiation induces desorption and partial dissociation of the molecule, which is dependant on the nature of the surface. The electronic excitations, induced in the material by UV absorption, play a major role in the photodestruction of the absorbed molecules on the solid surfaces.

  6. Adenine adlayers on Cu(111): XPS and NEXAFS study

    SciTech Connect

    Tsud, Nataliya; Bercha, Sofiia; Ševčíková, Klára; Matolín, Vladimír; Acres, Robert G.; Prince, Kevin C.

    2015-11-07

    The adsorption of adenine on Cu(111) was studied by photoelectron and near edge x-ray absorption fine structure spectroscopy. Disordered molecular films were deposited by means of physical vapor deposition on the substrate at room temperature. Adenine chemisorbs on the Cu(111) surface with strong rehybridization of the molecular orbitals and the Cu 3d states. Annealing at 150 °C caused the desorption of weakly bonded molecules accompanied by formation of a short-range ordered molecular adlayer. The interface is characterized by the formation of new states in the valence band at 1.5, 7, and 9 eV. The present work complements and refines existing knowledge of adenine interaction with this surface. The coverage is not the main parameter that defines the adenine geometry and adsorption properties on Cu(111). Excess thermal energy can further rearrange the molecular adlayer and, independent of the initial coverage, the flat lying stable molecular adlayer is formed.

  7. Why Flavins Are not Competitors of Chlorophyll in the Evolution of Biological Converters of Solar Energy

    PubMed Central

    Kritsky, Mikhail S.; Telegina, Taisiya A.; Vechtomova, Yulia L.; Buglak, Andrey A.

    2013-01-01

    Excited flavin molecules can photocatalyze reactions, leading to the accumulation of free energy in the products, and the data accumulated through biochemical experiments and by modeling prebiological processes suggest that flavins were available in the earliest stages of evolution. Furthermore, model experiments have shown that abiogenic flavin conjugated with a polyamino acid matrix, a pigment that photocatalyzes the phosphorylation of ADP to form ATP, could have been present in the prebiotic environment. Indeed, excited flavin molecules play key roles in many photoenzymes and regulatory photoreceptors, and the substantial structural differences between photoreceptor families indicate that evolution has repeatedly used flavins as chromophores for photoreceptor proteins. Some of these photoreceptors are equipped with a light-harvesting antenna, which transfers excitation energy to chemically reactive flavins in the reaction center. The sum of the available data suggests that evolution could have led to the formation of a flavin-based biological converter to convert light energy into energy in the form of ATP. PMID:23271372

  8. Rate enhancement of bacterial extracellular electron transport involves bound flavin semiquinones

    PubMed Central

    Okamoto, Akihiro; Hashimoto, Kazuhito; Nealson, Kenneth H.; Nakamura, Ryuhei

    2013-01-01

    Extracellular redox-active compounds, flavins and other quinones, have been hypothesized to play a major role in the delivery of electrons from cellular metabolic systems to extracellular insoluble substrates by a diffusion-based shuttling two-electron-transfer mechanism. Here we show that flavin molecules secreted by Shewanella oneidensis MR-1 enhance the ability of its outer-membrane c-type cytochromes (OM c-Cyts) to transport electrons as redox cofactors, but not free-form flavins. Whole-cell differential pulse voltammetry revealed that the redox potential of flavin was reversibly shifted more than 100 mV in a positive direction, in good agreement with increasing microbial current generation. Importantly, this flavin/OM c-Cyts interaction was found to facilitate a one-electron redox reaction via a semiquinone, resulting in a 103- to 105-fold faster reaction rate than that of free flavin. These results are not consistent with previously proposed redox-shuttling mechanisms but suggest that the flavin/OM c-Cyts interaction regulates the extent of extracellular electron transport coupled with intracellular metabolic activity. PMID:23576738

  9. UbiX is a flavin prenyltransferase required for bacterial ubiquinone biosynthesis

    NASA Astrophysics Data System (ADS)

    White, Mark D.; Payne, Karl A. P.; Fisher, Karl; Marshall, Stephen A.; Parker, David; Rattray, Nicholas J. W.; Trivedi, Drupad K.; Goodacre, Royston; Rigby, Stephen E. J.; Scrutton, Nigel S.; Hay, Sam; Leys, David

    2015-06-01

    Ubiquinone (also known as coenzyme Q) is a ubiquitous lipid-soluble redox cofactor that is an essential component of electron transfer chains. Eleven genes have been implicated in bacterial ubiquinone biosynthesis, including ubiX and ubiD, which are responsible for decarboxylation of the 3-octaprenyl-4-hydroxybenzoate precursor. Despite structural and biochemical characterization of UbiX as a flavin mononucleotide (FMN)-binding protein, no decarboxylase activity has been detected. Here we report that UbiX produces a novel flavin-derived cofactor required for the decarboxylase activity of UbiD. UbiX acts as a flavin prenyltransferase, linking a dimethylallyl moiety to the flavin N5 and C6 atoms. This adds a fourth non-aromatic ring to the flavin isoalloxazine group. In contrast to other prenyltransferases, UbiX is metal-independent and requires dimethylallyl-monophosphate as substrate. Kinetic crystallography reveals that the prenyltransferase mechanism of UbiX resembles that of the terpene synthases. The active site environment is dominated by π systems, which assist phosphate-C1' bond breakage following FMN reduction, leading to formation of the N5-C1' bond. UbiX then acts as a chaperone for adduct reorientation, via transient carbocation species, leading ultimately to formation of the dimethylallyl C3'-C6 bond. Our findings establish the mechanism for formation of a new flavin-derived cofactor, extending both flavin and terpenoid biochemical repertoires.

  10. Time- and spectrally resolved characteristics of flavin fluorescence in U87MG cancer cells in culture

    NASA Astrophysics Data System (ADS)

    Horilova, Julia; Cunderlikova, Beata; Marcek Chorvatova, Alzbeta

    2015-05-01

    Early detection of cancer is crucial for the successful diagnostics of its presence and its subsequent treatment. To improve cancer detection, we tested the progressive multimodal optical imaging of U87MG cells in culture. A combination of steady-state spectroscopic methods with the time-resolved approach provides a new insight into the native metabolism when focused on endogenous tissue fluorescence. In this contribution, we evaluated the metabolic state of living U87MG cancer cells in culture by means of endogenous flavin fluorescence. Confocal microscopy and time-resolved fluorescence imaging were employed to gather spectrally and time-resolved images of the flavin fluorescence. We observed that flavin fluorescence in U87MG cells was predominantly localized outside the cell nucleus in mitochondria, while exhibiting a spectral maximum under 500 nm and fluorescence lifetimes under 1.4 ns, suggesting the presence of bound flavins. In some cells, flavin fluorescence was also detected inside the cell nuclei in the nucleoli, exhibiting longer fluorescence lifetimes and a red-shifted spectral maximum, pointing to the presence of free flavin. Extra-nuclear flavin fluorescence was diminished by 2-deoxyglucose, but failed to increase with 2,4-dinitrophenol, the uncoupler of oxidative phosphorylation, indicating that the cells use glycolysis, rather than oxidative phosphorylation for functioning. These gathered data are the first step toward monitoring the metabolic state of U87MG cancer cells.

  11. The two-photon excitation cross section of 6MAP, a fluorescent adenine analogue.

    PubMed

    Stanley, Robert J; Hou, Zhanjia; Yang, Aiping; Hawkins, Mary E

    2005-03-01

    6MAP is a fluorescent analogue of adenine that undergoes Watson-Crick base pairing and base stacking in double-stranded DNA. The one-photon absorption spectrum of 6MAP is characterized by a maximum around 330 nm with moderate quantum yield fluorescence centered at about 420 nm. To take advantage of this probe for confocal and single-molecule microscopy, it would be advantageous to be able to excite the analogue via two photons. We report the first determination of the two-photon excitation cross section and spectrum for 6MAP from 614 to 700 nm. The power dependence of the fluorescence indicates that emission results from the absorption of two photons. The one-photon and two-photon emission line shapes are identical within experimental error. A study of the concentration dependence of the fluorescence yield for one-photon excitation shows no measurable quenching up to about 5 microM. The maximum in the two-photon excitation spectrum gives a two-photon cross section, delta(TPE), of 3.4 +/- 0.1 Goeppert-Mayer (G.M.) at 659 nm, which correlates well with the one-photon absorption maximum. This compares quite favorably with cross sections of various naturally fluorescent biological molecules such as flavins and nicotiamide. In addition, we have also obtained the two-photon-induced fluorescence emission spectrum of quinine sulfate. It is approximately the same as that for one-photon excitation, suggesting that two-photon excitation of quinine sulfate may be used for calibration purposes.

  12. The two-photon excitation cross section of 6MAP, a fluorescent adenine analogue.

    PubMed

    Stanley, Robert J; Hou, Zhanjia; Yang, Aiping; Hawkins, Mary E

    2005-03-01

    6MAP is a fluorescent analogue of adenine that undergoes Watson-Crick base pairing and base stacking in double-stranded DNA. The one-photon absorption spectrum of 6MAP is characterized by a maximum around 330 nm with moderate quantum yield fluorescence centered at about 420 nm. To take advantage of this probe for confocal and single-molecule microscopy, it would be advantageous to be able to excite the analogue via two photons. We report the first determination of the two-photon excitation cross section and spectrum for 6MAP from 614 to 700 nm. The power dependence of the fluorescence indicates that emission results from the absorption of two photons. The one-photon and two-photon emission line shapes are identical within experimental error. A study of the concentration dependence of the fluorescence yield for one-photon excitation shows no measurable quenching up to about 5 microM. The maximum in the two-photon excitation spectrum gives a two-photon cross section, delta(TPE), of 3.4 +/- 0.1 Goeppert-Mayer (G.M.) at 659 nm, which correlates well with the one-photon absorption maximum. This compares quite favorably with cross sections of various naturally fluorescent biological molecules such as flavins and nicotiamide. In addition, we have also obtained the two-photon-induced fluorescence emission spectrum of quinine sulfate. It is approximately the same as that for one-photon excitation, suggesting that two-photon excitation of quinine sulfate may be used for calibration purposes. PMID:16851408

  13. Production of flavin mononucleotide by metabolically engineered yeast Candida famata.

    PubMed

    Yatsyshyn, Valentyna Y; Ishchuk, Olena P; Voronovsky, Andriy Y; Fedorovych, Daria V; Sibirny, Andriy A

    2009-05-01

    Recombinant strains of the flavinogenic yeast Candida famata able to overproduce flavin mononucleotide (FMN) that contain FMN1 gene encoding riboflavin (RF) kinase driven by the strong constitutive promoter TEF1 (translation elongation factor 1alpha) were constructed. Transformation of these strains with the additional plasmid containing the FMN1 gene under the TEF1 promoter resulted in the 200-fold increase in the riboflavin kinase activity and 100-fold increase in FMN production as compared to the wild-type strain (last feature was found only in iron-deficient medium). Overexpression of the FMN1 gene in the mutant that has deregulated riboflavin biosynthesis pathway and high level of riboflavin production in iron-sufficient medium led to the 30-fold increase in the riboflavin kinase activity and 400-fold increase in FMN production of the resulted transformants. The obtained C. famata recombinant strains can be used for the further construction of improved FMN overproducers. PMID:19558965

  14. Control of redox reactivity of flavin and pterin coenzymes by metal ion coordination and hydrogen bonding.

    PubMed

    Fukuzumi, Shunichi; Kojima, Takahiko

    2008-03-01

    The electron-transfer activities of flavin and pterin coenzymes can be fine-tuned by coordination of metal ions, protonation and hydrogen bonding. Formation of hydrogen bonds with a hydrogen-bond receptor in metal-flavin complexes is made possible depending on the type of coordination bond that can leave the hydrogen-bonding sites. The electron-transfer catalytic functions of flavin and pterin coenzymes are described by showing a number of examples of both thermal and photochemical redox reactions, which proceed by controlling the electron-transfer reactivity of coenzymes with metal ion binding, protonation and hydrogen bonding.

  15. Chromatographic determination of riboflavin and its derivatives in food.

    PubMed

    Gliszczyńska-Swigło, A; Koziołowa, A

    2000-06-01

    Three elution methods on two different reversed-phase C18 columns were developed to determine flavin derivatives in raw egg white, raw egg yolk, egg powder, pasteurised milk, fermented milk products and liver (chicken, calf and pig). Additionally, 11 thin-layer chromatography solvent systems were used to confirm presence of flavins detected in assessed products. It was found that an Alphabond C18 column was not as effective as a Symmetry C18 column. Method A (mobile phase gradient of methanol-0.05 M ammonium acetate, pH 6.0 applied on an Alphabond C18 column) can be used for determination of flavin adenine dinucleotide, flavin mononucleotide, riboflavin 4',5'-cyclic phosphate, riboflavin, 10-formylmethylflavin and 10-hydroxyethylflavin in products that do not contain 7alpha-hydroxyriboflavin. Method B (mobile phase gradient of methanol-demineralized water, on an Alphabond C18 column) can be useful to separate flavin coenzymes from other flavin compounds or to confirm the presence of 7alpha-hydroxyriboflavin and 10-hydroxyethylflavin in analysed samples. Method C (mobile phase gradient of methanol-0.05 M ammonium acetate, pH 6.0, on a Symmetry C18 column) allows separation of all flavins detected in tested products: flavin adenine dinucleotide, flavin mononucleotide, riboflavin 4',5'-cyclic phosphate, riboflavin, 10-formylmethylflavin, 10-hydroxyethylflavin, 7alpha-hydroxyriboflavin, riboflavin-beta-D-galactoside and riboflavin-alpha-D-glucoside. PMID:10905712

  16. Thiamin and riboflavin vitamers in human milk: effects of lipid-based nutrient supplementation and stage of lactation on vitamer secretion and contributions to total vitamin content

    Technology Transfer Automated Retrieval System (TEKTRAN)

    While thiamin and riboflavin in breast milk have been analyzed for over 50 years, less attention has been given to the different forms of each vitamin. Thiamin-monophosphate (TMP) and free thiamin contribute to total thiamin content; flavin adenine-dinucleotide (FAD) and free riboflavin are the main...

  17. Effect of Exogenous Extracellular Nicotinamide Adenine Dinucleotide (NAD⁺) on Bioelectric Activity of the Pacemaker and Conduction System of the Heart.

    PubMed

    Pustovit, K B; Kuz'min, V S; Sukhova, G S

    2015-06-01

    In rat sinoatrial node, NAD(+) (10 μM) reduced the rate of spontaneous action potentials, duration of action potentials, and the velocity of slow diastolic depolarization, but the rate of action potential front propagation increases. In passed rabbit Purkinje fibers, NAD(+) (10 μM) reduced the duration of action potentials. Under conditions of spontaneous activity of Purkinje fibers, NAD(+) reduced the fi ring rate and the rate of slow diastolic depolarization. The effects of extracellular NAD(+) on bioelectric activity of the pacemaker (sinoatrial node) and conduction system of the heart (Purkinje fibers) are probably related to activation of P1 and P2 purinoceptors.

  18. Novel concept of enzyme selective nicotinamide adenine dinucleotide (NAD)-modified inhibitors based on enzyme taxonomy from the diphosphate conformation of NAD.

    PubMed

    Fujii, Mikio; Kitagawa, Yasuyuki; Iida, Shui; Kato, Keisuke; Ono, Machiko

    2015-11-15

    The dihedral angle θ of the diphosphate part of NAD(P) were investigated to distinguish the differences in the binding-conformation of NAD(P) to enzymes and to create an enzyme taxonomy. Furthermore, new inhibitors with fixed dihedral angles showed that enzymes could recognize the differences in the dihedral angle θ. We suggest the taxonomy and the dihedral angle θ are important values for chemists to consider when designing inhibitors and drugs that target enzymes.

  19. Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Cyclic ADP-Ribose (cADPR) Mediate Ca2+ Signaling in Cardiac Hypertrophy Induced by β-Adrenergic Stimulation

    PubMed Central

    Shawl, Asif Iqbal; Im, Soo-Yeul; Nam, Tae-Sik; Lee, Sun-Hwa; Ko, Jae-Ki; Jang, Kyu Yoon; Kim, Donghee; Kim, Uh-Hyun

    2016-01-01

    Ca2+ signaling plays a fundamental role in cardiac hypertrophic remodeling, but the underlying mechanisms remain poorly understood. We investigated the role of Ca2+-mobilizing second messengers, NAADP and cADPR, in the cardiac hypertrophy induced by β-adrenergic stimulation by isoproterenol. Isoproterenol induced an initial Ca2+ transients followed by sustained Ca2+ rises. Inhibition of the cADPR pathway with 8-Br-cADPR abolished only the sustained Ca2+ increase, whereas inhibition of the NAADP pathway with bafilomycin-A1 abolished both rapid and sustained phases of the isoproterenol-mediated signal, indicating that the Ca2+ signal is mediated by a sequential action of NAADP and cADPR. The sequential production of NAADP and cADPR was confirmed biochemically. The isoproterenol-mediated Ca2+ increase and cADPR production, but not NAADP production, were markedly reduced in cardiomyocytes obtained from CD38 knockout mice. CD38 knockout mice were rescued from chronic isoproterenol infusion-induced myocardial hypertrophy, interstitial fibrosis, and decrease in fractional shortening and ejection fraction. Thus, our findings indicate that β-adrenergic stimulation contributes to the development of maladaptive cardiac hypertrophy via Ca2+ signaling mediated by NAADP-synthesizing enzyme and CD38 that produce NAADP and cADPR, respectively. PMID:26959359

  20. Enhanced Reduced Nicotinamide Adenine Dinucleotide electrocatalysis onto multi-walled carbon nanotubes-decorated gold nanoparticles and their use in hybrid biofuel cell

    NASA Astrophysics Data System (ADS)

    Aquino Neto, S.; Almeida, T. S.; Belnap, D. M.; Minteer, S. D.; De Andrade, A. R.

    2015-01-01

    We report the preparation of Au nanoparticles synthetized by different protocols and supported on the surface of multi-walled carbon nanotubes containing different functional groups, focusing on their electrochemical performance towards NADH oxidation, ethanol bioelectrocatalysis, and ethanol/O2 biofuel cell. We describe four different synthesis protocols: microwave-assisted heating, water-in-oil, and dendrimer-encapsulated nanoparticles using acid or thiol species in the extraction step. The physical characterization of the metallic nanoparticles indicated that both the synthetic protocol as well as the type of functional groups on the carbon nanotubes affect the final particle size (varying from 13.4 to 2.4 nm) and their distribution onto the carbon surface. Moreover, the electrochemical data indicated that these two factors also influence their performance toward the electrooxidation of NADH. We observed that the samples containing Au nanoparticles with smaller size leads to higher catalytic currents and also shifts the oxidation potential of the targeted reaction, which varied from 0.13 to -0.06 V vs Ag/AgCl. Ethanol/O2 biofuel cell tests indicated that the hybrid bioelectrodes containing smaller and better distributed Au nanoparticles on the surface of carbon nanotubes generates higher power output, confirming that the electrochemical regeneration of NAD+ plays an important role in the overall biofuel cell performance.

  1. Induction of hepatoma carcinoma cell apoptosis through activation of the JNK-nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-ROS self-driven death signal circuit.

    PubMed

    Zeng, Ke-Wu; Song, Fang-Jiao; Wang, Ying-Hong; Li, Ning; Yu, Qian; Liao, Li-Xi; Jiang, Yong; Tu, Peng-Fei

    2014-10-28

    As an efficient method for inducing tumor cell apoptosis, ROS can be constantly formed and accumulated in NADPH oxidase overactivated-cells, resulting in further mitochondrial membrane damage and mitochondria-dependent apoptosis. In addition, JNK mitogen-activated protein kinase (JNK MAPK) signal also acts as a vital candidate pathway for inducing tumor cell apoptosis by targeting mitochondrial death pathway. However, the relationship between NADPH oxidase-ROS and JNK MAPK signal still remains unclear. Here, we discovered a novel self-driven signal circuit between NADPH oxidase-ROS and JNK MAPK, which was induced by a cytotoxic steroidal saponin (ASC) in hepatoma carcinoma cells. NADPH oxidase-dependent ROS production was markedly activated by ASC and directly led to JNK MAPK activation. Moreover, antioxidant, NADPH oxidase inhibitor and specific knock-out for p47 subunit of NADPH oxidase could effectively block NADPH oxidase-ROS-dependent JNK activation, suggesting that NADPH oxidase is an upstream regulator of JNK MAPK. Conversely, a specific JNK inhibitor could inhibit ASC-induced NADPH oxidase activation and down-regulate ROS levels as well, indicating that JNK might also regulate NADPH oxidase activity to some extent. These observations indicate that NADPH oxidase and JNK MAPK activate each other as a signal circuit. Furthermore, drug pretreatment experiments with ASC showed this signal circuit operated continuously via a self-driven mode and finally induced apoptosis in hepatoma carcinoma cells. Taken together, we provide a proof for inducing hepatoma carcinoma cell apoptosis by activating the JNK-NADPH oxidase-ROS-dependent self-driven signal circuit pathway. PMID:25064608

  2. The lactate dehydrogenase--reduced nicotinamide--adenine dinucleotide--pyruvate complex. Kinetics of pyruvate binding and quenching of coeznyme fluorescence.

    PubMed

    Südi, J

    1974-04-01

    The stopped-flow kinetic studies described in this and the following paper (Südi, 1974) demonstrate that a Haldane-type description of the reversible lactate dehydrogenase reaction presents an experimentally feasible task. Combined results of these two papers yield numerical values for the six rate constants defined by the following equilibrium scheme, where E represents lactate dehydrogenase: [Formula: see text] The experiments were carried out at pH8.4 at a relatively low temperature (6.3 degrees C) with the pig heart enzyme. Identification of the above two intermediates and determination of the corresponding rate constants actually involve four series of independent observations in these studies, since (a) the reaction can be followed in both directions, and (b) both the u.v. absorption and the fluorescence of the coenzymes are altered in the reaction, and it is shown that these two spectral changes do not occur simultaneously. Kinetic observations made in the reverse direction are reported in this paper. It is demonstrated that the fluorescence of NADH can no longer be observed in the ternary complex E(NADH) (Pyr). Even though the oxidation-reduction reaction rapidly follows the formation of this complex, the numerical values of k(-4) (8.33x10(5)m(-1).s(-1)) and k(+4) (222s(-1)) are easily obtained from a directly observed second-order reaction step in which fluorescent but not u.v.-absorbing material is disappearing. U.v.-absorption measurements do not clearly resolve the subsequent oxidation-reduction step from the dissociation of lactate. It is shown that this must be due partly to the instrumental dead time, and partly to a low transient concentration of E(NAD+) (Lac) in the two-step sequential reaction in which the detectable disappearance of u.v.-absorbing material takes place. It is estimated that about one-tenth of the total change in u.v. absorption is due to a ;burst reaction' in which E(NAD+) (Lac) is produced, and this estimation yields, from k(obs.)=120s(-1), k(-2)=1200s(-1). PMID:4377095

  3. Towards understanding the origins of the different specificities of binding the reduced (NADPH) and oxidised (NADP +) forms of nicotinamide adenine dinucleotide phosphate coenzyme to dihydrofolate reductase

    NASA Astrophysics Data System (ADS)

    Polshakov, Vladimir I.; Biekofsky, Rodolfo R.; Birdsall, Berry; Feeney, James

    2002-01-01

    Lactobacillus casei dihydrofolate reductase (DHFR) binds more than a thousand times tighter to NADPH than to NADP +. The origins of the difference in binding affinity to DHFR between NADPH and NADP + are investigated in the present study using experimental NMR data and hybrid density functional, B3LYP, calculations. Certain protein residues (Ala 6, Gln 7, Ile 13 and Gly 14) that are directly involved in hydrogen bonding with the nicotinamide carboxamide group show consistent differences in 1H and 15N chemical shift between NADPH and NADP + in a variety of ternary complexes. B3LYP calculations in model systems of protein-coenzyme interactions show differences in the H-bond geometry and differences in charge distribution between the oxidised and reduced forms of the nicotinamide ring. GIAO isotropic nuclear shieldings calculated for nuclei in these systems reproduce the experimentally observed trends in magnitudes and signs of the chemical shifts. The experimentally observed reduction in binding of NADP + compared with NADPH results partly from NADP + having to change its nicotinamide amide group from a cis- to a trans-conformation on binding and partly from the oxidised nicotinamide ring of NADP + being unable to take up its optimal hydrogen bonding geometry in its interactions with protein residues.

  4. The activity of uridine diphosphate-D-glucose: Nicotinamide-adenine dinucleotide oxidoreductase in cambial tissue and differentiating xylem isolated from sycamore trees.

    PubMed

    Rubery, P H

    1972-06-01

    The activity of UDPGlc: NAD oxidoreductase is measured in enzyme preparations obtained from sycamore cambium and xylem tissue. The activity of this enzyme is greater in xylem than in cambium whether expressed on a specific activity basis or on a per-cell basis. It is suggested that, in developing xylem, direct oxidation of UDPGlc may contribute significantly to the biosynthesis of polysaccharide precursors.

  5. Theoretical study on absorption and emission spectra of adenine analogues.

    PubMed

    Liu, Hongxia; Song, Qixia; Yang, Yan; Li, Yan; Wang, Haijun

    2014-04-01

    Fluorescent nucleoside analogues have attracted much attention in studying the structure and dynamics of nucleic acids in recent years. In the present work, we use theoretical calculations to investigate the structural and optical properties of four adenine analogues (termed as A1, A2, A3, and A4), and also consider the effects of aqueous solution and base pairing. The results show that the fluorescent adenine analogues can pair with thymine to form stable H-bonded WC base pairs. The excited geometries of both adenine analogues and WC base pairs are similar to the ground geometries. The absorption and emission maxima of adenine analogues are greatly red shifted compared with nature adenine, the oscillator strengths of A1 and A2 are stronger than A3 and A4 in both absorption and emission spectra. The calculated low-energy peaks in the absorption spectra are in good agreement with the experimental data. In general, the aqueous solution and base pairing can slightly red-shift both the absorption and emission maxima, and can increase the oscillator strengths of absorption spectra, but significantly decrease the oscillator strengths of A3 in emission spectra.

  6. DNA adenine hypomethylation leads to metabolic rewiring in Deinococcus radiodurans.

    PubMed

    Shaiwale, Nayana S; Basu, Bhakti; Deobagkar, Deepti D; Deobagkar, Dileep N; Apte, Shree K

    2015-08-01

    The protein encoded by DR_0643 gene from Deinococcus radiodurans was shown to be an active N-6 adenine-specific DNA methyltransferase (Dam). Deletion of corresponding protein reduced adenine methylation in the genome by 60% and resulted in slow-growth phenotype. Proteomic changes induced by DNA adenine hypomethylation were mapped by two-dimensional protein electrophoresis coupled with mass spectrometry. As compared to wild type D. radiodurans cells, at least 54 proteins were differentially expressed in Δdam mutant. Among these, 39 metabolic enzymes were differentially expressed in Δdam mutant. The most prominent change was DNA adenine hypomethylation induced de-repression of pyruvate dehydrogenase complex, E1 component (aceE) gene resulting in 10 fold increase in the abundance of corresponding protein. The observed differential expression profile of metabolic enzymes included increased abundance of enzymes involved in fatty acid and amino acid degradation to replenish acetyl Co-A and TCA cycle intermediates and diversion of phosphoenolpyruvate and pyruvate into amino acid biosynthesis, a metabolic rewiring attempt by Δdam mutant to restore energy generation via glycolysis-TCA cycle axis. This is the first report of DNA adenine hypomethylation mediated rewiring of metabolic pathways in prokaryotes.

  7. Molecular characterization of the gene cluster coxMSL encoding the molybdenum-containing carbon monoxide dehydrogenase of Oligotropha carboxidovorans.

    PubMed Central

    Schübel, U; Kraut, M; Mörsdorf, G; Meyer, O

    1995-01-01

    The CO dehydrogenase structural genes (cox) and orf4 are clustered in the transcriptional order coxM--> coxS--> coxL--> orf4 on the 128-kb megaplasmid pHCG3 of the carboxidotroph Oligotropha carboxidovorans OM5. Sequence analysis suggested association of molybdopterin cytosine dinucleotide and flavin adenine dinucleotide with CoxL and of the [2Fe-2S] clusters with CoxS. PMID:7721710

  8. Cerulenin-mediated apoptosis is involved in adenine metabolic pathway

    SciTech Connect

    Chung, Kyung-Sook; Sun, Nam-Kyu; Lee, Seung-Hee; Lee, Hyun-Jee; Choi, Shin-Jung; Kim, Sun-Kyung; Song, Ju-Hyun; Jang, Young-Joo; Song, Kyung-Bin; Yoo, Hyang-Sook; Simon, Julian . E-mail: jsimon@fhcrc.org; Won, Misun . E-mail: misun@kribb.re.kr

    2006-10-27

    Cerulenin, a fatty acid synthase (FAS) inhibitor, induces apoptosis of variety of tumor cells. To elucidate mode of action by cerulenin, we employed the proteomics approach using Schizosaccharomyces pombe. The differential protein expression profile of S. pombe revealed that cerulenin modulated the expressions of proteins involved in stresses and metabolism, including both ade10 and adk1 proteins. The nutrient supplementation assay demonstrated that cerulenin affected enzymatic steps transferring a phosphoribosyl group. This result suggests that cerulenin accumulates AMP and p-ribosyl-s-amino-imidazole carboxamide (AICAR) and reduces other necessary nucleotides, which induces feedback inhibition of enzymes and the transcriptional regulation of related genes in de novo and salvage adenine metabolic pathway. Furthermore, the deregulation of adenine nucleotide synthesis may interfere ribonucleotide reductase and cause defects in cell cycle progression and chromosome segregation. In conclusion, cerulenin induces apoptosis through deregulation of adenine nucleotide biosynthesis resulting in nuclear division defects in S. pombe.

  9. Possible prebiotic catalysts formed from adenine and aldehyde

    NASA Astrophysics Data System (ADS)

    Vergne, J.; Dumas, L.; Décout, J.-L.; Maurel, M.-C.

    2000-09-01

    Careful examination of the present metabolism and in vitro selection of various catalytic RNAs strongly support the "RNA World" hypothesis of the origin of life. However, in this scenario, the difficult prebiotic synthesis of ribose and consequently of nucleotides remain a major problem. In order to overcome this problem and obtain nucleoside analogs, we are investigating reactions of the nucleic acid base, adenine 1, with different aldehydes under presumably prebiotic conditions. In the reaction of adenine and pyruvaldehyde 2 in water, we report here the formation in high yield of two isomeric products. These compounds possessing alcohols functions as nucleosides result from condensation of two molecules of pyruvaldehyde on the 6-amino group of one adenine molecule. Their catalytic activities in the model hydrolysis of p-nitrophenylesters appeared interesting in the search of prebiotic catalysts.

  10. Structural basis for removal of adenine mispaired with 8-oxoguanine by MutY adenine DNA glycosylase.

    PubMed

    Fromme, J Christopher; Banerjee, Anirban; Huang, Susan J; Verdine, Gregory L

    2004-02-12

    The genomes of aerobic organisms suffer chronic oxidation of guanine to the genotoxic product 8-oxoguanine (oxoG). Replicative DNA polymerases misread oxoG residues and insert adenine instead of cytosine opposite the oxidized base. Both bases in the resulting A*oxoG mispair are mutagenic lesions, and both must undergo base-specific replacement to restore the original C*G pair. Doing so represents a formidable challenge to the DNA repair machinery, because adenine makes up roughly 25% of the bases in most genomes. The evolutionarily conserved enzyme adenine DNA glycosylase (called MutY in bacteria and hMYH in humans) initiates repair of A*oxoG to C*G by removing the inappropriately paired adenine base from the DNA backbone. A central issue concerning MutY function is the mechanism by which A*oxoG mispairs are targeted among the vast excess of A*T pairs. Here we report the use of disulphide crosslinking to obtain high-resolution crystal structures of MutY-DNA lesion-recognition complexes. These structures reveal the basis for recognizing both lesions in the A*oxoG pair and for catalysing removal of the adenine base. PMID:14961129

  11. Mature Microsatellites: Mechanisms Underlying Dinucleotide Microsatellite Mutational Biases in Human Cells

    PubMed Central

    Baptiste, Beverly A.; Ananda, Guruprasad; Strubczewski, Noelle; Lutzkanin, Andrew; Khoo, Su Jen; Srikanth, Abhinaya; Kim, Nari; Makova, Kateryna D.; Krasilnikova, Maria M.; Eckert, Kristin A.

    2013-01-01

    Dinucleotide microsatellites are dynamic DNA sequences that affect genome stability. Here, we focused on mature microsatellites, defined as pure repeats of lengths above the threshold and unlikely to mutate below it in a single mutational event. We investigated the prevalence and mutational behavior of these sequences by using human genome sequence data, human cells in culture, and purified DNA polymerases. Mature dinucleotides (≥10 units) are present within exonic sequences of >350 genes, resulting in vulnerability to cellular genetic integrity. Mature dinucleotide mutagenesis was examined experimentally using ex vivo and in vitro approaches. We observe an expansion bias for dinucleotide microsatellites up to 20 units in length in somatic human cells, in agreement with previous computational analyses of germ-line biases. Using purified DNA polymerases and human cell lines deficient for mismatch repair (MMR), we show that the expansion bias is caused by functional MMR and is not due to DNA polymerase error biases. Specifically, we observe that the MutSα and MutLα complexes protect against expansion mutations. Our data support a model wherein different MMR complexes shift the balance of mutations toward deletion or expansion. Finally, we show that replication fork progression is stalled within long dinucleotides, suggesting that mutational mechanisms within long repeats may be distinct from shorter lengths, depending on the biochemistry of fork resolution. Our work combines computational and experimental approaches to explain the complex mutational behavior of dinucleotide microsatellites in humans. PMID:23450065

  12. Mechanism of Action of a Flavin-Containing Monooxygenase

    SciTech Connect

    Eswaramoorthy,S.; Bonanno, J.; Burley, S.; Swaminathan, S.

    2006-01-01

    Elimination of nonnutritional and insoluble compounds is a critical task for any living organism. Flavin-containing monooxygenases (FMOs) attach an oxygen atom to the insoluble nucleophilic compounds to increase solubility and thereby increase excretion. Here we analyze the functional mechanism of FMO from Schizosaccharomyces pombe using the crystal structures of the wild type and protein-cofactor and protein-substrate complexes. The structure of the wild-type FMO revealed that the prosthetic group FAD is an integral part of the protein. FMO needs NADPH as a cofactor in addition to the prosthetic group for its catalytic activity. Structures of the protein-cofactor and protein-substrate complexes provide insights into mechanism of action. We propose that FMOs exist in the cell as a complex with a reduced form of the prosthetic group and NADPH cofactor, readying them to act on substrates. The 4{alpha}-hydroperoxyflavin form of the prosthetic group represents a transient intermediate of the monooxygenation process. The oxygenated and reduced forms of the prosthetic group help stabilize interactions with cofactor and substrate alternately to permit continuous enzyme turnover.

  13. KDM1 Class Flavin-Dependent Protein Lysine Demethylases

    PubMed Central

    Burg, Jonathan M.; Link, Jennifer E.; Morgan, Brittany S.; Heller, Frederick J.; Hargrove, Amanda E.; McCafferty, Dewey G.

    2015-01-01

    Flavin-dependent, lysine-specific protein demethylases (KDM1s) are a subfamily of amine oxidases that catalyze the selective posttranslational oxidative demethylation of methyllysine side chains within protein and peptide substrates. KDM1s participate in the widespread epigenetic regulation of both normal and disease state transcriptional programs. Their activities are central to various cellular functions, such as hematopoietic and neuronal differentiation, cancer proliferation and metastasis, and viral lytic replication and establishment of latency. Interestingly, KDM1s function as catalytic subunits within complexes with coregulatory molecules that modulate enzymatic activity of the demethylases and coordinate their access to specific substrates at distinct sites within the cell and chromatin. Although several classes of KDM1 -selective small molecule inhibitors have been recently developed, these pan-active site inhibition strategies lack the ability to selectively discriminate between KDM1 activity in specific, and occasionally opposing, functional contexts within these complexes. Here we review the discovery of this class of demethylases, their structures, chemical mechanisms, and specificity. Additionally, we review inhibition of this class of enzymes as well as emerging interactions with coregulatory molecules that regulate demethylase activity in highly specific functional contexts of biological and potential therapeutic importance. PMID:25787087

  14. Coenzyme Recognition and Gene Regulation by a Flavin Mononucleotide Riboswitch

    SciTech Connect

    Serganov, A.; Huang, L; Patel, D

    2009-01-01

    The biosynthesis of several protein cofactors is subject to feedback regulation by riboswitches. Flavin mononucleotide (FMN)-specific riboswitches also known as RFN elements, direct expression of bacterial genes involved in the biosynthesis and transport of riboflavin (vitamin B2) and related compounds. Here we present the crystal structures of the Fusobacterium nucleatum riboswitch bound to FMN, riboflavin and antibiotic roseoflavin. The FMN riboswitch structure, centred on an FMN-bound six-stem junction, does not fold by collinear stacking of adjacent helices, typical for folding of large RNAs. Rather, it adopts a butterfly-like scaffold, stapled together by opposingly directed but nearly identically folded peripheral domains. FMN is positioned asymmetrically within the junctional site and is specifically bound to RNA through interactions with the isoalloxazine ring chromophore and direct and Mg{sup 2+}-mediated contacts with the phosphate moiety. Our structural data, complemented by binding and footprinting experiments, imply a largely pre-folded tertiary RNA architecture and FMN recognition mediated by conformational transitions within the junctional binding pocket. The inherent plasticity of the FMN-binding pocket and the availability of large openings make the riboswitch an attractive target for structure-based design of FMN-like antimicrobial compounds. Our studies also explain the effects of spontaneous and antibiotic-induced deregulatory mutations and provided molecular insights into FMN-based control of gene expression in normal and riboflavin-overproducing bacterial strains.

  15. Mechanism of action of a flavin-containing monooxygenase

    PubMed Central

    Eswaramoorthy, Subramaniam; Bonanno, Jeffrey B.; Burley, Stephen K.; Swaminathan, Subramanyam

    2006-01-01

    Elimination of nonnutritional and insoluble compounds is a critical task for any living organism. Flavin-containing monooxygenases (FMOs) attach an oxygen atom to the insoluble nucleophilic compounds to increase solubility and thereby increase excretion. Here we analyze the functional mechanism of FMO from Schizosaccharomyces pombe using the crystal structures of the wild type and protein–cofactor and protein–substrate complexes. The structure of the wild-type FMO revealed that the prosthetic group FAD is an integral part of the protein. FMO needs NADPH as a cofactor in addition to the prosthetic group for its catalytic activity. Structures of the protein–cofactor and protein–substrate complexes provide insights into mechanism of action. We propose that FMOs exist in the cell as a complex with a reduced form of the prosthetic group and NADPH cofactor, readying them to act on substrates. The 4α-hydroperoxyflavin form of the prosthetic group represents a transient intermediate of the monooxygenation process. The oxygenated and reduced forms of the prosthetic group help stabilize interactions with cofactor and substrate alternately to permit continuous enzyme turnover. PMID:16777962

  16. Detection of electronically equivalent tautomers of adenine base: DFT study

    SciTech Connect

    Siddiqui, Shamoon Ahmad; Bouarissa, Nadir; Rasheed, Tabish; Al-Assiri, M.S.; Al-Hajry, A.

    2014-03-01

    Graphical abstract: - Highlights: • DFT calculations have been performed on adenine and its rare tautomer Cu{sup 2+} complexes. • Interaction of A-Cu{sup 2+} and rA-Cu{sup 2+} complexes with AlN modified fullerene (C{sub 60}) have been studied briefly. • It is found that AlN modified C{sub 60} could be used as a nanoscale sensor to detect these two A-Cu{sup 2+} and rA-Cu{sup 2+} complexes. - Abstract: In the present study, quantum chemical calculations were carried out to investigate the electronic structures and stabilities of adenine and its rare tautomer along with their Cu{sup 2+} complexes. Density Functional Theory (B3LYP method) was used in all calculations. The two Cu{sup 2+} complexes of adenine have almost similar energies and electronic structures; hence, their chemical differentiation is very difficult. For this purpose, interactions of these complexes with AlN modified fullerene (C{sub 60}) have been studied. Theoretical investigations reveal that AlN-doped C{sub 60} may serve as a potentially viable nanoscale sensor for detection of the two Cu{sup 2+} complexes of adenine.

  17. Thymine, adenine and lipoamino acid based gene delivery systems.

    PubMed

    Skwarczynski, Mariusz; Ziora, Zyta M; Coles, Daniel J; Lin, I-Chun; Toth, Istvan

    2010-05-14

    A novel class of thymine, adenine and lipoamino acid based non-viral carriers for gene delivery has been developed. Their ability to bind to DNA by hydrogen bonding was confirmed by NMR diffusion, isothermal titration calorimetry and transmission electron microscopy experiments.

  18. PolyAdenine cryogels for fast and effective RNA purification.

    PubMed

    Köse, Kazım; Erol, Kadir; Özgür, Erdoğan; Uzun, Lokman; Denizli, Adil

    2016-10-01

    Cryogels are used effectively for many diverse applications in a variety of fields. The isolation or purification of RNA, one of the potential utilizations for cryogels, is crucial due to their vital roles such as encoding, decoding, transcription and translation, and gene expression. RNA principally exists within every living thing, but their tendency to denaturation easily is still the most challenging issue. Herein, we aimed to develop adenine incorporated polymeric cryogels as an alternative sorbent for cost-friendly and fast RNA purification with high capacity. For this goal, we synthesized the polymerizable derivative of adenine called as adenine methacrylate (AdeM) through the substitution reaction between adenine and methacryloyl chloride. Then, 2-hydroxyethyl methacrylate (HEMA)-based cryogels were prepared in a partially frozen aqueous medium by copolymerization of monomers, AdeM, and HEMA. The cryogels were characterized by using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), surface area measurements, thermogravimetric analysis (TGA), and swelling tests. RNA adsorption experiments were performed via batch system while varying different conditions including pH, initial RNA concentration, temperature, and interaction time. We achieved high RNA adsorption capacity of cryogels, with the swelling ratio around 510%, as 11.86mg/g. The cryogels might be reused at least five times without significant decrease in adsorption capacity.

  19. PolyAdenine cryogels for fast and effective RNA purification.

    PubMed

    Köse, Kazım; Erol, Kadir; Özgür, Erdoğan; Uzun, Lokman; Denizli, Adil

    2016-10-01

    Cryogels are used effectively for many diverse applications in a variety of fields. The isolation or purification of RNA, one of the potential utilizations for cryogels, is crucial due to their vital roles such as encoding, decoding, transcription and translation, and gene expression. RNA principally exists within every living thing, but their tendency to denaturation easily is still the most challenging issue. Herein, we aimed to develop adenine incorporated polymeric cryogels as an alternative sorbent for cost-friendly and fast RNA purification with high capacity. For this goal, we synthesized the polymerizable derivative of adenine called as adenine methacrylate (AdeM) through the substitution reaction between adenine and methacryloyl chloride. Then, 2-hydroxyethyl methacrylate (HEMA)-based cryogels were prepared in a partially frozen aqueous medium by copolymerization of monomers, AdeM, and HEMA. The cryogels were characterized by using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), surface area measurements, thermogravimetric analysis (TGA), and swelling tests. RNA adsorption experiments were performed via batch system while varying different conditions including pH, initial RNA concentration, temperature, and interaction time. We achieved high RNA adsorption capacity of cryogels, with the swelling ratio around 510%, as 11.86mg/g. The cryogels might be reused at least five times without significant decrease in adsorption capacity. PMID:27434154

  20. Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase

    SciTech Connect

    Kamat, S.S.; Swaminathan, S.; Bagaria, A.; Kumaran, D.; Holmes-Hampton, G. P.; Fan, H.; Sali, A.; Sauder, J. M.; Burley, S. K.; Lindahl, P. A.; Raushel, F. M.

    2011-03-22

    Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (k{sub cat} = 2.0 s{sup -1}; k{sub cat}/K{sub m} = 2.5 x 10{sup 3} M{sup -1} s{sup -1}). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn{sup 2+} prior to induction, the purified enzyme was substantially more active for the deamination of adenine with kcat and kcat/Km values of 200 s{sup -1} and 5 x 10{sup 5} M{sup -1} s{sup -1}, respectively. The apoenzyme was prepared and reconstituted with Fe{sup 2+}, Zn{sup 2+}, or Mn{sup 2+}. In each case, two enzyme equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member of the deaminase subfamily of the amidohydrolase superfamily to utilize a binuclear metal center for the catalysis of a deamination reaction. [Fe{sup II}/Fe{sup II}]-ADE was oxidized to [Fe{sup III}/Fe{sup III}]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe{sup III}/Fe{sup III}]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential for catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Moessbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 {angstrom} resolution, and adenine was modeled into the active site on the basis of homology to other members of the amidohydrolase superfamily. On the basis of the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH-rate profiles, and solvent viscosity were utilized to propose a chemical reaction mechanism and the

  1. Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase

    SciTech Connect

    S Kamat; A Bagaria; D Kumaran; G Holmes-Hampton; H Fan; A Sali; J Sauder; S Burley; P Lindahl; et. al.

    2011-12-31

    Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (k{sub cat} = 2.0 s{sup -1}; k{sub cat}/K{sub m} = 2.5 x 10{sup 3} M{sup -1} s{sup -1}). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn{sup 2+} prior to induction, the purified enzyme was substantially more active for the deamination of adenine with k{sub cat} and k{sub cat}/K{sub m} values of 200 s{sup -1} and 5 x 10{sup 5} M{sup -1} s{sup -1}, respectively. The apoenzyme was prepared and reconstituted with Fe{sup 2+}, Zn{sup 2+}, or Mn{sup 2+}. In each case, two enzyme equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member of the deaminase subfamily of the amidohydrolase superfamily to utilize a binuclear metal center for the catalysis of a deamination reaction. [Fe{sup II}/Fe{sup II}]-ADE was oxidized to [Fe{sup III}/Fe{sup III}]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe{sup III}/Fe{sup III}]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential for catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Moessbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 {angstrom} resolution, and adenine was modeled into the active site on the basis of homology to other members of the amidohydrolase superfamily. On the basis of the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH-rate profiles, and solvent viscosity were utilized to propose a chemical reaction

  2. Dinucleotide Weight Matrices for Predicting Transcription Factor Binding Sites: Generalizing the Position Weight Matrix

    PubMed Central

    Siddharthan, Rahul

    2010-01-01

    Background Identifying transcription factor binding sites (TFBS) in silico is key in understanding gene regulation. TFBS are string patterns that exhibit some variability, commonly modelled as “position weight matrices” (PWMs). Though convenient, the PWM has significant limitations, in particular the assumed independence of positions within the binding motif; and predictions based on PWMs are usually not very specific to known functional sites. Analysis here on binding sites in yeast suggests that correlation of dinucleotides is not limited to near-neighbours, but can extend over considerable gaps. Methodology/Principal Findings I describe a straightforward generalization of the PWM model, that considers frequencies of dinucleotides instead of individual nucleotides. Unlike previous efforts, this method considers all dinucleotides within an extended binding region, and does not make an attempt to determine a priori the significance of particular dinucleotide correlations. I describe how to use a “dinucleotide weight matrix” (DWM) to predict binding sites, dealing in particular with the complication that its entries are not independent probabilities. Benchmarks show, for many factors, a dramatic improvement over PWMs in precision of predicting known targets. In most cases, significant further improvement arises by extending the commonly defined “core motifs” by about 10bp on either side. Though this flanking sequence shows no strong motif at the nucleotide level, the predictive power of the dinucleotide model suggests that the “signature” in DNA sequence of protein-binding affinity extends beyond the core protein-DNA contact region. Conclusion/Significance While computationally more demanding and slower than PWM-based approaches, this dinucleotide method is straightforward, both conceptually and in implementation, and can serve as a basis for future improvements. PMID:20339533

  3. Conformational Switching in the Fungal Light Sensor Vivid

    SciTech Connect

    Zoltowski,B.; Schwerdtgeger, C.; Widom, J.; Loros, J.; Bilwes, A.; Dunlap, J.; Crane, B.

    2007-01-01

    The Neurospora crassa photoreceptor Vivid tunes blue-light responses and modulates gating of the circadian clock. Crystal structures of dark-state and light-state Vivid reveal a light, oxygen, or voltage Per-Arnt-Sim domain with an unusual N-terminal cap region and a loop insertion that accommodates the flavin cofactor. Photoinduced formation of a cystein-flavin adduct drives flavin protonation to induce an N-terminal conformational change. A cysteine-to-serine substitution remote from the flavin adenine dinucleotide binding site decouples conformational switching from the flavin photocycle and prevents Vivid from sending signals in Neurospora. Key elements of this activation mechanism are conserved by other photosensors such as White Collar-1, ZEITLUPE, ENVOY, and flavin-binding, kelch repeat, F-BOX 1 (FKF1).

  4. A major integral protein of the plant plasma membrane binds flavin.

    PubMed

    Lorenz, Astrid; Kaldenhoff, Ralf; Hertel, Rainer

    2003-05-01

    Abundant flavin binding sites have been found in membranes of plants and fungi. With flavin mononucleotide-agarose affinity columns, riboflavin-binding activity from microsomes of Cucurbita pepoL. hypocotyls was purified and identified as a specific PIP1-homologous protein of the aquaporin family. Sequences such as gi|2149955 in Phaseolus vulgaris, PIP1b of Arabidopsis thaliana, and NtAQP1 of tobacco are closely related. The identification as a riboflavin-binding protein was confirmed by binding tests with an extract of Escherichia coli cells expressing the tobacco NtAQP1 as well as leaves of transgenic tobacco plants that overexpress NtAQP1 or were inhibited in PIP1 expression by antisense constructs. When binding was assayed in the presence of dithionite, the reduced flavin formed a relatively stable association with the protein. Upon dilution under oxidizing conditions, the adduct was resolved, and free flavin reappeared with a half time of about 30 min. Such an association can also be induced photochemically, with oxidized flavin by blue light at 450 nm, in the presence of an electron donor. Several criteria, localization in the plasma membrane, high abundance, affinity to roseoflavin, and photochemistry, argue for a role of the riboflavin-binding protein PIP1 as a photoreceptor. PMID:12768338

  5. Trichomonas vaginalis flavin reductase 1 and its role in metronidazole resistance.

    PubMed

    Leitsch, David; Janssen, Brian D; Kolarich, Daniel; Johnson, Patricia J; Duchêne, Michael

    2014-01-01

    The enzyme flavin reductase 1 (FR1) from Trichomonas vaginalis, formerly known as NADPH oxidase, was isolated and identified. Flavin reductase is part of the antioxidative defence in T. vaginalis and indirectly reduces molecular oxygen to hydrogen peroxide via free flavins. Importantly, a reduced or absent flavin reductase activity has been reported in metronidazole-resistant T. vaginalis, resulting in elevated intracellular oxygen levels and futile cycling of metronidazole. Interestingly, FR1 has no close homologue in any other sequenced genome, but seven full-length and three truncated isoforms exist in the T. vaginalis genome. However, out of these, only FR1 has an affinity for flavins, i.e. FMN, FAD and riboflavin, which is high enough to be of physiological relevance. Although there are no relevant changes in the gene sequence or any alterations of the predicted FR1-mRNA structure in any of the strains studied, FR1 is not expressed in highly metronidazole-resistant strains. Transfection of a metronidazole-resistant clinical isolate (B7268), which does not express any detectable amounts of FR, with a plasmid bearing a functional FR1 gene nearly completely restored metronidazole sensitivity. Our results indicate that FR1 has a significant role in the emergence of metronidazole resistance in T. vaginalis.

  6. Flavin Derivatives with Tailored Redox Properties: Synthesis, Characterization, and Electrochemical Behavior.

    PubMed

    Kormányos, Attila; Hossain, Mohammad S; Ghadimkhani, Ghazaleh; Johnson, Joe J; Janáky, Csaba; de Tacconi, Norma R; Foss, Frank W; Paz, Yaron; Rajeshwar, Krishnan

    2016-06-27

    This study establishes structure-property relationships for four synthetic flavin molecules as bioinspired redox mediators in electro- and photocatalysis applications. The studied flavin compounds were disubstituted with polar substituents at the N1 and N3 positions (alloxazine) or at the N3 and N10 positions (isoalloxazines). The electrochemical behavior of one such synthetic flavin analogue was examined in detail in aqueous solutions of varying pH in the range from 1 to 10. Cyclic voltammetry, used in conjunction with hydrodynamic (rotating disk electrode) voltammetry, showed quasi-reversible behavior consistent with freely diffusing molecules and an overall global 2e(-) , 2H(+) proton-coupled electron transfer scheme. UV/Vis spectroelectrochemical data was also employed to study the pH-dependent electrochemical behavior of this derivative. Substituent effects on the redox behavior were compared and contrasted for all the four compounds, and visualized within a scatter plot framework to afford comparison with prior knowledge on mostly natural flavins in aqueous media. Finally, a preliminary assessment of one of the synthetic flavins was performed of its electrocatalytic activity toward dioxygen reduction as a prelude to further (quantitative) studies of both freely diffusing and tethered molecules on various electrode surfaces. PMID:27243969

  7. Resolution of strongly competitive product channels with optimal dynamic discrimination: Application to flavins

    NASA Astrophysics Data System (ADS)

    Roslund, Jonathan; Roth, Matthias; Guyon, Laurent; Boutou, Véronique; Courvoisier, Francois; Wolf, Jean-Pierre; Rabitz, Herschel

    2011-01-01

    Fundamental molecular selectivity limits are probed by exploiting laser-controlled quantum interferences for the creation of distinct spectral signatures in two flavin molecules, erstwhile nearly indistinguishable via steady-state methods. Optimal dynamic discrimination (ODD) uses optimally shaped laser fields to transiently amplify minute molecular variations that would otherwise go unnoticed with linear absorption and fluorescence techniques. ODD is experimentally demonstrated by combining an optimally shaped UV pump pulse with a time-delayed, fluorescence-depleting IR pulse for discrimination amongst riboflavin and flavin mononucleotide in aqueous solution, which are structurally and spectroscopically very similar. Closed-loop, adaptive pulse shaping discovers a set of UV pulses that induce disparate responses from the two flavins and allows for concomitant flavin discrimination of ˜16σ. Additionally, attainment of ODD permits quantitative, analytical detection of the individual constituents in a flavin mixture. The successful implementation of ODD on quantum systems of such high complexity bodes well for the future development of the field and the use of ODD techniques in a variety of demanding practical applications.

  8. BluB cannibalizes flavin to form the lower ligand of vitamin B12

    PubMed Central

    Taga, Michiko E.; Larsen, Nicholas A.; Howard-Jones, Annaleise R.; Walsh, Christopher T.; Walker, Graham C.

    2009-01-01

    Vitamin B12 (cobalamin) is among the largest known non-polymeric natural products, and the only vitamin synthesized exclusively by microorganisms1. The biosynthesis of the lower ligand of vitamin B12, 5,6-dimethylbenzimidazole (DMB), is poorly understood1–3. Recently, we discovered that a Sinorhizobium meliloti gene, bluB, is necessary for DMB biosynthesis4. Here we show that BluB triggers the unprecedented fragmentation and contraction of the bound flavin mononucleotide cofactor and cleavage of the ribityl tail to form DMB and D-erythrose 4-phosphate. Our structural analysis shows that BluB resembles an NAD(P)H-flavin oxidoreductase, except that its unusually tight binding pocket accommodates flavin mononucleotide but not NAD(P)H. We characterize crystallographically an early intermediate along the reaction coordinate, revealing molecular oxygen poised over reduced flavin. Thus, BluB isolates and directs reduced flavin to activate molecular oxygen for its own cannibalization. This investigation of the biosynthesis of DMB provides clarification of an aspect of vitamin B12 that was otherwise incomplete, and may contribute to a better understanding of vitamin B12-related disease. PMID:17377583

  9. Flavin Charge Transfer Transitions Assist DNA Photolyase Electron Transfer

    NASA Astrophysics Data System (ADS)

    Skourtis, Spiros S.; Prytkova, Tatiana; Beratan, David N.

    2007-12-01

    This contribution describes molecular dynamics, semi-empirical and ab-initio studies of the primary photo-induced electron transfer reaction in DNA photolyase. DNA photolyases are FADH--containing proteins that repair UV-damaged DNA by photo-induced electron transfer. A DNA photolyase recognizes and binds to cyclobutatne pyrimidine dimer lesions of DNA. The protein repairs a bound lesion by transferring an electron to the lesion from FADH-, upon photo-excitation of FADH- with 350-450 nm light. We compute the lowest singlet excited states of FADH- in DNA photolyase using INDO/S configuration interaction, time-dependent density-functional, and time-dependent Hartree-Fock methods. The calculations identify the lowest singlet excited state of FADH- that is populated after photo-excitation and that acts as the electron donor. For this donor state we compute conformationally-averaged tunneling matrix elements to empty electron-acceptor states of a thymine dimer bound to photolyase. The conformational averaging involves different FADH--thymine dimer confromations obtained from molecular dynamics simulations of the solvated protein with a thymine dimer docked in its active site. The tunneling matrix element computations use INDO/S-level Green's function, energy splitting, and Generalized Mulliken-Hush methods. These calculations indicate that photo-excitation of FADH- causes a π→π* charge-transfer transition that shifts electron density to the side of the flavin isoalloxazine ring that is adjacent to the docked thymine dimer. This shift in electron density enhances the FADH--to-dimer electronic coupling, thus inducing rapid electron transfer.

  10. Characterization of Flavin-Containing Opine Dehydrogenase from Bacteria.

    PubMed

    Watanabe, Seiya; Sueda, Rui; Fukumori, Fumiyasu; Watanabe, Yasuo

    2015-01-01

    Opines, in particular nopaline and octopine, are specific compounds found in crown gall tumor tissues induced by infections with Agrobacterium species, and are synthesized by well-studied NAD(P)H-dependent dehydrogenases (synthases), which catalyze the reductive condensation of α-ketoglutarate or pyruvate with L-arginine. The corresponding genes are transferred into plant cells via a tumor-inducing (Ti) plasmid. In addition to the reverse oxidative reaction(s), the genes noxB-noxA and ooxB-ooxA are considered to be involved in opine catabolism as (membrane-associated) oxidases; however, their properties have not yet been elucidated in detail due to the difficulties associated with purification (and preservation). We herein successfully expressed Nox/Oox-like genes from Pseudomonas putida in P. putida cells. The purified protein consisted of different α-, β-, and γ-subunits encoded by the OdhA, OdhB, and OdhC genes, which were arranged in tandem on the chromosome (OdhB-C-A), and exhibited dehydrogenase (but not oxidase) activity toward nopaline in the presence of artificial electron acceptors such as 2,6-dichloroindophenol. The enzyme contained FAD, FMN, and [2Fe-2S]-iron sulfur as prosthetic groups. On the other hand, the gene cluster from Bradyrhizobium japonicum consisted of OdhB1-C-A-B2, from which two proteins, OdhAB1C and OdhAB2C, appeared through the assembly of each β-subunit together with common α- and γ-subunits. A poor phylogenetic relationship was detected between OdhB1 and OdhB2 in spite of them both functioning as octopine dehydrogenases, which provided clear evidence for the acquisition of novel functions by "subunit-exchange". To the best of our knowledge, this is the first study to have examined flavin-containing opine dehydrogenase. PMID:26382958

  11. Biotransformation of lepidocrocite in the presence of quinones and flavins

    NASA Astrophysics Data System (ADS)

    Bae, Sungjun; Lee, Woojin

    2013-08-01

    This study investigated the bioreduction of lepidocrocite (γ-FeIIIOOH) and its mineral transformation in the presence of exogenous (quinones) and endogenous (flavins) electron transfer mediators (ETMs) at low concentrations of the ETMs and bacterial cells (Shewanella putrefaciens CN32). It is very important to investigate the bioreduction of lepidocrocite in the presence of different ETMs because biotransformation of Fe(III)-containing minerals can be stimulated by ETMs and affect fate and transport of contaminants in contaminated environments. In the absence of phosphate, green rust formation was observed with fast Fe(II) production rate (0.44-0.56 mM d-1) during the bioreduction of lepidocrocite with exogenous ETMs, while goethite formed at slow Fe(II) production rate (0.24-0.29 mM d-1) with endogenous ETMs. In the presence of phosphate, formation of green rust and vivianite was observed with fast Fe(II) production rate (0.54-0.74 mM d-1) during the bioreduction of lepidocrocite with exogenous ETMs, while vivianite formed at moderate Fe(II) production rate (0.36-0.40 mM d-1) with endogenous ETMs. Vivianite formed in all experimental cases with phosphate in a broad range of Fe(II) production rates (0.23-0.74 mM d-1). Our results (1) suggest that exogenous and endogenous ETMs can significantly but differently affect the biotransformation of lepidocrocite, especially at low concentrations of the ETMs and bacterial cells, (2) highlight the importance of Fe(II) production rate to determine the formation of specific biogenic minerals, (3) provide additional evidence that phosphate can significantly affect the bioreduction rate and the mineral transformation, and (4) help to understand the basic knowledge about complex interactions among microbial cell, soil mineral, and ETM in natural environments and engineered systems.

  12. Reductive Dissolution of Goethite and Hematite by Reduced Flavins

    SciTech Connect

    Shi, Zhi; Zachara, John M.; Wang, Zheming; Shi, Liang; Fredrickson, Jim K.

    2013-10-02

    The abiotic reductive dissolution of goethite and hematite by the reduced forms of flavin mononucleotide (FMNH2) and riboflavin (RBFH2), electron transfer mediators (ETM) secreted by the dissimilatory iron-reducing bacterium Shewanella, was investigated under stringent anaerobic conditions. In contrast to the rapid redox reaction rate observed for ferrihydrite and lepidocrocite (Shi et al., 2012), the reductive dissolution of crystalline goethite and hematite was slower, with the extent of reaction limited by the thermodynamic driving force at circumneutral pH. Both the initial reaction rate and reaction extent increased with decreasing pH. On a unit surface area basis, goethite was less reactive than hematite between pH 4.0 and 7.0. AH2DS, the reduced form of the well-studied synthetic ETM anthraquinone-2,6-disulfonate (AQDS), yielded higher rates than FMNH2 under most reaction conditions, despite the fact that FMNH2 was a more effective reductant than AH2DS for ferryhydrite and lepidocrocite. Two additional model compounds, methyl viologen and benzyl viologen, were investigated under similar reaction conditions to explore the relationship between reaction rate and thermodynamic properties. Relevant kinetic data from the literature were also included in the analysis to span a broad range of half-cell potentials. Other conditions being equal, the surface area normalized initial reaction rate (ra) increased as the redox potential of the reductant became more negative. A non-linear, parabolic relationship was observed between log ra and the redox potential for eight reducants at pH 7.0, as predicted by Marcus theory for electron transfer. When pH and reductant concentration were fixed, log ra was positively correlated to the redox potential of four Fe(III) oxides over a wide pH range, following a non-linear parabolic relationship as well.

  13. Remaining challenges in cellular flavin cofactor homeostasis and flavoprotein biogenesis

    NASA Astrophysics Data System (ADS)

    Giancaspero, Teresa Anna; Colella, Matilde; Brizio, Carmen; Difonzo, Graziana; Fiorino, Giuseppina Maria; Leone, Piero; Brandsch, Roderich; Bonomi, Francesco; Iametti, Stefania; Barile, Maria

    2015-04-01

    The primary role of the water-soluble vitamin B2 (riboflavin) in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in energetic metabolism, epigenetics, protein folding, as well as in a number of diverse regulatory processes. The problem of localisation of flavin cofactor synthesis events and in particular of the FAD synthase (EC 2.7.7.2) in HepG2 cells is addressed here by confocal microscopy in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalysed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesising activity, hFADS is able to operate as a FAD "chaperone". The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear or a mitochondrial enzyme that is lysine specific demethylase 1 (LSD1, EC 1.-.-.-) and dimethylglycine dehydrogenase (Me2GlyDH, EC 1.5.8.4), respectively which carry out similar reactions of oxidative demethylation, assisted by tetrahydrofolate used to form 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells.

  14. Characterization of Flavin-Containing Opine Dehydrogenase from Bacteria

    PubMed Central

    Watanabe, Seiya; Sueda, Rui; Fukumori, Fumiyasu; Watanabe, Yasuo

    2015-01-01

    Opines, in particular nopaline and octopine, are specific compounds found in crown gall tumor tissues induced by infections with Agrobacterium species, and are synthesized by well-studied NAD(P)H-dependent dehydrogenases (synthases), which catalyze the reductive condensation of α-ketoglutarate or pyruvate with L-arginine. The corresponding genes are transferred into plant cells via a tumor-inducing (Ti) plasmid. In addition to the reverse oxidative reaction(s), the genes noxB-noxA and ooxB-ooxA are considered to be involved in opine catabolism as (membrane-associated) oxidases; however, their properties have not yet been elucidated in detail due to the difficulties associated with purification (and preservation). We herein successfully expressed Nox/Oox-like genes from Pseudomonas putida in P. putida cells. The purified protein consisted of different α-, β-, and γ-subunits encoded by the OdhA, OdhB, and OdhC genes, which were arranged in tandem on the chromosome (OdhB-C-A), and exhibited dehydrogenase (but not oxidase) activity toward nopaline in the presence of artificial electron acceptors such as 2,6-dichloroindophenol. The enzyme contained FAD, FMN, and [2Fe-2S]-iron sulfur as prosthetic groups. On the other hand, the gene cluster from Bradyrhizobium japonicum consisted of OdhB1-C-A-B2, from which two proteins, OdhAB1C and OdhAB2C, appeared through the assembly of each β-subunit together with common α- and γ-subunits. A poor phylogenetic relationship was detected between OdhB1 and OdhB2 in spite of them both functioning as octopine dehydrogenases, which provided clear evidence for the acquisition of novel functions by “subunit-exchange”. To the best of our knowledge, this is the first study to have examined flavin-containing opine dehydrogenase. PMID:26382958

  15. Copper-Adenine Complex Catalyst for O2 Production from

    NASA Astrophysics Data System (ADS)

    Vergne, Jacques; Bruston, F.; Calvayrac, R.; Grajcar, L.; Baron, M.-H.; Maurel, M.-C.

    The advent of oxygen-evolving photosynthesis is one of the central event in the development of life on earth. The early atmosphere has been midly reducing or neutral in overall redox balance and water photolysis by UV light can produce hydrogen peroxide. Before oxidation of water, intermediate stages are proposed in which H_2^O_2 was oxidized. The oxidation of H_2^O_2 to oxygen can be carried out by a modestly oxidizing species in which a metal-catalase like enzyme could extract electrons from H_2^O_2 producing the first oxygen-evolving complex. After what, modern photosynthesis with chlorophyll, to help transform H_2^O in O_2 was ready to come to light. In preliminary UV studies we were able to show that [Cu(adenine)2] system, containing copper coordinated to nitrogen activates H_2^O_2 disappearance. This was confirmed with the help of Raman and polarographic studies. Raman spectroscopy shows the formation of [Cu(adenine)2] complex in solution, quantifies H_2^O_2 consumption, polarography quantifies O_2 production. In both cases CuCl_2 addition entails H_2^O_2 disappearance. Without adenine, Cu_2^+ has only a weak catalytic effect. The molar activity of the [Cu(adenine)2] complex is much larger and concentration dependent. We emphasize that Cu(adenine)2 may have mimicked enzyme properties in the first stage of life evolution, in order to split H_2^O_2 into O_2 and H_2^O. Moreover, diluted copper and adenine, in small ephemeral prebiotic ponds , could have preserved biologically active entities from H_2^O_2 damage via dual properties: catalyzing H_2^O_2 disproportionation and also directly acting as a reductant complex. Finally, the present Mars surface is considered to be both reactive and embedded with oxydants. As it has been shown that the depth of diffusion for H_2^O_2 is less than 3 meters, it is important to study all the ways of H_2^O_2 consumption.

  16. Evidence that biliverdin-IX beta reductase and flavin reductase are identical.

    PubMed Central

    Shalloe, F; Elliott, G; Ennis, O; Mantle, T J

    1996-01-01

    A search of the database shows that human biliverdin-IX beta reductase and flavin reductase are identical. We have isolated flavin reductase from bovine erythrocytes and show that the activity co-elutes with biliverdin-IX beta reductase. Preparations of the enzyme that are electrophoretically homogeneous exhibit both flavin reductase and biliverdin-IX beta reductase activities; however, they are not capable of catalysing the reduction of biliverdin-IX alpha. Although there is little obvious sequence identity between biliverdin-IX alpha reductase (BVR-A) and biliverdin-IX beta reductase (BVR-B), they do show weak immunological cross-reactivity. Both enzymes bind to 2',5'-ADP-Sepharose. PMID:8687377

  17. Cyclic (di)nucleotides: the common language shared by microbe and host.

    PubMed

    Gao, Juyi; Tao, Jianli; Liang, Weili; Jiang, Zhengfan

    2016-04-01

    Fluency in a common language allows individuals to convey information and carry out complex activities that otherwise would be difficult or even impossible without the benefit of shared communication. Cyclic (di)nucleotides have recently been recognized as such an accessible language understood by both microbe and the host, ever since remarkable progresses have revealed the molecular details of these nucleotide second messengers used in cellular communication systems. Though undergoing separate evolutionary pathways in prokaryotes and eukaryotes, cyclic (di)nucleotides enable microbes to influence host cells immediately and fiercely by modulating a variety of cellular activities. Here we highlight recent insights in cyclic (di)nucleotides and focus on the balancing of these indispensable signaling molecules by synthases and phosphodiesterases.

  18. Typing dinucleotide repeat loci using microplate array diagonal gel electrophoresis: proof of principle.

    PubMed

    Rodríguez, Santiago; Chen, Xiao-He; Day, Ian N M

    2004-04-01

    Polymorphic dinucleotide repeat loci ('microsatellite markers') are found in varying abundance throughout the genomes of most organisms. They have been extensively used for genetic studies, but conventional techniques used for their genotyping require sophisticated equipment. Microplate array diagonal gel electrophoresis (MADGE) has previously been extended to economical high-throughput genotyping of trinucleotide and tetranucleotide microsatellite amplicons. However, the capability of this technique to resolve the alleles of dinucleotide repeat loci has not been explored previously. Here we show that a modified microsatellite-MADGE approach can provide sufficient resolution for dinucleotide repeat typing. This enables economical and convenient set up for analysis of single markers in many samples in parallel, suitable, for example, for population association studies.

  19. Synthesis of 10-Ethyl Flavin: A Multistep Synthesis Organic Chemistry Laboratory Experiment for Upper-Division Undergraduate Students

    ERIC Educational Resources Information Center

    Sichula, Vincent A.

    2015-01-01

    A multistep synthesis of 10-ethyl flavin was developed as an organic chemistry laboratory experiment for upper-division undergraduate students. Students synthesize 10-ethyl flavin as a bright yellow solid via a five-step sequence. The experiment introduces students to various hands-on experimental organic synthetic techniques, such as column…

  20. UbiX is a flavin prenyltransferase required for bacterial ubiquinone biosynthesis

    PubMed Central

    White, Mark D.; Payne, Karl A.P.; Fisher, Karl; Marshall, Stephen A.; Parker, David; Rattray, Nicholas J.W.; Trivedi, Drupad K.; Goodacre, Royston; Rigby, Stephen E.J.; Scrutton, Nigel S.; Hay, Sam; Leys, David

    2016-01-01

    Ubiquinone, or coenzyme Q, is a ubiquitous lipid-soluble redox cofactor that is an essential component of electron transfer chains1. Eleven genes have been implicated in bacterial ubiquinone biosynthesis, including ubiX and ubiD, which are responsible for decarboxylation of the 3-octaprenyl-4-hydroxybenzoate precursor2. Despite structural and biochemical characterization of UbiX as an FMN-binding protein, no decarboxylase activity has been detected3–4. We report here that UbiX produces a novel flavin-derived cofactor required for the decarboxylase activity of UbiD5. UbiX acts as a flavin prenyltransferase, linking a dimethylallyl moiety to the flavin N5 and C6 atoms. This adds a fourth non-aromatic ring to the flavin isoalloxazine group. In contrast to other prenyltransferases6–7, UbiX is metal-independent and requires dimethylallyl-monophosphate as substrate. Kinetic crystallography reveals that the prenyl transferase mechanism of UbiX resembles that of the terpene synthases8. The active site environment is dominated by π-systems, which assist phosphate-C1’ bond breakage following FMN reduction, leading to formation of the N5-C1’ bond. UbiX then acts as a chaperone for adduct reorientation, via transient carbocation species, leading ultimately to formation of the dimethylallyl C3’-C6 bond. The study establishes the mechanism for formation of a new flavin-derived cofactor, extending both flavin and terpenoid biochemical repertoire. PMID:26083743

  1. Resolution of overlapping skin auto-fluorescence for development of non-invasive applications

    NASA Astrophysics Data System (ADS)

    Su, Yu-Zheng; Lin, Li-Wu; Chen, Chuen-Yau; Hung, Min-Wei; Huang, Kuo-Cheng

    2010-08-01

    Skin auto-fluorescence spectra can provide useful biological information, but the obtained spectrum is overlapped and is difficult to distinguish each contributed component. We applied the genetic algorithm to decompose the overlapping spectrum. First, we simulate the overlapping spectral to confirm our feasible algorithm. The skin auto-fluorescence spectra were obtained from the normal human skin with 337 nm excitation light source. The nicotinamide adenine dinucleotid (NADH) and flavin adenine dinucleotide (FAD) are accurately decomposed and demonstrated. The developed algorithm can be widely applied to achieve qualitative and quantitative analysis for overlapping spectra.

  2. Reductive dissolution of goethite and hematite by reduced flavins

    NASA Astrophysics Data System (ADS)

    Shi, Zhi; Zachara, John M.; Wang, Zheming; Shi, Liang; Fredrickson, Jim K.

    2013-11-01

    The abiotic reductive dissolution of goethite and hematite by the reduced forms of flavin mononucleotide (FMNH2) and riboflavin (RBFH2), electron shuttles secreted by the dissimilatory iron-reducing bacterium Shewanella, was investigated under stringent anaerobic conditions. At pH 7.0, the reductive dissolution rates of goethite were 3.5 μmoles m-2 h-1 by 50 μM FMNH2 and 0.27 μmoles m-2 h-1 by 50 μM RBFH2; the reductive dissolution rates of hematite were 29 μmoles m-2 h-1 by 50 μM FMNH2 and 151 μmoles m-2 h-1 by 50 μM RBFH2. The extent of reaction was limited by the thermodynamic driving force at circumneutral pH. Both the initial reaction rate and reaction extent increased with decreasing pH. On a unit surface area basis, goethite was less reactive than hematite between pH 4.0 and 7.0. AH2DS, the reduced form of the well-studied synthetic electron shuttle anthraquinone-2,6-disulfonate, yielded higher rates than FMNH2 under most reaction conditions. Two additional model compounds, methyl viologen and benzyl viologen, were investigated under similar reaction conditions to explore the relationship between reaction rate and thermodynamic properties. Relevant kinetic data from the literature were also included in the analysis to span a large range in half-cell potentials. Other conditions being equal, the surface area normalized initial reaction rate (ra) increased as the redox potential of the reductant became more negative. A non-linear relationship was observed between log ra and the redox potential for eight reductants at pH 7.0. When pH and reductant concentration were fixed, log ra was positively correlated to the redox potential of four Fe(III) oxides over a wide pH range, following a non-linear relationship as well.

  3. Electrochemical sensor with flavin-containing monooxygenase for triethylamine solution.

    PubMed

    Saito, Hirokazu; Shirai, Takeshi; Kudo, Hiroyuki; Mitsubayashi, Kohji

    2008-06-01

    A bioelectronic sensor for triethylamine (TEA) was developed with a flavin-containing monooxygenase type 3 (FMO-3). The TEA biosensor consisted of a Clark-type dissolved-oxygen electrode and an FMO-3 immobilized membrane. The FMO-3 solution was mixed with a poly(vinyl alcohol) containing stilbazolium groups (PVA-SbQ), coated on to the dialysis membrane, and the membrane was irradiated with a fluorescent light to immobilize the enzyme. In order to amplify the biosensor output, a substrate regeneration cycle, obtained by coupling the monooxygenase with L-ascorbic acid (AsA) as reducing reagent system, was applied. The effect of pH on the determination of TEA was studied. The maximum response was achieved at pH >9.0. A drop of the phosphate buffer solution with the AsA was put on the sensing area of the oxygen electrode, and the FMO-3 immobilized membrane was placed on the oxygen electrode and covered with a supporting Nylon mesh net which was secured with a silicone O-ring. A measurement system for TEA solution was constructed using the FMO-3 biosensor, a personal computer, a computer-controlled potentiostat, and an A/D converter. The FMO-3 biosensor was used to measure TEA solution from 0.5 to 4.0 mmol L(-1) with 10.0 mmol L(-1) AsA. The biosensor also had good reproducibility, for example a 6.31% coefficient of variation for five measurements, and the output current was maintained over a few hours. In order to improve the selectivity of the TEA biosensor, three type of biosensor with FMO isomer types 1, 3, and 5 were constructed and used to measure nitrogen and sulfur compounds. The outputs of the isomer biosensors indicated individual patterns for each sample solution. The selectivity of TEA biosensor would be improved, and determination of sulfur and nitrogen compounds would be possible, by using the different output of biosensors prepared from different FMO isomers. PMID:18157663

  4. Dynamics and reactivity in Thermus aquaticus N6-adenine methyltransferase.

    PubMed

    Aranda, Juan; Zinovjev, Kirill; Roca, Maite; Tuñón, Iñaki

    2014-11-19

    M.TaqI is a DNA methyltransferase from Thermus aquaticus that catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the N6 position of an adenine, a process described only in prokaryotes. We have used full atomistic classical molecular dynamics simulations to explore the protein-SAM-DNA ternary complex where the target adenine is flipped out into the active site. Key protein-DNA interactions established by the target adenine in the active site are described in detail. The relaxed structure was used for a combined quantum mechanics/molecular mechanics exploration of the reaction mechanism using the string method. According to our free energy calculations the reaction takes place through a stepwise mechanism where the methyl transfer precedes the abstraction of the proton from the exocyclic amino group. The methyl transfer is the rate-determining step, and the obtained free energy barrier is in good agreement with the value derived from the experimental rate constant. Two possible candidates to extract the leftover proton have been explored: a water molecule found in the active site and Asn105, a residue activated by the hydrogen bonds formed through the amide hydrogens. The barrier for the proton abstraction is smaller when Asn105 acts as a base. The reaction mechanisms can be different in other N6-DNA-methyltransferases, as determined from the exploration of the reaction mechanism in the Asn105Asp M.TaqI mutant. PMID:25347783

  5. High resolution dissociative electron attachment to gas phase adenine

    SciTech Connect

    Huber, D.; Beikircher, M.; Denifl, S.; Zappa, F.; Matejcik, S.; Bacher, A.; Grill, V.; Maerk, T. D.; Scheier, P.

    2006-08-28

    The dissociative electron attachment to the gas phase nucleobase adenine is studied using two different experiments. A double focusing sector field mass spectrometer is utilized for measurements requiring high mass resolution, high sensitivity, and relative ion yields for all the fragment anions and a hemispherical electron monochromator instrument for high electron energy resolution. The negative ion mass spectra are discussed at two different electron energies of 2 and 6 eV. In contrast to previous gas phase studies a number of new negative ions are discovered in the mass spectra. The ion efficiency curves for the negative ions of adenine are measured for the electron energy range from about 0 to 15 eV with an electron energy resolution of about 100 meV. The total anion yield derived via the summation of all measured fragment anions is compared with the total cross section for negative ion formation measured recently without mass spectrometry. For adenine the shape of the two cross section curves agrees well, taking into account the different electron energy resolutions; however, for thymine some peculiar differences are observed.

  6. Fragmentation mechanisms of cytosine, adenine and guanine ionized bases.

    PubMed

    Sadr-Arani, Leila; Mignon, Pierre; Chermette, Henry; Abdoul-Carime, Hassan; Farizon, Bernadette; Farizon, Michel

    2015-05-01

    The different fragmentation channels of cytosine, adenine and guanine have been studied through DFT calculations. The electronic structure of bases, their cations, and the fragments obtained by breaking bonds provides a good understanding of the fragmentation process that can complete the experimental approach. The calculations allow assigning various fragments to the given peaks. The comparison between the energy required for the formation of fragments and the peak intensity in the mass spectrum is used. For cytosine and guanine the elimination of the HNCO molecule is a major route of dissociation, while for adenine multiple loss of HCN or HNC can be followed up to small fragments. For cytosine, this corresponds to the initial bond cleavage of N3-C4/N1-C2, which represents the main dissociation route. For guanine the release of HNCO is obtained through the N1-C2/C5-C6 bond cleavage (reverse order also possible) leading to the largest peak of the spectrum. The corresponding energies of 3.5 and 3.9 eV are typically in the range available in the experiments. The loss of NH3 or HCN is also possible but requires more energy. For adenine, fragmentation consists of multiple loss of the HCN molecule and the main route corresponding to HC8N9 loss is followed by the release of HC2N1. PMID:25869111

  7. Flavin-mediated dual oxidation controls an enzymatic Favorskii-type rearrangement

    PubMed Central

    Louie, Gordon; Noel, Joseph P.; Baran, Phil S.; Palfey, Bruce; Moore, Bradley S.

    2013-01-01

    Flavoproteins catalyze a diversity of fundamental redox reactions and are one of the most studied enzyme families1,2. As monooxygenases, they are universally thought to control oxygenation by means of a peroxyflavin species that transfers a single atom of molecular oxygen to an organic substrate1,3,4. Here we report that the bacterial flavoenzyme EncM5,6 catalyzes the peroxyflavin-independent oxygenation-dehydrogenation dual oxidation of a highly reactive poly(β-carbonyl). The crystal structure of EncM with bound substrate mimics coupled with isotope labeling studies reveal previously unknown flavin redox biochemistry. We show that EncM maintains an unanticipated stable flavin oxygenating species, proposed to be a flavin-N5-oxide, to promote substrate oxidation and trigger a rare Favorskii-type rearrangement that is central to the biosynthesis of the antibiotic enterocin. This work provides new insight into the fine-tuning of the flavin cofactor in offsetting the innate reactivity of a polyketide substrate to direct its efficient electrocyclization. PMID:24162851

  8. Flavin-mediated dual oxidation controls an enzymatic Favorskii-type rearrangement.

    PubMed

    Teufel, Robin; Miyanaga, Akimasa; Michaudel, Quentin; Stull, Frederick; Louie, Gordon; Noel, Joseph P; Baran, Phil S; Palfey, Bruce; Moore, Bradley S

    2013-11-28

    Flavoproteins catalyse a diversity of fundamental redox reactions and are one of the most studied enzyme families. As monooxygenases, they are universally thought to control oxygenation by means of a peroxyflavin species that transfers a single atom of molecular oxygen to an organic substrate. Here we report that the bacterial flavoenzyme EncM catalyses the peroxyflavin-independent oxygenation-dehydrogenation dual oxidation of a highly reactive poly(β-carbonyl). The crystal structure of EncM with bound substrate mimics and isotope labelling studies reveal previously unknown flavin redox biochemistry. We show that EncM maintains an unexpected stable flavin-oxygenating species, proposed to be a flavin-N5-oxide, to promote substrate oxidation and trigger a rare Favorskii-type rearrangement that is central to the biosynthesis of the antibiotic enterocin. This work provides new insight into the fine-tuning of the flavin cofactor in offsetting the innate reactivity of a polyketide substrate to direct its efficient electrocyclization. PMID:24162851

  9. A docking model of human ribonucleotide reductase with flavin and phenosafranine

    PubMed Central

    Priya, Panneerselvam Lakshmi; Shanmughavel, Piramanayagam

    2009-01-01

    Ribonucleotide Reductase (RNR) is an enzyme responsible for the reduction of ribonucleotides to their corresponding Deoxyribonucleotides (DNA), which is a building block for DNA replication and repair mechanisms. The key role of RNR in DNA synthesis and control in cell growth has made this an important target for anticancer therapy. Increased RNR activity has been associated with malignant transformation and tumor cell growth. In recent years, several RNR inhibitors, including Triapine, Gemcitabine and GTI-2040, have entered the clinical trials. Our current work focuses on an attempted to dock this inhibitors Flavin and Phenosafranine to curtail the action of human RNR2. The docked inhibitor Flavin and Phenosafranine binds at the active site with THR176, which are essential for free radical formation. The inhibitor must be a radical scavenger to destroy the tyrosyl radical or iron metal scavenger. The iron or radical site of R2 protein can react with one-electron reductants, whereby the tyrosyl radical is converted to a normal tyrosine residue. However, compounds such as Flavin and Phenosafranine were used in most of the cases to reduce the radical activity. The docking study was performed for the crystal structure of human RNR with the radical scavengers Flavin and Phenosafranine to inhibit the human RNR2. This helps to understand the functional aspects and also aids in the development of novel inhibitors for the human RNR2. PMID:20198185

  10. A docking model of human ribonucleotide reductase with flavin and phenosafranine.

    PubMed

    Priya, Panneerselvam Lakshmi; Shanmughavel, Piramanayagam

    2009-09-30

    Ribonucleotide Reductase (RNR) is an enzyme responsible for the reduction of ribonucleotides to their corresponding Deoxyribonucleotides (DNA), which is a building block for DNA replication and repair mechanisms. The key role of RNR in DNA synthesis and control in cell growth has made this an important target for anticancer therapy. Increased RNR activity has been associated with malignant transformation and tumor cell growth. In recent years, several RNR inhibitors, including Triapine, Gemcitabine and GTI-2040, have entered the clinical trials. Our current work focuses on an attempted to dock this inhibitors Flavin and Phenosafranine to curtail the action of human RNR2. The docked inhibitor Flavin and Phenosafranine binds at the active site with THR176, which are essential for free radical formation. The inhibitor must be a radical scavenger to destroy the tyrosyl radical or iron metal scavenger. The iron or radical site of R2 protein can react with one-electron reductants, whereby the tyrosyl radical is converted to a normal tyrosine residue. However, compounds such as Flavin and Phenosafranine were used in most of the cases to reduce the radical activity. The docking study was performed for the crystal structure of human RNR with the radical scavengers Flavin and Phenosafranine to inhibit the human RNR2. This helps to understand the functional aspects and also aids in the development of novel inhibitors for the human RNR2.

  11. Human Cryptochrome-1 Confers Light Independent Biological Activity in Transgenic Drosophila Correlated with Flavin Radical Stability

    PubMed Central

    Vieira, Jacqueline; Jones, Alex R.; Danon, Antoine; Sakuma, Michiyo; Hoang, Nathalie; Robles, David; Tait, Shirley; Heyes, Derren J.; Picot, Marie; Yoshii, Taishi; Helfrich-Förster, Charlotte; Soubigou, Guillaume; Coppee, Jean-Yves; Klarsfeld, André; Rouyer, Francois; Scrutton, Nigel S.; Ahmad, Margaret

    2012-01-01

    Cryptochromes are conserved flavoprotein receptors found throughout the biological kingdom with diversified roles in plant development and entrainment of the circadian clock in animals. Light perception is proposed to occur through flavin radical formation that correlates with biological activity in vivo in both plants and Drosophila. By contrast, mammalian (Type II) cryptochromes regulate the circadian clock independently of light, raising the fundamental question of whether mammalian cryptochromes have evolved entirely distinct signaling mechanisms. Here we show by developmental and transcriptome analysis that Homo sapiens cryptochrome - 1 (HsCRY1) confers biological activity in transgenic expressing Drosophila in darkness, that can in some cases be further stimulated by light. In contrast to all other cryptochromes, purified recombinant HsCRY1 protein was stably isolated in the anionic radical flavin state, containing only a small proportion of oxidized flavin which could be reduced by illumination. We conclude that animal Type I and Type II cryptochromes may both have signaling mechanisms involving formation of a flavin radical signaling state, and that light independent activity of Type II cryptochromes is a consequence of dark accumulation of this redox form in vivo rather than of a fundamental difference in signaling mechanism. PMID:22427812

  12. Stacking interactions in RNA and DNA: Roll-slide energy hyperspace for ten unique dinucleotide steps.

    PubMed

    Mukherjee, Sanchita; Kailasam, Senthilkumar; Bansal, Manju; Bhattacharyya, Dhananjay

    2015-03-01

    Understanding dinucleotide sequence directed structures of nuleic acids and their variability from experimental observation remained ineffective due to unavailability of statistically meaningful data. We have attempted to understand this from energy scan along twist, roll, and slide degrees of freedom which are mostly dependent on dinucleotide sequence using ab initio density functional theory. We have carried out stacking energy analysis in these dinucleotide parameter phase space for all ten unique dinucleotide steps in DNA and RNA using DFT-D by ωB97X-D/6-31G(2d,2p), which appears to satisfactorily explain conformational preferences for AU/AU step in our recent study. We show that values of roll, slide, and twist of most of the dinucleotide sequences in crystal structures fall in the low energy region. The minimum energy regions with large twist values are associated with the roll and slide values of B-DNA, whereas, smaller twist values correspond to higher stability to RNA and A-DNA like conformations. Incorporation of solvent effect by CPCM method could explain the preference shown by some sequences to occur in B-DNA or A-DNA conformations. Conformational preference of BII sub-state in B-DNA is preferentially displayed mainly by pyrimidine-purine steps and partly by purine-purine steps. The purine-pyrimidine steps show largest effect of 5-methyl group of thymine in stacking energy and the introduction of solvent reduces this effect significantly. These predicted structures and variabilities can explain the effect of sequence on DNA and RNA functionality.

  13. Enhanced photocatalytic activity of a self-stabilized synthetic flavin anchored on a TiO2 surface.

    PubMed

    Pandiri, Manjula; Hossain, Mohammad S; Foss, Frank W; Rajeshwar, Krishnan; Paz, Yaron

    2016-07-21

    Synthetic flavin molecules were anchored on Degussa P25 titanium dioxide (TiO2). The effect of their presence on the photocatalytic (PC) activity of TiO2 was studied. Under UV light, an increase in the degradation rate of ethanol was observed. This increase was accompanied by stabilization of the anchored flavin against self-degradation. The unprecedented stabilization effect was found also in the absence of a reducing agent such as ethanol. In contrast, under the less energetic visible light, fast degradation of the anchored flavin was observed. These rather surprising observations were attributed to the propensity for charge transport from excited flavin molecules to the semiconductor and to the role that such charge transfer may play in stabilizing the overall assembly. Anchored flavins excited by UV light to their S2, S3 electronic states were able to transfer the excited electrons to the TiO2 phase whereas anchored flavin molecules that were excited by visible light to the S1 state were less likely to transfer the photo-excited electrons and therefore were destabilized. These findings may be relevant not only to anchored flavins in general but to other functionalized photocatalysts, and may open up new vistas in the implementation of sensitizers in PC systems. PMID:27346787

  14. Enhanced photocatalytic activity of a self-stabilized synthetic flavin anchored on a TiO2 surface.

    PubMed

    Pandiri, Manjula; Hossain, Mohammad S; Foss, Frank W; Rajeshwar, Krishnan; Paz, Yaron

    2016-07-21

    Synthetic flavin molecules were anchored on Degussa P25 titanium dioxide (TiO2). The effect of their presence on the photocatalytic (PC) activity of TiO2 was studied. Under UV light, an increase in the degradation rate of ethanol was observed. This increase was accompanied by stabilization of the anchored flavin against self-degradation. The unprecedented stabilization effect was found also in the absence of a reducing agent such as ethanol. In contrast, under the less energetic visible light, fast degradation of the anchored flavin was observed. These rather surprising observations were attributed to the propensity for charge transport from excited flavin molecules to the semiconductor and to the role that such charge transfer may play in stabilizing the overall assembly. Anchored flavins excited by UV light to their S2, S3 electronic states were able to transfer the excited electrons to the TiO2 phase whereas anchored flavin molecules that were excited by visible light to the S1 state were less likely to transfer the photo-excited electrons and therefore were destabilized. These findings may be relevant not only to anchored flavins in general but to other functionalized photocatalysts, and may open up new vistas in the implementation of sensitizers in PC systems.

  15. 15N solid-state NMR provides a sensitive probe of oxidized flavin reactive sites.

    PubMed

    Koder, Ronald L; Walsh, Joseph D; Pometun, Maxim S; Dutton, P Leslie; Wittebort, Richard J; Miller, Anne-Frances

    2006-11-29

    Flavins are central to the reactivity of a wide variety of enzymes and electron transport proteins. There is great interest in understanding the basis for the different reactivities displayed by flavins in different protein contexts. We propose solid-state nuclear magnetic resonance (SS-NMR) as a tool for directly observing reactive positions of the flavin ring and thereby obtaining information on their frontier orbitals. We now report the SS-NMR signals of the redox-active nitrogens N1 and N5, as well as that of N3. The chemical shift tensor of N5 is over 720 ppm wide, in accordance with the predictions of theory and our calculations. The signal of N3 can be distinguished on the basis of coupling to 1H absent for N1 and N5, as well as the shift tensor span of only 170 ppm, consistent with N3's lower aromaticity and lack of a nonbonding lone pair. The isotropic shifts and spans of N5 and N1 reflect two opposite extremes of the chemical shift range for "pyridine-type" N's, consistent with their electrophilic and nucleophilic chemical reactivities, respectively. Upon flavin reduction, N5's chemical shift tensor contracts dramatically to a span of less than 110 ppm, and the isotropic chemical shift changes by approximately 300 ppm. Both are consistent with loss of N5's nonbonding lone pair and decreased aromaticity, and illustrate the responsiveness of the 15N chemical shift principal values to electronic structure. Thus. 15N chemical shift principal values promise to be valuable tools for understanding electronic differences that underlie variations in flavin reactivity, as well as the reactivities of other heterocyclic cofactors. PMID:17117871

  16. Influence of hydrogen bonding on the geometry of the adenine fragment

    NASA Astrophysics Data System (ADS)

    Słowikowska, Joanna Maria; Woźniak, Krzysztof

    1996-01-01

    The crystal structures of two adenine derivatives, N(6),9-dimethyl-8-butyladenine (I) and its hydrate (1 : 1) (II), have been determined by single-crystal X-ray diffraction. The geometrical features of both structures are discussed. The influence of protonation, substitution and hydrogen bond formation on the geometry of the adenine fragment was studied, based on data retrieved from the Cambridge Structural Database. Total correlation analysis showed mutual correlation between the structural parameters in the adenine ring system; partial correlation calculations for the adenine nucleoside fragments suggest intercorrelation between the parameters of the hydrogen bonding involved in base pairing and the N(adenine)-C(sugar) bond through the adenine fragment; few such correlations were found for fragments without the sugar substituent.

  17. Examination of tyrosine/adenine stacking interactions in protein complexes.

    PubMed

    Copeland, Kari L; Pellock, Samuel J; Cox, James R; Cafiero, Mauricio L; Tschumper, Gregory S

    2013-11-14

    The π-stacking interactions between tyrosine amino acid side chains and adenine-bearing ligands are examined. Crystalline protein structures from the protein data bank (PDB) exhibiting face-to-face tyrosine/adenine arrangements were used to construct 20 unique 4-methylphenol/N9-methyladenine (p-cresol/9MeA) model systems. Full geometry optimization of the 20 crystal structures with the M06-2X density functional theory method identified 11 unique low-energy conformations. CCSD(T) complete basis set (CBS) limit interaction energies were estimated for all of the structures to determine the magnitude of the interaction between the two ring systems. CCSD(T) computations with double-ζ basis sets (e.g., 6-31G*(0.25) and aug-cc-pVDZ) indicate that the MP2 method overbinds by as much as 3.07 kcal mol(-1) for the crystal structures and 3.90 kcal mol(-1) for the optimized structures. In the 20 crystal structures, the estimated CCSD(T) CBS limit interaction energy ranges from -4.00 to -6.83 kcal mol(-1), with an average interaction energy of -5.47 kcal mol(-1), values remarkably similar to the corresponding data for phenylalanine/adenine stacking interactions. Geometry optimization significantly increases the interaction energies of the p-cresol/9MeA model systems. The average estimated CCSD(T) CBS limit interaction energy of the 11 optimized structures is 3.23 kcal mol(-1) larger than that for the 20 crystal structures.

  18. A comparison of adenine and some derivatives on pig isolated tracheal muscle.

    PubMed Central

    Bach-Dieterle, Y.; Holden, W. E.; Junod, A. F.

    1983-01-01

    We studied the muscle relaxation induced by adenine and several adenine derivatives in strips of tracheal smooth muscle from pigs; in addition their metabolism by the tissue was examined. Adenine relaxed tissue which was contracted by carbachol, histamine, or KCl. Adenine's potency was similar to that of adenosine and ATP (threshold about 4 X 10(-5)M). In tissues with carbachol-induced tone, the adenine effect differed from adenosine and ATP by being slower in onset and in 'washout' time. Furthermore, neither dipyridamole nor theophylline modified the response to adenine. The relationship was examined between pharmacological effects and the metabolism of [3H]-adenosine and [3H]-adenine. Both substrates were taken up by the tissue and converted to nucleotides, but relaxation correlated with nucleotide accumulation only in the case of [3H]-adenine. We conclude that the site and mechanism of adenine-induced relaxation is different from that of adenosine and ATP in porcine tracheal muscle. PMID:6571222

  19. Immune-Stimulatory Dinucleotide at the 5′-End of Oligodeoxynucleotides Is Critical for TLR9-Mediated Immune Responses

    PubMed Central

    2013-01-01

    Oligodeoxynucleotides (ODNs) containing a CpG or certain synthetic dinucleotides, referred to as immune-stimulatory dinucleotides, induce Toll-like receptor 9 (TLR9)-mediated immune responses. Chemical modifications such as 2′-O-methylribonucleotides incorporated adjacent to the immune-stimulatory dinucleotide on the 5′-side abrogate TLR9-mediated immune responses. In this study, we evaluated the effect of the location of immune-stimulatory dinucleotides in ODNs on TLR9-mediated immune responses. We designed and synthesized ODNs with two immune-stimulatory dinucleotides, one placed toward the 5′-end region and the other toward the 3′-end region, incorporated 2′-O-methylribonucleotides selectively preceding the 5′- or 3′-immune-stimulatory dinucleotide or both, and studied TLR9-mediated immune responses of these compounds in cell-based assays and in vivo in mice. These studies showed that an immune-stimulatory dinucleotide located closer to the 5′-end is critical for and dictates TLR9-mediated immune responses. These studies provide insights for the use of ODNs when employed as TLR9 agonists and antagonists or antisense agents. PMID:24900663

  20. Modelling proton tunnelling in the adenine-thymine base pair.

    PubMed

    Godbeer, A D; Al-Khalili, J S; Stevenson, P D

    2015-05-21

    The energies of the canonical (standard, amino-keto) and tautomeric (non-standard, imino-enol) charge-neutral forms of the adenine-thymine base pair (A-T and A*-T*, respectively) are calculated using density functional theory. The reaction pathway is then computed using a transition state search to provide the asymmetric double-well potential minima along with the barrier height and shape, which are combined to create the potential energy surface using a polynomial fit. The influence of quantum tunnelling on proton transfer within a base pair H-bond (modelled as the DFT deduced double-well potential) is then investigated by solving the time-dependent master equation for the density matrix. The effect on a quantum system by its surrounding water molecules is explored via the inclusion of a dissipative Lindblad term in the master equation, in which the environment is modelled as a heat bath of harmonic oscillators. It is found that quantum tunnelling, due to transitions to higher energy eigenstates with significant amplitudes in the shallow (tautomeric) side of the potential, is unlikely to be a significant mechanism for the creation of adenine-thymine tautomers within DNA, with thermally assisted coupling of the environment only able to boost the tunnelling probability to a maximum of 2 × 10(-9). This is barely increased for different choices of the starting wave function or when the geometry of the potential energy surface is varied.

  1. Nonselective enrichment for yeast adenine mutants by flow cytometry

    NASA Technical Reports Server (NTRS)

    Bruschi, C. V.; Chuba, P. J.

    1988-01-01

    The expression of certain adenine biosynthetic mutations in the yeast Saccharomyces cerevisiae results in a red colony color. This phenomenon has historically provided an ideal genetic marker for the study of mutation, recombination, and aneuploidy in lower eukaryotes by classical genetic analysis. In this paper, it is reported that cells carrying ade1 and/or ade2 mutations exhibit primary fluorescence. Based on this observation, the nonselective enrichment of yeast cultures for viable adenine mutants by using the fluorescence-activated cell sorter has been achieved. The advantages of this approach over conventional genetic analysis of mutation, recombination, and mitotic chromosomal stability include speed and accuracy in acquiring data for large numbers of clones. By using appropriate strains, the cell sorter has been used for the isolation of both forward mutations and chromosomal loss events in S. cerevisiae. The resolving power of this system and its noninvasiveness can easily be extended to more complex organisms, including mammalian cells, in which analogous metabolic mutants are available.

  2. Human augmenter of liver regeneration: probing the catalytic mechanism of a flavin-dependent sulfhydryl oxidase.

    PubMed

    Schaefer-Ramadan, Stephanie; Gannon, Shawn A; Thorpe, Colin

    2013-11-19

    Augmenter of liver regeneration is a member of the ERV family of small flavin-dependent sulfhydryl oxidases that contain a redox-active CxxC disulfide bond in redox communication with the isoalloxazine ring of bound FAD. These enzymes catalyze the oxidation of thiol substrates with the reduction of molecular oxygen to hydrogen peroxide. This work studies the catalytic mechanism of the short, cytokine form of augmenter of liver regeneration (sfALR) using model thiol substrates of the enzyme. The redox potential of the proximal disulfide in sfALR was found to be approximately 57 mV more reducing than the flavin chromophore, in agreement with titration experiments. Rapid reaction studies show that dithiothreitol (DTT) generates a transient mixed disulfide intermediate with sfALR signaled by a weak charge-transfer interaction between the thiolate of C145 and the oxidized flavin. The subsequent transfer of reducing equivalents to the flavin ring is relatively slow, with a limiting apparent rate constant of 12.4 s(-1). However, reoxidation of the reduced flavin by molecular oxygen is even slower (2.3 s(-1) at air saturation) and thus largely limits turnover at 5 mM DTT. The nature of the charge-transfer complexes observed with DTT was explored using a range of simple monothiols to mimic the initial nucleophilic attack on the proximal disulfide. While β-mercaptoethanol is a very poor substrate of sfALR (∼0.3 min(-1) at 100 mM thiol), it rapidly generates a mixed disulfide intermediate allowing the thiolate of C145 to form a strong charge-transfer complex with the flavin. Unlike the other monothiols tested, glutathione is unable to form charge-transfer complexes and is an undetectable substrate of the oxidase. These data are rationalized on the basis of the stringent steric requirements for thiol-disulfide exchange reactions. The inability of the relatively bulky glutathione to attain the in-line geometry required for efficient disulfide exchange in sfALR may be

  3. X-ray Crystallography Reveals a Reduced Substrate Complex of UDP-Galactopyranose Mutase Poised for Covalent Catalysis by Flavin

    SciTech Connect

    Gruber, Todd D.; Westler, William M.; Kiessling, Laura L.; Forest, Katrina T.

    2009-11-04

    The flavoenzyme uridine 5'-diphosphate galactopyranose mutase (UGM or Glf) catalyzes the interconversion of UDP-galactopyranose and UDP-galactofuranose. The latter is a key building block for cell wall construction in numerous pathogens, including Mycobacterium tuberculosis. Mechanistic studies of UGM suggested a novel role for the flavin, and we previously provided evidence that the catalytic mechanism proceeds through a covalent flavin-galactose iminium. Here, we describe 2.3 and 2.5 {angstrom} resolution X-ray crystal structures of the substrate-bound enzyme in oxidized and reduced forms, respectively. In the latter, C1 of the substrate is 3.6 {angstrom} from the nucleophilic flavin N5 position. This orientation is consistent with covalent catalysis by flavin.

  4. Correction: Enhanced photocatalytic activity of a self-stabilized synthetic flavin anchored on a TiO2 surface.

    PubMed

    Pandiri, Manjula; Shaham-Waldmann, Nurit; Hossain, Mohammad S; Foss, Frank W; Rajeshwar, Krishnan; Paz, Yaron

    2016-09-14

    Correction for 'Enhanced photocatalytic activity of a self-stabilized synthetic flavin anchored on a TiO2 surface' by Manjula Pandiri et al., Phys. Chem. Chem. Phys., 2016, 18, 18575-18583. PMID:27509005

  5. Distortion of Flavin Geometry Is Linked to Ligand Binding in Cholesterol Oxidase

    SciTech Connect

    Lyubimov, A.Y.; Heard, K.; Tang, H.; Sampson, N.S.; Vrielink, A.

    2009-06-03

    Two high-resolution structures of a double mutant of bacterial cholesterol oxidase in the presence or absence of a ligand, glycerol, are presented, showing the trajectory of glycerol as it binds in a Michaelis complex-like position in the active site. A group of three aromatic residues forces the oxidized isoalloxazine moiety to bend along the N5-N10 axis as a response to the binding of glycerol in the active site. Movement of these aromatic residues is only observed in the glycerol-bound structure, indicating that some tuning of the FAD redox potential is caused by the formation of the Michaelis complex during regular catalysis. This structural study suggests a possible mechanism of substrate-assisted flavin activation, improves our understanding of the interplay between the enzyme, its flavin cofactor and its substrate, and is of use to the future design of effective cholesterol oxidase inhibitors.

  6. Photoactivation mechanisms of flavin-binding photoreceptors revealed through ultrafast spectroscopy and global analysis methods.

    PubMed

    Mathes, Tilo; van Stokkum, Ivo H M; Kennis, John T M

    2014-01-01

    Flavin-binding photoreceptor proteins use the isoalloxazine moiety of flavin cofactors to absorb light in the blue/UV-A wavelength region and subsequently translate it into biological information. The underlying photochemical reactions and protein structural dynamics are delicately tuned by the protein environment and represent fundamental reactions in biology and chemistry. Due to their photo-switchable nature, these proteins can be studied efficiently with laser-flash induced transient absorption and emission spectroscopy with temporal precision down to the femtosecond time domain. Here, we describe the application of both visible and mid-IR ultrafast transient absorption and time-resolved fluorescence methods in combination with sophisticated global analysis procedures to elucidate the photochemistry and signal transduction of BLUF (Blue light receptors using FAD) and LOV (Light oxygen voltage) photoreceptor domains.

  7. Use of flavins as catalyst for the remediation of halogenated compounds.

    PubMed

    Rathore, Deepshikha; Singh, Ram; Geetanjali; Srivastava, Richa

    2014-10-01

    Flavin-containing monooxygenases (FMOs) are an important monooxygenase system present in living organisms starting from eukaryotes to human beings. They are involved in catalysing wide variety of oxygenation reactions including bioremediation process. The central reaction in these enzymes is always the formation of a peroxyflavin intermediate by reaction of reduced flavin with molecular oxygen. The microenvironment of the peroxyflavin regulates the reactive character of the peroxyflavin. Utilizing this aspect of the biology, chemremediation of aromatic halogenated phenols have been initiated and achieved using flavinium perchlorate salt as catalyst in 38 % yield. The flavinium perchlorate during the reaction gets converted to peroxyflavin with H2O2. This method will be useful in the removal of halogens from aromatic halogenated phenols.

  8. Adenine nucleotides as allosteric effectors of pea seed glutamine synthetase.

    PubMed

    Knight, T J; Langston-Unkefer, P J

    1988-08-15

    The effects of adenine nucleotides on pea seed glutamine synthetase (EC 6.3.1.2) activity were examined as a part of our investigation of the regulation of this octameric plant enzyme. Saturation curves for glutamine synthetase activity versus ATP with ADP as the changing fixed inhibitor were not hyperbolic; greater apparent Vmax values were observed in the presence of added ADP than the Vmax observed in the absence of ADP. Hill plots of data with ADP present curved upward and crossed the plot with no added ADP. The stoichiometry of adenine nucleotide binding to glutamine synthetase was examined. Two molecules of [gamma-32P]ATP were bound per subunit in the presence of methionine sulfoximine. These ATP molecules were bound at an allosteric site and at the active site. One molecule of either [gamma-32P]ATP or [14C]ADP bound per subunit in the absence of methionine sulfoximine; this nucleotide was bound at an allosteric site. ADP and ATP compete for binding at the allosteric site, although ADP was preferred. ADP binding to the allosteric site proceeded in two kinetic phases. A Vmax value of 1.55 units/mg was measured for glutamine synthetase with one ADP tightly bound per enzyme subunit; a Vmax value of 0.8 unit/mg was measured for enzyme with no adenine nucleotide bound at the allosteric site. The enzyme activation caused by the binding of ADP to the allosteric sites was preceded by a lag phase, the length of which was dependent on the ADP concentration. Enzyme incubated in 10 mM ADP bound approximately 4 mol of ADP/mol of native enzyme before activation was observed; the activation was complete when 7-8 mol of ADP were bound per mol of the octameric, native enzyme. The Km for ATP (2 mM) was not changed by ADP binding to the allosteric sites. ADP was a simple competitive inhibitor (Ki = 0.05 mM) of ATP for glutamine synthetase with eight molecules of ADP tightly bound to the allosteric sites of the octamer. Binding of ATP to the allosteric sites led to marked

  9. Role of Valine 464 in the Flavin Oxidation Reaction Catalyzed by Choline Oxidase

    SciTech Connect

    Finnegan, Steffan; Agniswamy, Johnson; Weber, Irene T.; Gadda, Giovanni

    2010-11-03

    The oxidation of reduced flavin cofactors by oxygen is a very important reaction that is central to the chemical versatility of hundreds of flavoproteins classified as monooxygenases and oxidases. These enzymes are characterized by bimolecular rate constants {ge} 10{sup 5} M{sup -1} s{sup -1} and produce water and hydrogen peroxide, respectively. A hydrophobic cavity close to the reactive flavin C(4a) atom has been previously identified in the 3D structure of monooxygenases but not in flavoprotein oxidases. In the present study, we have investigated by X-ray crystallography, mutagenesis, steady-state, and rapid reaction approaches the role of Val464, which is <6 {angstrom} from the flavin C(4a) atom in choline oxidase. The 3D structure of the Val464Ala enzyme was essentially identical to that of the wild-type enzyme as shown by X-ray crystallography. Time-resolved anaerobic substrate reduction of the enzymes showed that replacement of Val464 with alanine or threonine did not affect the reductive half-reaction. Steady-state and rapid kinetics as well as enzyme-monitored turnovers indicated that the oxidative half-reaction in the Ala464 and Thr464 enzymes was decreased by 50-fold with respect to the wild-type enzyme. We propose that the side chain of Val464 in choline oxidase provides a nonpolar site that is required to guide oxygen in proximity of the C(4a) atom of the flavin, where it will subsequently react via electrostatic catalysis. Visual analysis of available structures suggests that analogous nonpolar sites are likely present in most flavoprotein oxidases. Mechanistic considerations provide rationalization for the differences between sites in monooxygenases and oxidases.

  10. Major and minor groove conformations of DNA trimers modified on guanine or adenine by 4-aminobiphenyl: Adenine adducts favor the minor groove

    SciTech Connect

    Shapiro, R.; Ellis, S.; Hingerty, B.E.

    1995-01-01

    We have studied the conformational effects of 4-aminobiphenyl modification at C-8 of guanine or adenine on double-stranded DNA trimers. We used sequences with the modified purine at the central base pair and all 16 possible neighboring sequences at the outer pairs. Minimized potential energy calculations were carried out using the molecular mechanics program DUPLEX to survey the conformation space of these adducts, using a total of 1280 starting structures both in the modified guanine series and in the modified adenine series. Conformer families in which the bound 4-aminobiphenyl was located in the DNA major groove, and in the minor groove, were located for both adenine and guanine modification. In the modified guanine series, the major and minor groove families were roughly comparable in energy, and the sequence context determined which was more stable in a particular case. In the modified adenine series, however, the minor groove structure was more that 10 kcal/mol more stable than the major groove for all sequences. As a result, minor groove adducts provided most of the global minima in the adenine-modified series. This result may be relevant to a previous mutagenesis study [Lasko et al. (1988) J. Biol. Chem. 263, 15429-15435] in which the hot spot of most frequent occurrence was located at an adenine, in the sequence GAT. 25 refs., 9 figs., 4 tabs.

  11. Modifying the collagen framework of costal cartilage under the impact of UV and a flavin mononucleotide

    NASA Astrophysics Data System (ADS)

    Ignat'eva, N. Yu.; Zakharkina, O. L.; Semchishen, V. A.; Molchanov, M. D.; Lunin, V. V.; Bagratashvili, V. N.

    2016-03-01

    Modifications of the matrix of the tissue of costal cartilage under the impact of UV (λ = 365 nm) and a flavin mononucleotide (FMN) is studied. The changes in the macroscopic properties of the tissue are detected by means of differential scanning calorimetry and under the conditions of uniaxial compression during mechanical testing. The endothermic effects of the denaturation of the collagen framework of the tissue and the Young's modulus are determined. It is shown that the presence of a flavin mononucleotide in the interstitial fluid leads lowers the temperature of collagen denaturation by 2.5°C and doubles the Young's modulus. It is found that the temperature of denaturation and the Young's modulus grow gradually after treating the tissue with the UV radiation, and their values ultimately exceed by far the corresponding values for intact samples. It is concluded that the obtained data indicate the possibility of stabilizing the framework of the matrix of costal cartilage under the impact of UV radiation and a flavin mononucleotide.

  12. FAD synthesis and degradation in the nucleus create a local flavin cofactor pool.

    PubMed

    Giancaspero, Teresa Anna; Busco, Giovanni; Panebianco, Concetta; Carmone, Claudia; Miccolis, Angelica; Liuzzi, Grazia Maria; Colella, Matilde; Barile, Maria

    2013-10-01

    FAD is a redox cofactor ensuring the activity of many flavoenzymes mainly located in mitochondria but also relevant for nuclear redox activities. The last enzyme in the metabolic pathway producing FAD is FAD synthase (EC 2.7.7.2), a protein known to be localized both in cytosol and in mitochondria. FAD degradation to riboflavin occurs via still poorly characterized enzymes, possibly belonging to the NUDIX hydrolase family. By confocal microscopy and immunoblotting experiments, we demonstrate here the existence of FAD synthase in the nucleus of different experimental rat models. HPLC experiments demonstrated that isolated rat liver nuclei contain ∼300 pmol of FAD·mg(-1) protein, which was mainly protein-bound FAD. A mean FAD synthesis rate of 18.1 pmol·min(-1)·mg(-1) protein was estimated by both HPLC and continuous coupled enzymatic spectrophotometric assays. Rat liver nuclei were also shown to be endowed with a FAD pyrophosphatase that hydrolyzes FAD with an optimum at alkaline pH and is significantly inhibited by adenylate-containing nucleotides. The coordinate activity of these FAD forming and degrading enzymes provides a potential mechanism by which a dynamic pool of flavin cofactor is created in the nucleus. These data, which significantly add to the biochemical comprehension of flavin metabolism and its subcellular compartmentation, may also provide the basis for a more detailed comprehension of the role of flavin homeostasis in biologically and clinically relevant epigenetic events. PMID:23946482

  13. FAD Synthesis and Degradation in the Nucleus Create a Local Flavin Cofactor Pool*

    PubMed Central

    Giancaspero, Teresa Anna; Busco, Giovanni; Panebianco, Concetta; Carmone, Claudia; Miccolis, Angelica; Liuzzi, Grazia Maria; Colella, Matilde; Barile, Maria

    2013-01-01

    FAD is a redox cofactor ensuring the activity of many flavoenzymes mainly located in mitochondria but also relevant for nuclear redox activities. The last enzyme in the metabolic pathway producing FAD is FAD synthase (EC 2.7.7.2), a protein known to be localized both in cytosol and in mitochondria. FAD degradation to riboflavin occurs via still poorly characterized enzymes, possibly belonging to the NUDIX hydrolase family. By confocal microscopy and immunoblotting experiments, we demonstrate here the existence of FAD synthase in the nucleus of different experimental rat models. HPLC experiments demonstrated that isolated rat liver nuclei contain ∼300 pmol of FAD·mg−1 protein, which was mainly protein-bound FAD. A mean FAD synthesis rate of 18.1 pmol·min−1·mg−1 protein was estimated by both HPLC and continuous coupled enzymatic spectrophotometric assays. Rat liver nuclei were also shown to be endowed with a FAD pyrophosphatase that hydrolyzes FAD with an optimum at alkaline pH and is significantly inhibited by adenylate-containing nucleotides. The coordinate activity of these FAD forming and degrading enzymes provides a potential mechanism by which a dynamic pool of flavin cofactor is created in the nucleus. These data, which significantly add to the biochemical comprehension of flavin metabolism and its subcellular compartmentation, may also provide the basis for a more detailed comprehension of the role of flavin homeostasis in biologically and clinically relevant epigenetic events. PMID:23946482

  14. Binding of the Covalent Flavin Assembly Factor to the Flavoprotein Subunit of Complex II.

    PubMed

    Maklashina, Elena; Rajagukguk, Sany; Starbird, Chrystal A; McDonald, W Hayes; Koganitsky, Anna; Eisenbach, Michael; Iverson, Tina M; Cecchini, Gary

    2016-02-01

    Escherichia coli harbors two highly conserved homologs of the essential mitochondrial respiratory complex II (succinate:ubiquinone oxidoreductase). Aerobically the bacterium synthesizes succinate:quinone reductase as part of its respiratory chain, whereas under microaerophilic conditions, the quinol:fumarate reductase can be utilized. All complex II enzymes harbor a covalently bound FAD co-factor that is essential for their ability to oxidize succinate. In eukaryotes and many bacteria, assembly of the covalent flavin linkage is facilitated by a small protein assembly factor, termed SdhE in E. coli. How SdhE assists with formation of the covalent flavin bond and how it binds the flavoprotein subunit of complex II remain unknown. Using photo-cross-linking, we report the interaction site between the flavoprotein of complex II and the SdhE assembly factor. These data indicate that SdhE binds to the flavoprotein between two independently folded domains and that this binding mode likely influences the interdomain orientation. In so doing, SdhE likely orients amino acid residues near the dicarboxylate and FAD binding site, which facilitates formation of the covalent flavin linkage. These studies identify how the conserved SdhE assembly factor and its homologs participate in complex II maturation.

  15. Dynamic changes in nicotinamide pyridine dinucleotide content in normal human epidermal keratinocytes and their effect on retinoic acid biosynthesis

    SciTech Connect

    Pinkas-Sarafova, Adriana . E-mail: apinkassaraf@notes.cc.sunysb.edu; Markova, N.G. . E-mail: nmarkova@notes.cc.sunysb.edu; Simon, M. . E-mail: marsimon@notes.cc.sunysb.edu

    2005-10-21

    The function of many enzymes that regulate metabolism and transcription depends critically on the nicotinamide pyridine dinucleotides. To understand the role of NAD(P)(H) in physiology and pathophysiology, it is imperative to estimate both their amount and ratios in a given cell type. In human epidermis and in cultured epidermal keratinocytes, we found that the total dinucleotide content is in the low millimolar range. The dinucleotide pattern changes during proliferation and maturation of keratinocytes in culture. Differences in the concentrations of NAD(P)(H) of 1.5- to 12-fold were observed. This resulted in alteration of the NAD(P)H/NAD(P) ratio, which could impact the differential regulation of both transcriptional and metabolic processes. In support of this notion, we provide evidence that the two-step oxidation of retinol to retinoic acid, a nuclear hormone critical for epidermal homeostasis, can be regulated by the relative physiological amounts of the pyridine dinucleotides.

  16. Interaction of sulfanilamide and sulfamethoxazole with bovine serum albumin and adenine: spectroscopic and molecular docking investigations.

    PubMed

    Rajendiran, N; Thulasidhasan, J

    2015-06-01

    Interaction between sulfanilamide (SAM) and sulfamethoxazole (SMO) with BSA and DNA base (adenine) was investigated by UV-visible, fluorescence, cyclic voltammetry and molecular docking studies. Stern-Volmer fluorescence quenching constant (Ka) suggests SMO is more quenched with BSA/adenine than that of SAM. The distance r between donor (BSA/adenine) and acceptor (SAM and SMO) was obtained according to fluorescence resonance energy transfer (FRET). The results showed that hydrophobic forces, electrostatic interactions, and hydrogen bonds played vital roles in the SAM and SMO with BSA/adenine binding interaction. During the interaction, sulfa drugs could insert into the hydrophobic pocket, where the non-radioactive energy transfer from BSA/adenine to sulfa drugs occurred with high possibility. Cyclic voltammetry results suggested that when the drug concentration is increased, the anodic electrode potential deceased. The docking method indicates aniline group is interacted with the BSA molecules. PMID:25754395

  17. Interaction of sulfanilamide and sulfamethoxazole with bovine serum albumin and adenine: Spectroscopic and molecular docking investigations

    NASA Astrophysics Data System (ADS)

    Rajendiran, N.; Thulasidhasan, J.

    2015-06-01

    Interaction between sulfanilamide (SAM) and sulfamethoxazole (SMO) with BSA and DNA base (adenine) was investigated by UV-visible, fluorescence, cyclic voltammetry and molecular docking studies. Stern-Volmer fluorescence quenching constant (Ka) suggests SMO is more quenched with BSA/adenine than that of SAM. The distance r between donor (BSA/adenine) and acceptor (SAM and SMO) was obtained according to fluorescence resonance energy transfer (FRET). The results showed that hydrophobic forces, electrostatic interactions, and hydrogen bonds played vital roles in the SAM and SMO with BSA/adenine binding interaction. During the interaction, sulfa drugs could insert into the hydrophobic pocket, where the non-radioactive energy transfer from BSA/adenine to sulfa drugs occurred with high possibility. Cyclic voltammetry results suggested that when the drug concentration is increased, the anodic electrode potential deceased. The docking method indicates aniline group is interacted with the BSA molecules.

  18. Hydrogen-1, carbon-13, and nitrogen-15 NMR spectroscopy of Anabaena 7120 flavodoxin: Assignment of. beta. -sheet and flavin binding site resonances and analysis of protein-flavin interactions

    SciTech Connect

    Stockman, B.J.; Krezel, A.M.; Markley, J.L. ); Leonhardt, K.G.; Straus, N.A. )

    1990-10-01

    Sequence-specific {sup 1}H and {sup 13}C NMR assignments have been made for residues that form the five-stranded parallel {beta}-sheet and the flavin mononucleotide (FMN) binding site of oxidized Anabaena 7120 flavodoxin. Interstrand nuclear Overhauser enhancements (NOEs) indicate that the {beta}-sheet arrangement is similar to that observed in the crystal structure of the 70% homologous long-chain flavodoxin from Anacystis nidulans. A total of 62 NOEs were identified: 8 between protons of bound FMN, 29 between protons of the protein in the flavin binding site, and 25 between protons of bound FMN and protons of the protein. These constraints were used to determine the localized solution structure of the FMN binding site. The electronic environment and conformation of the protein-bound flavin isoalloxazine ring were investigated by determining {sup 13}C-{sup 1}H coupling constants. The carbonyl edge of the flavin ring was found to be slightly polarized. The xylene ring was found to be nonplanar. Tyrosine 94, located adjacent to the flavin isoalloxazine ring, was shown to have a hindered aromatic ring flip rate.

  19. Association of poly(N-isopropylacrylamide) containing nucleobase multiple hydrogen bonding of adenine for DNA recognition

    NASA Astrophysics Data System (ADS)

    Yang, Hsiu-Wen; Chen, Jem-Kun; Cheng, Chih-Chia; Kuo, Shiao-Wei

    2013-04-01

    In this study we used the poly(N-isopropylacrylamide) (PNIPAAm) as a medium to generate PNIPAAm-adenine supramolecular complexes. A nucleobase-like hydrogen bonding (NLHB) between PNIPAAm and adenine was found that changed the morphology, crystalline structure, and temperature responsiveness of PNIPAAm microgels relatively to the adenine concentrations. With increasing the adenine concentration, the PNIPAAm-adenine supramolecular complexes gradually altered their morphologies from microgel particles to thin film structures and suppressed the thermodynamical coil-to-globule transition of PNIPAAm because of the NLHB existed between the PNIPAAm amide and ester groups and the adenine amide groups (Cdbnd O⋯Hsbnd N and Nsbnd H⋯Nsbnd R), verified by FTIR spectral analysis. NLHB was also diverse and extensive upon increasing the temperature; therefore, the thermoresponsive behavior of the complexes was altered with the NLBH intensity, evaluated by the inter-association equilibrium constant (Ka) above and below their LCST. Therefore, PNIPAAm can be as a medium to recognize adenine in various concentrations, which could potentially be applied in DNA recognition.

  20. Renoprotective effect of the xanthine oxidoreductase inhibitor topiroxostat on adenine-induced renal injury.

    PubMed

    Kamijo-Ikemori, Atsuko; Sugaya, Takeshi; Hibi, Chihiro; Nakamura, Takashi; Murase, Takayo; Oikawa, Tsuyoshi; Hoshino, Seiko; Hisamichi, Mikako; Hirata, Kazuaki; Kimura, Kenjiro; Shibagaki, Yugo

    2016-06-01

    The aim of the present study was to reveal the effect of a xanthine oxidoreductase (XOR) inhibitor, topiroxostat (Top), compared with another inhibitor, febuxostat (Feb), in an adenine-induced renal injury model. We used human liver-type fatty acid-binding protein (L-FABP) chromosomal transgenic mice, and urinary L-FABP, a biomarker of tubulointerstitial damage, was used to evaluate tubulointerstitial damage. Male transgenic mice (n = 24) were fed a 0.2% (wt/wt) adenine-containing diet. Two weeks after the start of this diet, renal dysfunction was confirmed, and the mice were divided into the following four groups: the adenine group was given only the diet containing adenine, and the Feb, high-dose Top (Top-H), and low-dose Top (Top-L) groups were given diets containing Feb (3 mg/kg), Top-H (3 mg/kg), and Top-L (1 mg/kg) in addition to adenine for another 2 wk. After withdrawal of the adenine diet, each medication was continued for 2 wk. Serum creatinine levels, the degree of macrophage infiltration, tubulointerstitial damage, renal fibrosis, urinary 15-F2t-isoprostane levels, and renal XOR activity were significantly attenuated in the kidneys of the Feb, Top-L, and Top-H groups compared with the adenine group. Serum creatinine levels in the Top-L and Top-H groups as well as renal XOR in the Top-H group were significantly lower than those in the Feb group. Urinary excretion of L-FABP in both the Top-H and Top-L groups was significantly lower than in the adenine and Feb groups. In conclusion, Top attenuated renal damage in an adenine-induced renal injury model. PMID:27029427

  1. Renoprotective effect of the xanthine oxidoreductase inhibitor topiroxostat on adenine-induced renal injury.

    PubMed

    Kamijo-Ikemori, Atsuko; Sugaya, Takeshi; Hibi, Chihiro; Nakamura, Takashi; Murase, Takayo; Oikawa, Tsuyoshi; Hoshino, Seiko; Hisamichi, Mikako; Hirata, Kazuaki; Kimura, Kenjiro; Shibagaki, Yugo

    2016-06-01

    The aim of the present study was to reveal the effect of a xanthine oxidoreductase (XOR) inhibitor, topiroxostat (Top), compared with another inhibitor, febuxostat (Feb), in an adenine-induced renal injury model. We used human liver-type fatty acid-binding protein (L-FABP) chromosomal transgenic mice, and urinary L-FABP, a biomarker of tubulointerstitial damage, was used to evaluate tubulointerstitial damage. Male transgenic mice (n = 24) were fed a 0.2% (wt/wt) adenine-containing diet. Two weeks after the start of this diet, renal dysfunction was confirmed, and the mice were divided into the following four groups: the adenine group was given only the diet containing adenine, and the Feb, high-dose Top (Top-H), and low-dose Top (Top-L) groups were given diets containing Feb (3 mg/kg), Top-H (3 mg/kg), and Top-L (1 mg/kg) in addition to adenine for another 2 wk. After withdrawal of the adenine diet, each medication was continued for 2 wk. Serum creatinine levels, the degree of macrophage infiltration, tubulointerstitial damage, renal fibrosis, urinary 15-F2t-isoprostane levels, and renal XOR activity were significantly attenuated in the kidneys of the Feb, Top-L, and Top-H groups compared with the adenine group. Serum creatinine levels in the Top-L and Top-H groups as well as renal XOR in the Top-H group were significantly lower than those in the Feb group. Urinary excretion of L-FABP in both the Top-H and Top-L groups was significantly lower than in the adenine and Feb groups. In conclusion, Top attenuated renal damage in an adenine-induced renal injury model.

  2. Isolation and characterization of dinucleotide microsatellite loci in the Asian elephant (Elephas maximus).

    PubMed

    Kongrit, C; Siripunkaw, C; Brockelman, W Y; Akkarapatumwong, V; Wright, T F; Eggert, L S

    2008-01-01

    The endangered Asian elephant is found today primarily in protected areas. We characterized 18 dinucleotide microsatellite loci in this species. Allelic diversity ranged from three to eight per locus, and observed heterozygosity ranged from 0.200 to 0.842 in a wild population. All loci were in Hardy-Weinberg equilibrium, but linkage disequilibrium was detected between two loci in the wild, but not in the zoo elephants. These loci will be useful for the population-level studies of this species.

  3. N6-methyl-adenine: an epigenetic signal for DNA-protein interactions.

    PubMed

    Wion, Didier; Casadesús, Josep

    2006-03-01

    N(6)-methyl-adenine is found in the genomes of bacteria, archaea, protists and fungi. Most bacterial DNA adenine methyltransferases are part of restriction-modification systems. Certain groups of Proteobacteria also harbour solitary DNA adenine methyltransferases that provide signals for DNA-protein interactions. In gamma-proteobacteria, Dam methylation regulates chromosome replication, nucleoid segregation, DNA repair, transposition of insertion elements and transcription of specific genes. In Salmonella, Haemophilus, Yersinia and Vibrio species and in pathogenic Escherichia coli, Dam methylation is required for virulence. In alpha-proteobacteria, CcrM methylation regulates the cell cycle in Caulobacter, Rhizobium and Agrobacterium, and has a role in Brucella abortus infection.

  4. Adenine Phosphoribosyltransferase in Plant Tissues: Some Effects of Kinetin on Enzymic Activity 1

    PubMed Central

    Nicholls, P. B.; Murray, A. W.

    1968-01-01

    Adenine phosphoribosyltransferase activity was measured in extracts of soybean (Glycine max var. Acme) callus and of senescing barley leaves (Hordeum distichon c.v. Prior). The enzyme from soybean callus had Michaelis constants for adenine and 5-phosphoribosyl pyrophosphate of 1.5 and 7.5 μm respectively and was inhibited by AMP and stimulated by ATP. The presence of kinetin was found to considerably increase the activity of adenine phosphoribosyltransferase in extracts of soybean callus and senescing barley leaves. PMID:16656820

  5. Structure-Function Analysis of Escherichia coli MnmG (GidA), a Highly Conserved tRNA-Modifying Enzyme

    SciTech Connect

    Shi, Rong; Villarroya, Magda; Ruiz-Partida, Rafael; Li, Yunge; Proteau, Ariane; Prado, Silvia; Moukadiri, Ismaïl; Benítez-Páez, Alfonso; Lomas, Rodrigo; Wagner, John; Matte, Allan; Velázquez-Campoy, Adrián; Armengod, M.-Eugenia; Cygler, Miroslaw

    2010-01-12

    The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of Escherichia coli MnmG at 2.4-{angstrom} resolution, mutated highly conserved residues with putative roles in flavin adenine dinucleotide (FAD) or tRNA binding and MnmE interaction, and analyzed the effects of these mutations in vivo and in vitro. Limited trypsinolysis of MnmG suggests significant conformational changes upon FAD binding.

  6. Structure-function Analysis of Escherichia coli MnmG (GidA) a Highly Conserved tRNA-modifying Enzyme

    SciTech Connect

    R Shi; M Villarroya; R Ruiz-Partida; Y Li; A Proteau; S prado; I Moukafiri; A Benatez-Paez; R Lomas; et al.

    2011-12-31

    The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of Escherichia coli MnmG at 2.4-{angstrom} resolution, mutated highly conserved residues with putative roles in flavin adenine dinucleotide (FAD) or tRNA binding and MnmE interaction, and analyzed the effects of these mutations in vivo and in vitro. Limited trypsinolysis of MnmG suggests significant conformational changes upon FAD binding.

  7. Fabrication of submicron proteinaceous structures by direct laser writing

    SciTech Connect

    Serien, Daniela; Takeuchi, Shoji

    2015-07-06

    In this paper, we provide a characterization of truly free-standing proteinaceous structures with submicron feature sizes depending on the fabrication conditions by model-based analysis. Protein cross-linking of bovine serum albumin is performed by direct laser writing and two-photon excitation of flavin adenine dinucleotide. We analyze the obtainable fabrication resolution and required threshold energy for polymerization. The applied polymerization model allows prediction of fabrication conditions and resulting fabrication size, alleviating the application of proteinaceous structure fabrication.

  8. Production of a covalent flavin linkage in lipoamide dehydrogenase. Reaction with 8-Cl-FAD.

    PubMed

    Moore, E G; Cardemil, E; Massey, V

    1978-09-25

    A method is described for preparation of apolipoamide dehydrogenase which gives quantitative removal of FAD. Active holoenzyme can be reconstituted by incubation with FAD. Reconstitution of apoenzyme with 8-Cl-FAD results in the fixation of most of the flavin to the protein in a covalently bound form. The portion noncovalently bound was shown to be unmodified 8-Cl-FAD. The covalently bound flavin has an absorption spectrum quite different from that of 8-Cl-FAD. It has a single band in the visible with a maximum at 459 nm (extinction coefficient of 22 mM-1 cm-1) and a shoulder at 480 nm. Model reactions between 8-Cl-Flavin (riboflavin or FAD) and organic thiols (thiophenol, beta-mercaptoethanol, or N-acetylcysteine) give products with spectra which are similar to that of FAD covalently bound to lipoamide dehydrogenase. The products of the model reactions have a single visible band with a maximum at 480 nm (extinction coefficient of 23.6 mM-1 cm-1 to 28.4 mM-1 cm-1) and a shoulder at 460 nm. The products of the model reaction and the covalently bound FAD of lipoamide dehydrogenase appear to be the result of a nucleophilic attack on the carbon at position 8 of the flavin ring by a thiolate anion, displacing the chloride. Thus, the product of the model reaction is 8-(RS)-flavin, and the product of the reaction between 8-Cl-FAD and protein probably has a cysteinyl residue covalently attacked at position 8 of FAD. Reconstitution of apoliopoamide dehydrogenase with 8-Cl-FAD gives two enzyme products which are fractionated by ammonium sulfate. Enzyme fractionating between 20% and 45% ammonium sulfate is monomeric and contains covanently bound FAD. Enzyme fractionating between 55% and 75% ammonium sulfate is dimeric and contains both covalently bound FAD and noncovalently bound 8-Cl-FAD. Both protein fractions contain one FAD per protein subunit and both are active with physiological substrates with Km values for NAD and dihydrolipoamide similar to those of native lipoamide

  9. Production of a covalent flavin linkage in lipoamide dehydrogenase. Reaction with 8-Cl-FAD.

    PubMed

    Moore, E G; Cardemil, E; Massey, V

    1978-09-25

    A method is described for preparation of apolipoamide dehydrogenase which gives quantitative removal of FAD. Active holoenzyme can be reconstituted by incubation with FAD. Reconstitution of apoenzyme with 8-Cl-FAD results in the fixation of most of the flavin to the protein in a covalently bound form. The portion noncovalently bound was shown to be unmodified 8-Cl-FAD. The covalently bound flavin has an absorption spectrum quite different from that of 8-Cl-FAD. It has a single band in the visible with a maximum at 459 nm (extinction coefficient of 22 mM-1 cm-1) and a shoulder at 480 nm. Model reactions between 8-Cl-Flavin (riboflavin or FAD) and organic thiols (thiophenol, beta-mercaptoethanol, or N-acetylcysteine) give products with spectra which are similar to that of FAD covalently bound to lipoamide dehydrogenase. The products of the model reactions have a single visible band with a maximum at 480 nm (extinction coefficient of 23.6 mM-1 cm-1 to 28.4 mM-1 cm-1) and a shoulder at 460 nm. The products of the model reaction and the covalently bound FAD of lipoamide dehydrogenase appear to be the result of a nucleophilic attack on the carbon at position 8 of the flavin ring by a thiolate anion, displacing the chloride. Thus, the product of the model reaction is 8-(RS)-flavin, and the product of the reaction between 8-Cl-FAD and protein probably has a cysteinyl residue covalently attacked at position 8 of FAD. Reconstitution of apoliopoamide dehydrogenase with 8-Cl-FAD gives two enzyme products which are fractionated by ammonium sulfate. Enzyme fractionating between 20% and 45% ammonium sulfate is monomeric and contains covanently bound FAD. Enzyme fractionating between 55% and 75% ammonium sulfate is dimeric and contains both covalently bound FAD and noncovalently bound 8-Cl-FAD. Both protein fractions contain one FAD per protein subunit and both are active with physiological substrates with Km values for NAD and dihydrolipoamide similar to those of native lipoamide

  10. Flavin-derived self-organization and chirality separation of single-walled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Ju, Sang-Yong

    2008-07-01

    Formed by rolling up a two-dimensional sheet of one or more layer of graphite, graphene, carbon nanotubes (SWNTs) are the marvel materials of modern materials science. They are phenomenally strong and stiff, and have the unusual property of being excellent conductors of heat along the tube's axis, but good thermal insulators across it. But it is their electrical characteristics that excite the most interest. Especially, single-walled carbon nanotube (SWNTs), formed by one layer of cylindrical graphene, has better physical properties over multi-walled carbon nanotubes (MWNTs) having over two layer of graphene. Depending on the precise way they are rolled up, which is defined by ( n,m) vector, SWNTs can be made into either metals or semiconductors. So far, SWNTs can generally only be fabricated in batches that vary widely, both in the diameter of the individual tubes and in the orientation of their graphene lattice relative to the tube axis, the property known as chirality. Separating out these various conformations is a challenging, but one that must be solved if nanotubes are ever to fulfill their electrifying potential in devices. This thesis presents that flavin-based helical self-assembly can impart multi degrees of SWNTs separation (i.e., metallicity, diameter, chirality, and handedness). As opening chapters for carbon nanotube and flavin derivative, Chapter 1 provide the introduction of carbon nanotubes, especially single-walled tubes, and the current state-of-the-art nanotube separation. Also, Chapter 1 presents a variety of naturally-occurring flavin derivatives, their redox behavior, and their biological utilization as cofactors for various proteins. Motivated by chemoluminescence of flavin mononucleotide (FMN, phosphorylated form of Vitamin B2) with bacterial luciferase, Chapter 2 discuss about the synthesis and covalent attachment of flavin mononucleotide (FMN, phosphorylated form of Vitamin B2) analogue to oxidized SWNTs. Along with nine step synthesis

  11. DNA methylation on N6-adenine in C. elegans

    PubMed Central

    Greer, Eric Lieberman; Blanco, Mario Andres; Gu, Lei; Sendinc, Erdem; Liu, Jianzhao; Aristizábal-Corrales, David; Hsu, Chih-Hung; Aravind, L.; He, Chuan; Shi, Yang

    2015-01-01

    Summary In mammalian cells, DNA methylation on the 5th position of cytosine (5mC) plays an important role as an epigenetic mark. However, DNA methylation was considered to be absent in C. elegans because of the lack of detectable 5mC as well as homologs of the cytosine DNA methyltransferases. Here, using multiple approaches, we demonstrate the presence of adenine N6-methylation (6mA) in C. elegans DNA. We further demonstrate that this modification increases trans-generationally in a paradigm of epigenetic inheritance. Importantly, we identify a DNA demethylase, NMAD-1, and a potential DNA methyltransferase, DAMT-1, which regulate 6mA levels and crosstalk between methylation of histone H3K4me2 and 6mA, and control the epigenetic inheritance of phenotypes associated with the loss of the H3K4me2 demethylase spr-5. Together, these data identify a DNA modification in C. elegans and raise the exciting possibility that 6mA may be a carrier of heritable epigenetic information in eukaryotes. PMID:25936839

  12. The structure of flavin-dependent tryptophan 7-halogenase RebH

    SciTech Connect

    Bitto, Eduard; Huang, Yu; Bingman, Craig A.; Singh, Shanteri; Thorson, Jon S.; Phillips, Jr., George N.

    2010-02-19

    Enzyme catalyzed regio- and stereo-specific halogenations influence the biological activity of a diverse array of therapeutically important natural products, including the antibiotics vancomycin and chloramphenicol as well as the anticancer agents calicheamicin and rebeccamycin. The major class of enzymes responsible for this challenging synthetic reaction, the flavin-dependent halogenases, catalyzes the formation of carbon-halogen bonds using flavin, a halide ion (Cl{sup -}, Br{sup -} or I{sup -}), and O{sub 2}. Recent mechanistic and structural advances achieved with the model flavin-dependent tryptophan 7-halogenases PrnA and RebH have greatly enhanced the level of understanding of this unique reaction. According to these studies, the mechanism for tryptophan halogenation proceeds via FAD(C4a)-OOH activation of a chloride ion into the transient chlorinating species HOCl. The key evidence for the requirement of a transient chlorinating species is the discovery that a {approx}10-{angstrom}-long tunnel separates FAD and tryptophan in the ligand-bound form of PrnA. In a recent compelling study to elucidate the strategy by which RebH controls this highly reactive and indiscriminant oxidant, a Lys79-{var_epsilon}NH-Cl chloramine intermediate was implicated as the actual chlorinating species within RebH and a structural investigation of RebH was reported. Here we report our independent structural analysis of Lechevalieria aerocolonigenes RebH (Uni-Prot accession number Q8KHZ8, 530 amino acids) in its apo-form as well as in a complex with both tryptophan and FAD.

  13. Sub-millitesla magnetic field effects on the recombination reaction of flavin and ascorbic acid radicals.

    PubMed

    Evans, Emrys W; Kattnig, Daniel R; Henbest, Kevin B; Hore, P J; Mackenzie, Stuart R; Timmel, Christiane R

    2016-08-28

    Even though the interaction of a <1 mT magnetic field with an electron spin is less than a millionth of the thermal energy at room temperature (kBT), it still can have a profound effect on the quantum yields of radical pair reactions. We present a study of the effects of sub-millitesla magnetic fields on the photoreaction of flavin mononucleotide with ascorbic acid. Direct control of the reaction pathway is achieved by varying the rate of electron transfer from ascorbic acid to the photo-excited flavin. At pH 7.0, we verify the theoretical prediction that, apart from a sign change, the form of the magnetic field effect is independent of the initial spin configuration of the radical pair. The data agree well with model calculations based on a Green's function approach that allows multinuclear spin systems to be treated including the diffusive motion of the radicals, their spin-selective recombination reactions, and the effects of the inter-radical exchange interaction. The protonation states of the radicals are uniquely determined from the form of the magnetic field-dependence. At pH 3.0, the effects of two chemically distinct radical pair complexes combine to produce a pronounced response to ∼500 μT magnetic fields. These findings are relevant to the magnetic responses of cryptochromes (flavin-containing proteins proposed as magnetoreceptors in birds) and may aid the evaluation of effects of weak magnetic fields on other biologically relevant electron transfer processes. PMID:27586950

  14. Sub-millitesla magnetic field effects on the recombination reaction of flavin and ascorbic acid radicals

    NASA Astrophysics Data System (ADS)

    Evans, Emrys W.; Kattnig, Daniel R.; Henbest, Kevin B.; Hore, P. J.; Mackenzie, Stuart R.; Timmel, Christiane R.

    2016-08-01

    Even though the interaction of a <1 mT magnetic field with an electron spin is less than a millionth of the thermal energy at room temperature (kBT), it still can have a profound effect on the quantum yields of radical pair reactions. We present a study of the effects of sub-millitesla magnetic fields on the photoreaction of flavin mononucleotide with ascorbic acid. Direct control of the reaction pathway is achieved by varying the rate of electron transfer from ascorbic acid to the photo-excited flavin. At pH 7.0, we verify the theoretical prediction that, apart from a sign change, the form of the magnetic field effect is independent of the initial spin configuration of the radical pair. The data agree well with model calculations based on a Green's function approach that allows multinuclear spin systems to be treated including the diffusive motion of the radicals, their spin-selective recombination reactions, and the effects of the inter-radical exchange interaction. The protonation states of the radicals are uniquely determined from the form of the magnetic field-dependence. At pH 3.0, the effects of two chemically distinct radical pair complexes combine to produce a pronounced response to ˜500 μT magnetic fields. These findings are relevant to the magnetic responses of cryptochromes (flavin-containing proteins proposed as magnetoreceptors in birds) and may aid the evaluation of effects of weak magnetic fields on other biologically relevant electron transfer processes.

  15. Aspergillus fumigatus SidA is a highly specific ornithine hydroxylase with bound flavin cofactor.

    PubMed

    Chocklett, Samuel W; Sobrado, Pablo

    2010-08-10

    Ferrichrome is a hydroxamate-containing siderophore produced by the pathogenic fungus Aspergillus fumigatus under iron-limiting conditions. This siderophore contains N(5)-hydroxylated l-ornithines essential for iron binding. A. fumigatus siderophore A (Af SidA) catalyzes the flavin- and NADPH-dependent hydroxylation of l-ornithine in ferrichrome biosynthesis. Af SidA was recombinantly expressed and purified as a soluble tetramer and is the first member of this class of flavin monooxygenases to be isolated with a bound flavin cofactor. The enzyme showed typical saturation kinetics with respect to l-ornithine while substrate inhibition was observed at high concentrations of NADPH and NADH. Increasing amounts of hydrogen peroxide were measured as a function of reduced nicotinamide coenzyme concentration, indicating that inhibition was caused by increased uncoupling. Af SidA is highly specific for its amino acid substrate, only hydroxylating l-ornithine. An 8-fold preference in the catalytic efficiency was determined for NADPH compared to NADH. In the absence of substrate, Af SidA can be reduced by NADPH, and a C4a-(hydro)peroxyflavin intermediate is observed. The decay of this intermediate is accelerated by l-ornithine binding. This intermediate was only stabilized by NADPH and not by NADH, suggesting a role for NADP(+) in the stabilization of intermediates in the reaction of Af SidA. NADP(+) is a competitive inhibitor with respect to NADPH, demonstrating that Af SidA forms a ternary complex with NADP(+) and l-ornithine during catalysis. The data suggest that Af SidA likely proceeds by a sequential kinetic mechanism.

  16. Coordinated regulation of accessory genetic elements produces cyclic di-nucleotides for V. cholerae virulence.

    PubMed

    Davies, Bryan W; Bogard, Ryan W; Young, Travis S; Mekalanos, John J

    2012-04-13

    The function of the Vibrio 7(th) pandemic island-1 (VSP-1) in cholera pathogenesis has remained obscure. Utilizing chromatin immunoprecipitation sequencing and RNA sequencing to map the regulon of the master virulence regulator ToxT, we identify a TCP island-encoded small RNA that reduces the expression of a previously unrecognized VSP-1-encoded transcription factor termed VspR. VspR modulates the expression of several VSP-1 genes including one that encodes a novel class of di-nucleotide cyclase (DncV), which preferentially synthesizes a previously undescribed hybrid cyclic AMP-GMP molecule. We show that DncV is required for efficient intestinal colonization and downregulates V. cholerae chemotaxis, a phenotype previously associated with hyperinfectivity. This pathway couples the actions of previously disparate genomic islands, defines VSP-1 as a pathogenicity island in V. cholerae, and implicates its occurrence in 7(th) pandemic strains as a benefit for host adaptation through the production of a regulatory cyclic di-nucleotide.

  17. Separation and characterization of oxaliplatin dinucleotides from DNA using HPLC-ESI ion trap mass spectrometry.

    PubMed

    Mowaka, Shereen; Linscheid, Michael

    2008-11-01

    Oxaliplatin is a third-generation platinum complex, and has a broad spectrum of antitumor activity. Such platinum complexes with the DACH carrier ligand have recently received increasing attention since they show efficacy against cisplatin-resistant cell lines. As the foremost indication of antitumor activity of platinum drugs is the formation of adducts with genomic DNA, calf thymus DNA-oxaliplatin adducts were the major target in this study. Calf thymus DNA was incubated with oxaliplatin, resulting in the formation of a large number of platinum-DNA adducts. Treated DNA was digested into the dinucleotides with a combination of enzymes, namely, benzonase, alkaline phosphatase, and nuclease S1. Using a high-performance liquid chromatography, we carried out the separation of individual platinum-DNA adducts which were concurrently identified using electrospray ionization ion trap mass spectrometry (MS). Both 1,2-intrastrand and 1,2-interstrand cross-linked adducts were found; however, those of the intrastrand nature have a considerably higher abundance than those of the interstrand cross-links. Among them, d(GpG)-oxaliplatin was the most abundant bifuctional adduct. To a lesser extent, a few monofunctional adducts were detected as well. MS(n) experiments served to ascertain the detailed structures of oxaliplatin adducts of dinucleoside monophosphates and of dinucleotides.

  18. Base-boronated dinucleotides: synthesis and effect of N7-cyanoborane substitution on the base protons.

    PubMed Central

    Hasan, A; Li, H; Tomasz, J; Shaw, B R

    1996-01-01

    Boron-modified nucleic acids comprise a new set of DNA mimics that have potential biological and therapeutic applications. A series of nine dinucleotides containing N7-cyanoborane-2'-deoxyguanosine ((7b)dG) at the 3', 5' or both positions of the phosphodiester linkage have been synthesized using solution phase phosphoramidite chemistry. Fmoc was used as the 5'-protecting group because of incompatibility of the cyanoborane moiety with 5'-DMT cations generated during the deprotection step. The presence of the cyanoborane group was confirmed on the basis of Fab-MS and 1H NMR spectroscopy. The H-8 proton of (7b)dG in the dinucleotides shifted 0.35-0.80 p.p.m. downfield relative to that of unmodified dG. A comparison of the D20 exchange kinetics of the H-8 proton at 60 degrees C showed that H-8 of (7b)dG is very labile relative to unmodified dG, indicating that the N7-cyanoborane modification increases the acidity of the H-8 proton of (7b)dG. These studies illustrate the feasibility of synthesizing boron-containing oligonucleotides which are modified at the N7-guanine to block Hoogsteen pairing in the DNA major groove. PMID:8668548

  19. Structural Flexibility and Conformation Features of Cyclic Dinucleotides in Aqueous Solutions.

    PubMed

    Che, Xing; Zhang, Jun; Zhu, Yanyu; Yang, Lijiang; Quan, Hui; Gao, Yi Qin

    2016-03-17

    Cyclic dinucleotides are able to trigger the innate immune system by activating STING. It was found that the binding affinity of asymmetric 2'3'-cGAMP to symmetric dimer of STING is 3 orders of magnitude higher than that of the symmetric 3'3'-cyclic dinucleotides. Such a phenomenon has not been understood yet. Here we show that the subtle changes in phosphodiester linkage of CDNs lead to their distinct structural properties which correspond to the varied binding affinities. 2'-5' and/or 3'-5' linked CDNs adopt specific while different types of ribose puckers and backbone conformations. That ribose conformations and base types have different propensities for anti or syn glycosidic conformations further affects the overall flexibility of CDNs. The counterbalance between backbone ring tension and electrostatic repulsion, both affected by the ring size, also contributes to the different flexibility of CDNs. Our calculations reveal that the free energy cost for 2'3'-cGAMP to adopt the STING-bound structure is smaller than that for 3'3'-cGAMP and cyclic-di-GMP. These findings may serve as a reference for design of CDN-analogues as vaccine adjuvants. Moreover, the cyclization pattern of CDNs closely related to their physiological roles suggests the importance of understanding structural properties in the study of protein-ligand interactions.

  20. A cluster of highly polymorphic dinucleotide repeats in intron 17b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

    PubMed Central

    Zielenski, J; Markiewicz, D; Rininsland, F; Rommens, J; Tsui, L C

    1991-01-01

    A cluster of highly polymorphic dinucleotide repeats has been detected in intron 17b of the CFTR gene, 200 bp downstream from the preceding exon. At least 24 alleles, with sizes ranging from 7 to 56 units of a TA repeat, have been identified in a panel of 92 unrelated carriers of cystic fibrosis (CF). The common ones are those with 7, 30, and 31 dinucleotide units, with frequencies of .22, .19, and .12, respectively, among the non-CF chromosomes. Mendelian, codominant segregation of the alleles has been demonstrated in family studies, as expected. A less polymorphic dinucleotide (CA repeat) cluster has also been detected in a region 167 bp downstream from the TA repeat. The length of the CA repeat cluster varies from 11 to 17 dinucleotide units, and it appears to have an inverse relationship to that of the TA repeats. These dinucleotide repeats should be useful in genetic linkage studies, in counseling for CF families with unknown mutations, and in tracing the origins of the various mutant CF alleles. Images Figure 2 Figure 3 PMID:1720926

  1. Binding of scandium ions to metalloporphyrin-flavin complexes for long-lived charge separation.

    PubMed

    Kojima, Takahiko; Kobayashi, Ryosuke; Ishizuka, Tomoya; Yamakawa, Shinya; Kotani, Hiroaki; Nakanishi, Tatsuaki; Ohkubo, Kei; Shiota, Yoshihito; Yoshizawa, Kazunari; Fukuzumi, Shunichi

    2014-11-17

    A porphyrin-flavin-linked dyad and its zinc and palladium complexes (MPor-Fl: 2-M, M=2 H, Zn, and Pd) were newly synthesized and the X-ray crystal structure of 2-Pd was determined. The photodynamics of 2-M were examined by femto- and nanosecond laser flash photolysis measurements. Photoinduced electron transfer (ET) in 2-H2 occurred from the singlet excited state of the porphyrin moiety (H2 Por) to the flavin (Fl) moiety to produce the singlet charge-separated (CS) state (1) (H2 Por(.+) -Fl(.-) ), which decayed through back ET (BET) to form (3) [H2 Por]*-Fl with rate constants of 1.2×10(10) and 1.2×10(9)  s(-1) , respectively. Similarly, photoinduced ET in 2-Pd afforded the singlet CS state, which decayed through BET to form (3) [PdPor]*Fl with rate constants of 2.1×10(11) and 6.0×10(10)  s(-1) , respectively. The rate constant of photoinduced ET and BET of 2-M were related to the ET and BET driving forces by using the Marcus theory of ET. One and two Sc(3+) ions bind to the flavin moiety to form the Fl-Sc(3+) and Fl-(Sc(3+) )2 complexes with binding constants of K1 =2.2×10(5)  M(-1) and K2 =1.8×10(3)  M(-1) , respectively. Other metal ions, such as Y(3+) , Zn(2+) , and Mg(2+) , form only 1:1 complexes with flavin. In contrast to 2-M and the 1:1 complexes with metal ions, which afforded the short-lived singlet CS state, photoinduced ET in 2-Pd⋅⋅⋅Sc(3+) complexes afforded the triplet CS state ((3) [PdPor(.+) -Fl(.-) (Sc(3+) )2 ]), which exhibited a remarkably long lifetime of τ=110 ms (kBET =9.1 s(-1) ).

  2. Flavin and porphyrin-micro optical fibre biosensor: analysis and design

    NASA Astrophysics Data System (ADS)

    Velazquez-Gonzalez, J. S.; Mujica-Ascencio, S.; Aguilar Morales, A. I.; Marrujo-Garcia, S.; Alvarez-Chavez, J. A.; Martinez-Pinon, F.

    2014-05-01

    Micro Optical Fibre Biosensors (MOFBs) are emerging as one of the most sensitive bio-detection system technologies which do not require of labelling or amplification of the analyte. In these devices, a short region of the fibre core is exposed to the external environment so that the evanescent field can interact with biological species such as cells, proteins, and DNA. In order to increase the sensitivity and selectivity, MOFBs are often used in combination with other optical transduction mechanisms such as changes in refractive index, absorption, fluorescence and surface plasmon resonance. In this work we present the full characteristics, analysis and design of a MOFBs for Flavin and Porphyrin detection.

  3. [Flavin pool in Propionibacterium shermanii and the formation of corrinoids by bacterial cultures].

    PubMed

    Datsiuk, N M; Kucheras, R V; Rurik, V D; Gudz', S P; Kostruba, M F; Kremenetskaia, S Ia

    1978-01-01

    The content of flavins and vitamin B12 was studied in the cultures of Propionibacterium shermanii. The limited ability of propionic bacteria to synthesize 5,6-dimethylbenzimidazole, the nucleotide base in the true forms of vitamin B12, was found to be caused by a deficiency of the biogenetic precursor (riboflavin). Exogenous 5,6-dimethylbenzimidazole had no effect on the processes of flavinogenesis. In the absence of the nucleotide base, the yield of the true forms of vitamin B12 increased upon aeration of cultures which were grown for four days on an iron-deficient medium in the anaerobic conditions.

  4. Single Amino Acid Switch between a Flavin-Dependent Dehalogenase and Nitroreductase.

    PubMed

    Mukherjee, Arnab; Rokita, Steven E

    2015-12-16

    A single mutation within a flavoprotein is capable of switching the catalytic activity of a dehalogenase into a nitroreductase. This change in function correlates with a destabilization of the one-electron-reduced flavin semiquinone that is differentially expressed in the nitro-FMN reductase superfamily during redox cycling. The diversity of function within such a superfamily therefore has the potential to arise from rapid evolution, and its members should provide a convenient basis for developing new catalysts with an altered specificity of choice. PMID:26616824

  5. Spin-dependent electron transport in zinc- and manganese-doped adenine molecules

    SciTech Connect

    Simchi, Hamidreza; Esmaeilzadeh, Mahdi Mazidabadi, Hossein

    2014-01-28

    The spin-dependent electron transport properties of zinc- and manganese-doped adenine molecules connected to zigzag graphene leads are studied in the zero bias regime using the non-equilibrium Green's function method. The conductance of the adenine molecule increased and became spin-dependent when a zinc or manganese atom was doped into the molecules. The effects of a transverse electric field on the spin-polarization of the transmitted electrons were investigated and the spin-polarization was controlled by changing the transverse electric field. Under the presence of a transverse electric field, both the zinc- and manganese-doped adenine molecules acted as spin-filters. The maximum spin-polarization of the manganese-doped adenine molecule was greater than the molecule doped with zinc.

  6. Identification of a mitochondrial ATP synthase-adenine nucleotide translocator complex in Leishmania.

    PubMed

    Detke, Siegfried; Elsabrouty, Rania

    2008-01-01

    The ATP synthasome is a macromolecular complex consisting of ATP synthase, adenine nucleotide translocator and phosphate carrier. To determine if this complex is evolutionary old or young, we searched for its presence in Leishmania, a mitochondria containing protozoan which evolved from the main eukaryote line soon after eukaryotes split from prokaryotes. Sucrose gradient centrifugation showed that the distribution of ANT among the fractions coincided with the distribution of ATP synthase. In addition, ATP synthase co-precipitated with FLAG tagged and wild type adenine nucleotide translocator isolated with anti FLAG and anti adenine nucleotide translocator antibodies, respectively. These data indicate that the adenine nucleotide translocator interacted with the ATP synthase to form a stable structure referred to as the ATP synthasome. The presence of the ATP synthasome in Leishmania, an organism branching off the main line of eukaryotes early in the development of eukaryotes, as well as in higher eukaryotes suggests that the ATP synthasome is a phylogenetically ancient structure. PMID:17920025

  7. Expression, Purification, and Characterization of a Recombinant Flavin Reductase from the Luminescent Marine Bacterium "Photobacterium Leiognathi": A Set of Exercises for Students

    ERIC Educational Resources Information Center

    Crowley, Thomas E.

    2010-01-01

    In "Photobacterium," the flavin reductase encoded by "lux"G regenerates the reduced form of flavin mononucleotide (FMN). Reduced FMN is one of the substrates of the luciferase enzyme that catalyzes a light-emitting reaction. A set of experiments, that employs a "lux"G-expression plasmid construct (pGhis) and is suitable for an undergraduate…

  8. Benchmark Thermochemistry for Biologically Relevant Adenine and Cytosine. A Combined Experimental and Theoretical Study.

    PubMed

    Emel'yanenko, Vladimir N; Zaitsau, Dzmitry H; Shoifet, Evgeni; Meurer, Florian; Verevkin, Sergey P; Schick, Christoph; Held, Christoph

    2015-09-17

    The thermochemical properties available in the literature for adenine and cytosine are in disarray. A new condensed phase standard (p° = 0.1 MPa) molar enthalpy of formation at T = 298.15 K was measured by using combustion calorimetry. New molar enthalpies of sublimation were derived from the temperature dependence of vapor pressure measured by transpiration and by the quarz-crystal microbalance technique. The heat capacities of crystalline adenine and cytosine were measured by temperature-modulated DSC. Thermodynamic data on adenine and cytosine available in the literature were collected, evaluated, and combined with our experimental results. Thus, the evaluated collection of data together with the new experimental results reported here has helped to resolve contradictions in the available enthalpies of formation. A set of reliable thermochemical data is recommended for adenine and cytosine for further thermochemical calculations. Quantum-chemical calculations of the gas phase molar enthalpies of formation of adenine and cytosine have been performed by using the G4 method and results were in excellent agreement with the recommended experimental data. The standard molar entropies of formation and the standard molar Gibbs functions of formation in crystal and gas state have been calculated. Experimental vapor-pressure data measured in this work were used to estimate pure-component PC-SAFT parameters. This allowed modeling solubility of adenine and cytosine in water over the temperature interval 278-310 K. PMID:26317826

  9. Benchmark Thermochemistry for Biologically Relevant Adenine and Cytosine. A Combined Experimental and Theoretical Study.

    PubMed

    Emel'yanenko, Vladimir N; Zaitsau, Dzmitry H; Shoifet, Evgeni; Meurer, Florian; Verevkin, Sergey P; Schick, Christoph; Held, Christoph

    2015-09-17

    The thermochemical properties available in the literature for adenine and cytosine are in disarray. A new condensed phase standard (p° = 0.1 MPa) molar enthalpy of formation at T = 298.15 K was measured by using combustion calorimetry. New molar enthalpies of sublimation were derived from the temperature dependence of vapor pressure measured by transpiration and by the quarz-crystal microbalance technique. The heat capacities of crystalline adenine and cytosine were measured by temperature-modulated DSC. Thermodynamic data on adenine and cytosine available in the literature were collected, evaluated, and combined with our experimental results. Thus, the evaluated collection of data together with the new experimental results reported here has helped to resolve contradictions in the available enthalpies of formation. A set of reliable thermochemical data is recommended for adenine and cytosine for further thermochemical calculations. Quantum-chemical calculations of the gas phase molar enthalpies of formation of adenine and cytosine have been performed by using the G4 method and results were in excellent agreement with the recommended experimental data. The standard molar entropies of formation and the standard molar Gibbs functions of formation in crystal and gas state have been calculated. Experimental vapor-pressure data measured in this work were used to estimate pure-component PC-SAFT parameters. This allowed modeling solubility of adenine and cytosine in water over the temperature interval 278-310 K.

  10. Phosphonate analogues of dinucleotides as substrates for DNA-dependent RNA polymerase from Escherichia coli in primed abortive initiation reaction.

    PubMed

    Cvekl, A; Horská, K; Sebesta, K; Rosenberg, I; Holý, A

    1989-02-01

    Dinucleotides (3'-5')-ApU and UpA and their 3'-O-phosphonylmethyl and 5'-O-phosphonylmethyl analogues were studied as substrates in the primed abortive synthesis catalysed by Escherichia coli DNA-dependent RNA polymerase on poly[d(A-T)] template. All phosphonate analogues of dinucleotides containing the anomalous sugar-phosphate backbone are substrates for the holoenzyme as verified by RNase A and RNase T2 digestion of the trinucleotide analogues obtained. The finding that phosphonate dinucleotides act as primers for transcription indicates that steric requirements at the initiation site are not as specific as previously supposed. Analysis of kinetic constants of ordered bibi reaction Kia, KmA, KmB and Vmax suggests that the instability of short RNA-DNA hybrids contributes to the abortive release of trinucleotides formed.

  11. Labeling of mitochondrial adenine nucleotides of bovine sperm

    SciTech Connect

    Cheetham, J.; Lardy, H.A.

    1986-05-01

    Incorporation of /sup 32/P/sub i/ into the adenine nucleotide pool of intact bovine spermatozoa utilizing endogenous substrates results in a specific activity (S.A.) ratio ATP/ADP of 0.3 to 0.5, suggesting compartmentation of nucleotide pools or a pathway for phosphorylation of AMP in addition to the myokinase reaction. Incubation of filipin-permeabilized cells with pyruvate, acetylcarnitine, or ..cap alpha..-ketoglutarate (..cap alpha..KG) resulted in ATP-ADP S.A. ratios of 0.5, 0.8, and 1.6, respectively, for mitochondrial nucleotides. However, when malate was included with pyruvate or acetylcarnitine, the ATP/ADP S.A. ratio increased by 400% to 2.0 for pyruvate/malate and by 290% to 2.8 for acetylcarnitine/malate, while the ATP/ADP ratio increased by less than 100% in both cases. These results may indicate that under conditions of limited flux through the citric acid cycle a pathway for phosphorylation of AMP from a precursor other than ATP exists or that ATP is compartmented within the mitochondrion. In the presence of uncoupler and oligomycin with ..cap alpha..KG, pyruvate/malate, or acetylcarnitine/malate, /sup 32/P/sub i/ is incorporated primarily into ATP, resulting in an ATP/ADP S.A. ratio of 4.0 for ..cap alpha..KG, 2.7 for pyruvate/malate, and 2.8 for acetylcarnitine/malate. These data are consistent with phosphorylation of ADP during substrate level phosphorylation in the citric acid cycle.

  12. Phenotype and Genotype Characterization of Adenine Phosphoribosyltransferase Deficiency

    PubMed Central

    Bollée, Guillaume; Dollinger, Cécile; Boutaud, Lucile; Guillemot, Delphine; Bensman, Albert; Harambat, Jérôme; Deteix, Patrice; Daudon, Michel; Knebelmann, Bertrand

    2010-01-01

    Adenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive disorder causing 2,8-dihydroxyadenine stones and renal failure secondary to intratubular crystalline precipitation. Little is known regarding the clinical presentation of APRT deficiency, especially in the white population. We retrospectively reviewed all 53 cases of APRT deficiency (from 43 families) identified at a single institution between 1978 and 2009. The median age at diagnosis was 36.3 years (range 0.5 to 78.0 years). In many patients, a several-year delay separated the onset of symptoms and diagnosis. Of the 40 patients from 33 families with full clinical data available, 14 (35%) had decreased renal function at diagnosis. Diagnosis occurred in six (15%) patients after reaching ESRD, with five diagnoses made at the time of disease recurrence in a renal allograft. Eight (20%) patients reached ESRD during a median follow-up of 74 months. Thirty-one families underwent APRT sequencing, which identified 54 (87%) mutant alleles on the 62 chromosomes analyzed. We identified 18 distinct mutations. A single T insertion in a splice donor site in intron 4 (IVS4 + 2insT), which produces a truncated protein, accounted for 40.3% of the mutations. We detected the IVS4 + 2insT mutation in two (0.98%) of 204 chromosomes of healthy newborns. This report, which is the largest published series of APRT deficiency to date, highlights the underdiagnosis and potential severity of this disease. Early diagnosis is crucial for initiation of effective treatment with allopurinol and for prevention of renal complications. PMID:20150536

  13. Thermodynamics of the quasi-epitaxial flavin assembly around various-chirality carbon nanotubes.

    PubMed

    Sharifi, Roholah; Samaraweera, Milinda; Gascón, José A; Papadimitrakopoulos, Fotios

    2014-05-21

    Establishing methods to accurately assess and model the binding strength of surfactants around a given-chirality single-walled carbon nanotube (SWNT) are crucial for selective enrichment, targeted functionalization, and spectrally sharp nanodevices. Unlike surfactant exchange, which is subject to interferences from the second surfactant, we herein introduce a thermal dissociation method based on reversible H(+)/O2 doping to determine SWNT/surfactant thermodynamic stability values with greater fidelity. Thermodynamic values were reproduced using molecular mechanics augmented by ab initio calculations in order to better assess π-π interactions. This afforded detailed quantification of the flavin binding strength in terms of π-π stacking (55-58%), with the remaining portion roughly split 3:1 between electrostatic plus van der Waals flavin mononucleotide (FMN) interdigitation and H-bonding interactions, respectively. Quasi-epitaxial π-π alignment between the near-armchair FMN helix and the underlying nanotube lattice plays a crucial role in stabilizing these assemblies. The close resemblance of the thermal dissociation method to helix-coil and ligand-binding transitions of DNA opens up a unique insight into the molecular engineering of self-organizing surfactants around various-chirality nanotubes.

  14. Revealing the moonlighting role of NADP in the structure of a flavin-containing monooxygenase.

    PubMed

    Alfieri, Andrea; Malito, Enrico; Orru, Roberto; Fraaije, Marco W; Mattevi, Andrea

    2008-05-01

    Flavin-containing monooxygenases (FMOs) are, after cytochromes P450, the most important monooxygenase system in humans and are involved in xenobiotics metabolism and variability in drug response. The x-ray structure of a soluble prokaryotic FMO from Methylophaga sp. strain SK1 has been solved at 2.6-A resolution and is now the protein of known structure with the highest sequence similarity to human FMOs. The structure possesses a two-domain architecture, with both FAD and NADP(+) well defined by the electron density maps. Biochemical analysis shows that the prokaryotic enzyme shares many functional properties with mammalian FMOs, including substrate specificity and the ability to stabilize the hydroperoxyflavin intermediate that is crucial in substrate oxygenation. On the basis of their location in the structure, the nicotinamide ring and the adjacent ribose of NADP(+) turn out to be an integral part of the catalytic site being actively engaged in the stabilization of the oxygenating intermediate. This feature suggests that NADP(H) has a moonlighting role, in that it adopts two binding modes that allow it to function in both flavin reduction and oxygen reactivity modulation, respectively. We hypothesize that a relative domain rotation is needed to bring NADP(H) to these distinct positions inside the active site. Localization of mutations in human FMO3 that are known to cause trimethylaminuria (fish-odor syndrome) in the elucidated FMO structure provides a structural explanation for their biological effects.

  15. Revealing the moonlighting role of NADP in the structure of a flavin-containing monooxygenase

    PubMed Central

    Alfieri, Andrea; Malito, Enrico; Orru, Roberto; Fraaije, Marco W.; Mattevi, Andrea

    2008-01-01

    Flavin-containing monooxygenases (FMOs) are, after cytochromes P450, the most important monooxygenase system in humans and are involved in xenobiotics metabolism and variability in drug response. The x-ray structure of a soluble prokaryotic FMO from Methylophaga sp. strain SK1 has been solved at 2.6-Å resolution and is now the protein of known structure with the highest sequence similarity to human FMOs. The structure possesses a two-domain architecture, with both FAD and NADP+ well defined by the electron density maps. Biochemical analysis shows that the prokaryotic enzyme shares many functional properties with mammalian FMOs, including substrate specificity and the ability to stabilize the hydroperoxyflavin intermediate that is crucial in substrate oxygenation. On the basis of their location in the structure, the nicotinamide ring and the adjacent ribose of NADP+ turn out to be an integral part of the catalytic site being actively engaged in the stabilization of the oxygenating intermediate. This feature suggests that NADP(H) has a moonlighting role, in that it adopts two binding modes that allow it to function in both flavin reduction and oxygen reactivity modulation, respectively. We hypothesize that a relative domain rotation is needed to bring NADP(H) to these distinct positions inside the active site. Localization of mutations in human FMO3 that are known to cause trimethylaminuria (fish-odor syndrome) in the elucidated FMO structure provides a structural explanation for their biological effects. PMID:18443301

  16. The role of threonine 37 in flavin reactivity of the old yellow enzyme

    PubMed Central

    Xu, Dong; Kohli, Rahul M.; Massey, Vincent

    1999-01-01

    Threonine 37 is conserved among all the members of the old yellow enzyme (OYE) family. The hydroxyl group of this residue forms a hydrogen bond with the C-4 oxygen atom of the FMN reaction center of the enzyme [Fox, K. M. & Karplus, P. A. (1994) Structure 2, 1089–1105]. The position of Thr-37 and its interaction with flavin allow for speculations about its role in enzyme activity. This residue was mutated to alanine and the mutant enzyme was studied and compared with the wild-type OYE1 to evaluate its mechanistic function. The mutation has different effects on the two separate half-reactions of the enzyme. The mutant enzyme has enhanced activity in the oxidative half-reaction but the reductive half-reaction is slowed down by more than one order of magnitude. The peaks of the absorption spectra for enzyme bound with phenolic compounds are shifted toward shorter wavelengths than those of wild-type OYE1, consistent with its lower redox potential. It is suggested that Thr-37 in the wild-type OYE1 increases the redox potential of the enzyme by stabilizing the negative charge of the reduced flavin through hydrogen bonding with it. PMID:10097075

  17. Detecting horizontally transferred and essential genes based on dinucleotide relative abundance.

    PubMed

    Baran, Robert H; Ko, Hanseok

    2008-10-01

    Various methods have been developed to detect horizontal gene transfer in bacteria, based on anomalous nucleotide composition, assuming that compositional features undergo amelioration in the host genome. Evolutionary theory predicts the inevitability of false positives when essential sequences are strongly conserved. Foreign genes could become more detectable on the basis of their higher order compositions if such features ameliorate more rapidly and uniformly than lower order features. This possibility is tested by comparing the heterogeneities of bacterial genomes with respect to strand-independent first- and second-order features, (i) G + C content and (ii) dinucleotide relative abundance, in 1 kb segments. Although statistical analysis confirms that (ii) is less inhomogeneous than (i) in all 12 species examined, extreme anomalies with respect to (ii) in the Escherichia coli K12 genome are typically co-located with essential genes.

  18. Sequence-dependent dynamics of duplex DNA: the applicability of a dinucleotide model.

    PubMed Central

    Okonogi, T M; Alley, S C; Reese, A W; Hopkins, P B; Robinson, B H

    2002-01-01

    The short-time (submicrosecond) bending dynamics of duplex DNA were measured to determine the effect of sequence on dynamics. All measurements were obtained from a single site on duplex DNA, using a single, site-specific modified base containing a rigidly tethered, electron paramagnetic resonance active spin probe. The observed dynamics are interpreted in terms of single-step sequence-dependent bending force constants, determined from the mean squared amplitude of bending relative to the end-to-end vector using the modified weakly bending rod model. The bending dynamics at a single site are a function of the sequence of the nucleotides constituting the duplex DNA. We developed and examined several dinucleotide-based models for flexibility. The models indicate that the dominant feature of the dynamics is best explained in terms of purine- and pyrimidine-type steps, although distinction is made among all 10 unique steps: It was found that purine-purine steps (which are the same as pyrimidine-pyrimidine steps) were near average in flexibility, but the pyrimidine-purine steps (5' to 3') were nearly twice as flexible, whereas purine-pyrimidine steps were more than half as flexible as average DNA. Therefore, the range of stepwise flexibility is approximately fourfold and is characterized by both the type of base pair step (pyrimidine/purine combination) and the identity of the bases within the pair (G, A, T, or C). All of the four models considered here underscore the complexity of the dependence of dynamics on DNA sequence with certain sequences not satisfactorily explainable in terms of any dinucleotide model. These findings provide a quantitative basis for interpreting the dynamics and kinetics of DNA-sequence-dependent biological processes, including protein recognition and chromatin packaging. PMID:12496111

  19. Dissection of the PHO pathway in Schizosaccharomyces pombe using epistasis and the alternate repressor adenine.

    PubMed

    Estill, Molly; Kerwin-Iosue, Christine L; Wykoff, Dennis D

    2015-05-01

    In Saccharomyces cerevisiae, intracellular phosphate levels are maintained by the PHO pathway, activation of which is assayed by increased phosphatase activity. The PHO pathway of Schizosaccharomyces pombe upregulates phosphatase activity (encoded by pho1 (+)) during low extracellular phosphate levels, but the underlying mechanism is poorly understood. We utilized an alternate repressor of pho1 (+) expression (adenine supplementation) along with epistasis analysis to develop a model of how S. pombe PHO pathway components interact. Analyzing Pho1 activity in S. pombe PHO pathway deletion mutants during adenine starvation, we observed most mutants with a phosphatase defect in phosphate starvation also had a defect in adenine starvation. Pho7, a transcription factor in the PHO pathway, is necessary for an adenine starvation-mediated increase in Pho1 activity. Comparing adenine starvation to phosphate starvation, there are differences in the degree to which individual mutants regulate the two responses. Through epistasis studies, we identified two positive regulatory arms and one repressive arm of the PHO pathway. PKA activation is a positive regulator of Pho1 activity under both environmental conditions and is critical for transducing adenine concentrations in the cell. The synthesis of IP7 also appears critical for the induction of Pho1 activity during adenine starvation, but IP7 is not critical during phosphate starvation, which differs from S. cerevisiae. Finally, Csk1 is critical for repression of pho1 (+) expression during phosphate starvation. We believe all of these regulatory arms converge to increase transcription of pho1 (+) and some of the regulation acts through pho7 (+).

  20. Flavin-derived self-organization and chirality separation of single-walled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Ju, Sang-Yong

    2008-07-01

    Formed by rolling up a two-dimensional sheet of one or more layer of graphite, graphene, carbon nanotubes (SWNTs) are the marvel materials of modern materials science. They are phenomenally strong and stiff, and have the unusual property of being excellent conductors of heat along the tube's axis, but good thermal insulators across it. But it is their electrical characteristics that excite the most interest. Especially, single-walled carbon nanotube (SWNTs), formed by one layer of cylindrical graphene, has better physical properties over multi-walled carbon nanotubes (MWNTs) having over two layer of graphene. Depending on the precise way they are rolled up, which is defined by ( n,m) vector, SWNTs can be made into either metals or semiconductors. So far, SWNTs can generally only be fabricated in batches that vary widely, both in the diameter of the individual tubes and in the orientation of their graphene lattice relative to the tube axis, the property known as chirality. Separating out these various conformations is a challenging, but one that must be solved if nanotubes are ever to fulfill their electrifying potential in devices. This thesis presents that flavin-based helical self-assembly can impart multi degrees of SWNTs separation (i.e., metallicity, diameter, chirality, and handedness). As opening chapters for carbon nanotube and flavin derivative, Chapter 1 provide the introduction of carbon nanotubes, especially single-walled tubes, and the current state-of-the-art nanotube separation. Also, Chapter 1 presents a variety of naturally-occurring flavin derivatives, their redox behavior, and their biological utilization as cofactors for various proteins. Motivated by chemoluminescence of flavin mononucleotide (FMN, phosphorylated form of Vitamin B2) with bacterial luciferase, Chapter 2 discuss about the synthesis and covalent attachment of flavin mononucleotide (FMN, phosphorylated form of Vitamin B2) analogue to oxidized SWNTs. Along with nine step synthesis

  1. Assignment of the Gene for Adenine Phosphoribosyltransferase to Human Chromosome 16 by Mouse-Human Somatic Cell Hybridization

    PubMed Central

    Tischfield, Jay A.; Ruddle, Frank H.

    1974-01-01

    A series of mouse-human hybrids was prepared from mouse cells deficient in adenine phosphoribosyltransferase (EC 2.4.2.7) and normal human cells. The hybrids were made in medium containing adenine and alanosine, an antimetabolite known to inhibit de novo adenylic acid biosynthesis. The mouse cells, unable to utilize exogenous adenine, were killed in this medium, but the hybrids proliferated as a consequence of their retaining the human aprt gene. The hybrids were then exposed to the adenine analogs 2,6-diaminopurine and 2-fluoroadenine to select for cells that had lost this gene. Before exposure to the adenine analogs, the expression of human adenine phosphoribosyltransferase by the hybrids was strongly associated only with the presence of human chromosome 16, and afterwards this was the only human chromosome consistently lost. This observation suggests that the human aprt gene can be assigned to chromosome 16. Images PMID:4129802

  2. Dual role of NADP(H) in the reaction of a flavin dependent N-hydroxylating monooxygenase.

    PubMed

    Romero, Elvira; Fedkenheuer, Michael; Chocklett, Samuel W; Qi, Jun; Oppenheimer, Michelle; Sobrado, Pablo

    2012-06-01

    Aspergillus fumigatus siderophore A (Af SidA) is a flavin-dependent monooxygenase that catalyzes the hydroxylation of ornithine, producing N(5)-hydroxyornithine. This is the first step in the biosynthesis of hydroxamate-containing siderophores in A. fumigatus. Af SidA is essential for virulence, validating this enzyme as a drug target. Af SidA can accept reducing equivalents from either NADPH or NADH and displays similar kinetic parameters when using either coenzyme. When the enzyme is reduced with NADPH and reacted with molecular oxygen, a C4a-hydroperoxyflavin intermediate is observed. When the enzyme is reduced with NADH, the intermediate is 2-fold less stable. Steady-state kinetic isotope effect values of 3 and 2 were determined for NADPH and NADH, respectively. The difference in the isotope effect values is due to differences in the rate of flavin reduction by these coenzymes. A difference in the binding mode between these coenzymes was observed by monitoring flavin fluorescence. Limited proteolysis studies show that NADP(+), and not NAD(+), protects Af SidA from proteolysis, suggesting that it induces conformational changes upon binding. Together, these results are consistent with NADPH having a role in flavin reduction and in the modulation of conformational changes, which positions NADP(+) to also play a role in stabilization of the C4a-hydroperoxyflavin.

  3. Active mammalian replication origins are associated with a high-density cluster of mCpG dinucleotides.

    PubMed Central

    Rein, T; Zorbas, H; DePamphilis, M L

    1997-01-01

    ori-beta is a well-characterized origin of bidirectional replication (OBR) located approximately 17 kb downstream of the dihydrofolate reductase gene in hamster cell chromosomes. The approximately 2-kb region of ori-beta that exhibits greatest replication initiation activity also contains 12 potential methylation sites in the form of CpG dinucleotides. To ascertain whether DNA methylation might play a role at mammalian replication origins, the methylation status of these sites was examined with bisulfite to chemically distinguish cytosine (C) from 5-methylcytosine (mC). All of the CpGs were methylated, and nine of them were located within 356 bp flanking the minimal OBR, creating a high-density cluster of mCpGs that was approximately 10 times greater than average for human DNA. However, the previously reported densely methylated island in which all cytosines were methylated regardless of their dinucleotide composition was not detected and appeared to be an experimental artifact. A second OBR, located at the 5' end of the RPS14 gene, exhibited a strikingly similar methylation pattern, and the organization of CpG dinucleotides at other mammalian origins revealed the potential for high-density CpG methylation. Moreover, analysis of bromodeoxyuridine-labeled nascent DNA confirmed that active replication origins were methylated. These results suggest that a high-density cluster of mCpG dinucleotides may play a role in either the establishment or the regulation of mammalian replication origins. PMID:8972222

  4. Joint functions of protein residues and NADP(H) in oxygen activation by flavin-containing monooxygenase.

    PubMed

    Orru, Roberto; Pazmiño, Daniel E Torres; Fraaije, Marco W; Mattevi, Andrea

    2010-11-01

    The reactivity of flavoenzymes with dioxygen is at the heart of a number of biochemical reactions with far reaching implications for cell physiology and pathology. Flavin-containing monooxygenases are an attractive model system to study flavin-mediated oxygenation. In these enzymes, the NADP(H) cofactor is essential for stabilizing the flavin intermediate, which activates dioxygen and makes it ready to react with the substrate undergoing oxygenation. Our studies combine site-directed mutagenesis with the usage of NADP(+) analogues to dissect the specific roles of the cofactors and surrounding protein matrix. The highlight of this "double-engineering" approach is that subtle alterations in the hydrogen bonding and stereochemical environment can drastically alter the efficiency and outcome of the reaction with oxygen. This is illustrated by the seemingly marginal replacement of an Asn to Ser in the oxygen-reacting site, which inactivates the enzyme by effectively converting it into an oxidase. These data rationalize the effect of mutations that cause enzyme deficiency in patients affected by the fish odor syndrome. A crucial role of NADP(+) in the oxygenation reaction is to shield the reacting flavin N5 atom by H-bond interactions. A Tyr residue functions as backdoor that stabilizes this crucial binding conformation of the nicotinamide cofactor. A general concept emerging from this analysis is that the two alternative pathways of flavoprotein-oxygen reactivity (oxidation versus monooxygenation) are predicted to have very similar activation barriers. The necessity of fine tuning the hydrogen-bonding, electrostatics, and accessibility of the flavin will represent a challenge for the design and development of oxidases and monoxygenases for biotechnological applications.

  5. Determination of adenine based on the fluorescence recovery of the L-Tryptophan-Cu(2+) complex.

    PubMed

    Duan, Ruilin; Li, Chunyan; Liu, Shaopu; Liu, Zhongfang; Li, Yuanfang; Yuan, Yusheng; Hu, Xiaoli

    2016-01-01

    A simple and sensitive method for determination of adenine was developed based on fluorescence quenching and recovery of L-Tryptophan (L-Trp). The fluorescence of L-Trp could efficiently quenched by copper ion compared with other common metal ions. Upon addition of adenine (Ade) in L-Trp-Cu(II) system, the fluorescence was reoccurred. Under the optimum conditions, the recovery fluorescence intensity was linearly correlated with the concentration of adenine in the range from 0.34 to 25.0μmolL(-1), with a correlation coefficient (R(2)) of 0.9994. The detection limit (3σ/k) was 0.046μmolL(-1), indicating that this method could applied to detect trace adenine. In this study, amino acids including L-Trp, D-Trp, L-Tyr, D-Tyr, L-Phe, D-Phe were investigated and only L-Trp could well chelated copper ion. Additionally, the mechanism of quench and recovery also were discussed and the method was successfully applied to detect the adenine in DNA with satisfactory results.

  6. Determination of adenine based on the fluorescence recovery of the L-Tryptophan-Cu2+ complex

    NASA Astrophysics Data System (ADS)

    Duan, Ruilin; Li, Chunyan; Liu, Shaopu; Liu, Zhongfang; Li, Yuanfang; Yuan, Yusheng; Hu, Xiaoli

    2016-01-01

    A simple and sensitive method for determination of adenine was developed based on fluorescence quenching and recovery of L-Tryptophan (L-Trp). The fluorescence of L-Trp could efficiently quenched by copper ion compared with other common metal ions. Upon addition of adenine (Ade) in L-Trp-Cu(II) system, the fluorescence was reoccurred. Under the optimum conditions, the recovery fluorescence intensity was linearly correlated with the concentration of adenine in the range from 0.34 to 25.0 μmol L-1, with a correlation coefficient (R2) of 0.9994. The detection limit (3σ/k) was 0.046 μmol L-1, indicating that this method could applied to detect trace adenine. In this study, amino acids including L-Trp, D-Trp, L-Tyr, D-Tyr, L-Phe, D-Phe were investigated and only L-Trp could well chelated copper ion. Additionally, the mechanism of quench and recovery also were discussed and the method was successfully applied to detect the adenine in DNA with satisfactory results.

  7. Adenine: an important drug scaffold for the design of antiviral agents

    PubMed Central

    Wang, Changyuan; Song, Zhendong; Yu, Haiqing; Liu, Kexin; Ma, Xiaodong

    2015-01-01

    Adenine derivatives, in particular the scaffold bearing the acyclic nucleoside phosphonates (ANPS), possess significant antiviral and cytostatic activity. Till now, several effective adenine derivatives have been marketed for the treatment of HIV, HBV, CMV and other virus-infected diseases. These compounds are represented by tenofovir (PMPA), a medicine for both HIV and HBV, and adefovir as an anti-HBV agent. More than this, other analogs, such as GS9148, GS9131, and GS7340, are also well-known anti-viral agents that have been progressed to the clinical studies for their excellent activity. In general, the structures of these compounds include an adenine nucleobase linked to a phosphonate side chain. Considerable structural modifications on the scaffold itself and the peripheral sections were made. The structure-activity relationships (SARs) of this skeleton will provide valuable clues to identify more effective adenine derivatives as antiviral drugs. Here, we systematically summarized the SARs of the adenine derivatives, and gave important information for further optimizing this template. PMID:26579473

  8. Enhanced energy transfer in respiratory-deficient endothelial cells probed by microscopic fluorescence excitation spectroscopy

    NASA Astrophysics Data System (ADS)

    Schneckenburger, Herbert; Gschwend, Michael H.; Bauer, Manfred; Strauss, Wolfgang S. L.; Steiner, Rudolf W.

    1996-12-01

    Mitochondrial malfunction may be concomitant with changes of the redox states of the coenzymes nicotinamide adenine dinucleotide (NAD+/NADH), as well as flavin.mononucleotide or dinucleotide. The intrinsic fluorescence of these coenzymes was therefore proposed to be a measure of malfunction. Since mitochondrial fluorescence is strongly superposed by autofluorescence from various cytoplasmatic fluorophores, cultivated endothelial cells were incubated with the mitochondrial marker rhodamine 123 (R123), and after excitation of flavin molecules, energy transfer to R123 was investigated. Due to spectral overlap of flavin and R123 fluorescence, energy transfer flavin yields R123 could not be detected from their emission spectra. Therefore, the method of microscopic fluorescence excitation spectroscopy was established. When detecting R123 fluorescence, excitation maxima at 370 - 390 nm and 420-460 nm were assigned to flavins, whereas a pronounced excitation band at 465 - 490 nm was attributed to R123. Therefore, excitation at 475 nm reflected the intracellular concentration of R123, whereas excitation at 385 nm reflected flavin excitation with a subsequent energy transfer to R123 molecules. An enhanced energy transfer after inhibition of specific enzyme complexes of the respiratory chain is discussed in the present article.

  9. Cell nonautonomous activation of flavin-containing monooxygenase promotes longevity and health span.

    PubMed

    Leiser, Scott F; Miller, Hillary; Rossner, Ryan; Fletcher, Marissa; Leonard, Alison; Primitivo, Melissa; Rintala, Nicholas; Ramos, Fresnida J; Miller, Dana L; Kaeberlein, Matt

    2015-12-11

    Stabilization of the hypoxia-inducible factor 1 (HIF-1) increases life span and health span in nematodes through an unknown mechanism. We report that neuronal stabilization of HIF-1 mediates these effects in Caenorhabditis elegans through a cell nonautonomous signal to the intestine, which results in activation of the xenobiotic detoxification enzyme flavin-containing monooxygenase-2 (FMO-2). This prolongevity signal requires the serotonin biosynthetic enzyme TPH-1 in neurons and the serotonin receptor SER-7 in the intestine. Intestinal FMO-2 is also activated by dietary restriction (DR) and is necessary for DR-mediated life-span extension, which suggests that this enzyme represents a point of convergence for two distinct longevity pathways. FMOs are conserved in eukaryotes and induced by multiple life span-extending interventions in mice, which suggests that these enzymes may play a critical role in promoting health and longevity across phyla.

  10. Cell Non-Autonomous Activation of Flavin-containing Monooxygenase Promotes Longevity and Healthspan

    PubMed Central

    Leiser, Scott F.; Fletcher, Marissa; Leonard, Alison; Primitivo, Melissa; Rintala, Nicholas; Ramos, Fresnida J.; Miller, Dana L.; Kaeberlein, Matt

    2016-01-01

    Stabilization of the hypoxia-inducible factor-1 (HIF-1) increases lifespan and healthspan in nematodes through an unknown mechanism. We report that neuronal stabilization of HIF-1 mediates these effects in C. elegans through a cell non-autonomous signal to the intestine resulting in activation of the xenobiotic detoxification enzyme flavin-containing monooxygenase-2 (FMO-2). This pro-longevity signal requires the serotonin biosynthetic enzyme TPH-1 in neurons and the serotonin receptor SER-7 in the intestine. Intestinal FMO-2 is also activated by dietary restriction (DR) and necessary for DR-mediated lifespan extension, suggesting that this enzyme represents a point of convergence for two distinct longevity pathways. FMOs are conserved in eukaryotes and induced by multiple lifespan-extending interventions in mice, suggesting that these enzymes may play a critical role in promoting health and longevity across phyla. PMID:26586189

  11. A prebiotic role for 8-oxoguanosine as a flavin mimic in pyrimidine dimer photorepair.

    PubMed

    Nguyen, Khiem Van; Burrows, Cynthia J

    2011-09-21

    Redox-active enzyme cofactors derived from ribonucleotides have been called "fossils of the RNA world," suggesting that early catalysts employed modified nucleobases to facilitate redox chemistry in primitive metabolism. Here, we show that the common oxidative damage product 8-oxo-7,8-dihydroguanine (OG), when incorporated into a DNA or RNA strand in proximity to a cyclobutane pyrimidine dimer, can mimic the function of a flavin in photorepair. The OG nucleotide acts catalytically in a mechanism consistent with that of photolyase in which the photoexcited state of the purine donates an electron to a pyrimidine dimer to initiate bond cleavage; subsequent back electron transfer regenerates OG. This unusual example of one form of DNA damage, oxidation, functioning to repair another, photodimerization, may provide insight into the origins of prebiotic redox processes. PMID:21877686

  12. Biosynthesis of flavin cofactors in man: implications in health and disease.

    PubMed

    Barile, Maria; Giancaspero, Teresa Anna; Brizio, Carmen; Panebianco, Concetta; Indiveri, Cesare; Galluccio, Michele; Vergani, Lodovica; Eberini, Ivano; Gianazza, Elisabetta

    2013-01-01

    The primary role of the water-soluble vitamin B2, i.e. riboflavin, in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, reductases and oxidases involved in energetic metabolism, redox homeostasis and protein folding as well as in diverse regulatory events. Deficiency of riboflavin in men and experimental animal models has been linked to several diseases, including neuromuscular and neurological disorders and cancer. Riboflavin at pharmacological doses has been shown to play unexpected and incompletely understood regulatory roles. Besides a summary on riboflavin uptake and a survey on riboflavin-related diseases, the main focus of this review is on discovery and characterization of FAD synthase (EC 2.7.7.2) and other components of the cellular networks that ensure flavin cofactor homeostasis.Special attention is devoted to the problem of sub-cellular compartmentalization of cofactor synthesis in eukaryotes, made possible by the existence of different FAD synthase isoforms and specific molecular components involved in flavin trafficking across sub-cellular membranes.Another point addressed in this review is the mechanism of cofactor delivery to nascent apo-proteins, especially those localized into mitochondria, where they integrate FAD in a process that involves additional mitochondrial protein(s) still to be identified. Further efforts are necessary to elucidate the role of riboflavin/FAD network in human pathologies and to exploit the structural differences between human and microbial/fungal FAD synthase as the rational basis for developing novel antibiotic/antimycotic drugs. PMID:23116402

  13. Temperature-dependent self-assembly of adenine derivative on HOPG.

    PubMed

    Mu, Zhongcheng; Rubner, Oliver; Bamler, Markus; Blömker, Tobias; Kehr, Gerald; Erker, Gerhard; Heuer, Andreas; Fuchs, Harald; Chi, Lifeng

    2013-08-27

    Temperature-dependent self-assembly formed by the adsorption of the nucleobase adenine derivative on a graphite surface were investigated by in situ scanning tunneling microscopy (STM). The high-resolution STM images reveal two types of structures, α phase and β phase, which are mainly driven by either hydrogen bonding or aromatic π-π interactions between adenine bases, respectively, as well as the interactions of alkyl chains. α-Phase structures can be transformed into β-phase structures by increasing temperature. The reverse is true for decreasing temperature. This reflects structural stabilities resulting from the different interactions. Density functional theory (DFT) calculations were performed to characterize possible arrangements of adjacent adenine moieties systematically in terms of binding energies and structural properties. Via a systematic search algorithm, all possible network structures were determined on a microscopic level. In this way, it is possible to rationalize the structural parameters as found in the STM images.

  14. Deep-UV surface-enhanced resonance Raman scattering of adenine on aluminum nanoparticle arrays.

    PubMed

    Jha, Shankar K; Ahmed, Zeeshan; Agio, Mario; Ekinci, Yasin; Löffler, Jörg F

    2012-02-01

    We report the ultrasensitive detection of adenine using deep-UV surface-enhanced resonance Raman scattering on aluminum nanostructures. Well-defined Al nanoparticle arrays fabricated over large areas using extreme-UV interference lithography exhibited sharp and tunable plasmon resonances in the UV and deep-UV wavelength ranges. Theoretical modeling based on the finite-difference time-domain method was used to understand the near-field and far-field optical properties of the nanoparticle arrays. Raman measurements were performed on adenine molecules coated uniformly on the Al nanoparticle arrays at a laser excitation wavelength of 257.2 nm. With this technique, less than 10 amol of label-free adenine molecules could be detected reproducibly in real time. Zeptomole (~30,000 molecules) detection sensitivity was readily achieved proving that deep-UV surface-enhanced resonance Raman scattering is an extremely sensitive tool for the detection of biomolecules.

  15. Counterregulation of nuclear 3,5,3'-triiodo-L-thyronine (T3) binding by oxidized and reduced-nicotinamide adenine dinucleotide phosphates in the presence of cytosolic T3-binding protein in vitro

    SciTech Connect

    Hashizume, K.; Miyamoto, T.; Yamauchi, K.; Ichikawa, K.; Kobayashi, M.; Ohtsuka, H.; Sakurai, A.; Suzuki, S.; Yamada, T.

    1989-04-01

    The role of cytosolic T3-binding protein (CTBP) in the regulation of nuclear T3 binding was studied in vitro. Nuclear (125I)T3 binding was observed in the presence of 1.0 mM dithiothreitol (DTT). When the nuclei prepared from rat kidney were incubated with inactive form of CTBP which was also prepared from rat kidney, (125I)T3 binding to nuclei was not affected. When the nuclei were incubated with inactive form of CTBP in the presence of NADP, (125I)T3 binding to nuclei was increased, whereas binding was diminished when nuclei were incubated with CTBP in the presence of NADPH. The inactive form of CTBP was activated by NADPH. NADP also activated CTBP in the presence of DTT. Both active forms of CTBP were again inactivated by extraction with charcoal, and these inactive forms were reactivated by NADPH or by NADP and DTT, but not by NADP alone. Although the nuclei treated with 0.3 M NaCl lost the binding activity for (125I)T3 in the absence of NADP, the nuclei retained the binding activity for (125I)T3 in the presence of NADP and the inactive form of CTBP. Treatment of the nuclei with 0.5 M NaCl lost the binding activity for (125I)T3 not only in the absence but also in the presence of NADP and CTBP. These results suggested that NADP and NADPH play roles as counterregulatory factors for nuclear T3 binding in the presence of CTBP. Further, it was speculated that binding sites for the T3-CTBP complex, which is generated in the presence of NADP and DTT, are present in nuclei, and that binding sites for the complex are different from nuclear T3 receptors.

  16. DNA with adenine tracts contains poly(dA).poly(dT) conformational features in solution.

    PubMed

    Brahms, S; Brahms, J G

    1990-03-25

    The conformation of DNA's with adenine-thymine tracts exhibiting retardation in electrophoretic migration and considered as curved were investigated in solution by CD and RAMAN spectroscopy. The following curved multimers with adenine tracts but of different flanking sequences d(CA5TGCC)n, d(TCTCTA6TATATA5)n, d(GA4T4C)n yield CD spectroscopic features indicating a non-B structure of the dA.dT tract with similarities to polyd(A).polyd(T). We suggest that adenine-thymine bases in these multimers contain some of the distinctive conformational features of poly(A).polyd(T) probably with large propeller twist found by NMR (Behling and Kearns, 1987) and by X-ray diffraction on oligonucleotides containing a tract of adenines (Nelson et al. 1987, Coll et al; 1987; DiGabriele et al. 1989). Some elements of distinctive CD features of the contiguous adenines run are also observed in the straight multi-9-mer d(CA5GCC)n which lacks in-phase relation to the helical repeat. Despite the presence of the TpA step in the straight multimer d(GT4A4)n, the altered dA.dT conformation is not completely destroyed. Interruption of adenine tract by a guanine in d(CAAGAATGCC)n leads to a B-like conformation and to a normal electrophoretic mobility. The Raman spectra reveal a rearrangement of the sugar-phosphate backbone of dA.dT tract in the multimer d(CA5TGCC)n with respect to that of polydA.polydT. This is reflected in the presence of an unique Raman band associated to C2'-endo sugar with a predominant contribution of C1'-exo puckering which is exhibited by the multimer whereas two distinct Raman bands characterize poly(dA).poly(dT) backbone conformation.

  17. Solution structure of a five-adenine bulge loop within a DNA duplex.

    PubMed

    Dornberger, U; Hillisch, A; Gollmick, F A; Fritzsche, H; Diekmann, S

    1999-09-28

    The three-dimensional solution structure of a DNA molecule of the sequence 5'-d(GCATCGAAAAAGCTACG)-3' paired with 5'-d(CGTAGCCGATGC)-3' containing a five-adenine bulge loop (dA(5)-bulge) between two double helical stems was determined by 2D (1)H and (31)P NMR, infrared, and Raman spectroscopy. The DNA in both stems adopt a classical B-form double helical structure with Watson-Crick base pairing and C2'-endo sugar conformation. In addition, the two dG/dC base pairs framing the dA(5)-bulge loop are formed and are stable at least up to 30 degrees C. The five adenine bases of the bulge loop are localized at intrahelical positions within the double helical stems. Stacking on the double helical stem is continued for the first four 5'-adenines in the bulge loop. The total rise (the height) of these four stacked adenines roughly equals the diameter of the double helical stem. The stacking interactions are broken between the last of these four 5'-adenines and the fifth loop adenine at the 3'-end. This 3'-adenine partially stacks on the other stem. The angle between the base planes of the two nonstacking adenines (A10 and A11) in the bulge loop reflects the kinking angle of the global DNA structure. The neighboring cytosines opposite the dA(5)-bulge (being parts of the bulge flanking base pairs) do not stack on one another. This disruption of stacking is characterized by a partial shearing of these bases, such that certain sequential NOEs for this base step are preserved. In the base step opposite the loop, an extraordinary hydrogen bond is observed between the phosphate backbone of the 5'-dC and the amino proton of the 3'-dC in about two-thirds of the conformers. This hydrogen bond probably contributes to stabilizing the global DNA structure. The dA(5)-bulge induces a local kink into the DNA molecule of about 73 degrees (+/-11 degrees ). This kinking angle and the mutual orientation of the two double helical stems agree well with results from fluorescence resonance energy

  18. Comparative study of spontaneous deamination of adenine and cytosine in unbuffered aqueous solution at room temperature

    NASA Astrophysics Data System (ADS)

    Wang, Shiliang; Hu, Anguang

    2016-06-01

    Adenine in unbuffered nanopure water at a concentration of 2 mM is completely deaminated (>99%) to hypoxanthine at room temperature in ca. 10 weeks, with an estimated half-life (t1/2) less than 10 days, about six orders of magnitude faster than previously reported. Cytosine is not deaminated under the same condition, even after 3 years. This is in contrast to previous observations that cytosine deaminates 20-40 times faster than adenine free base, in nucleoside, in nucleotide and in single-stranded DNA in buffered neutral aqueous solutions.

  19. Copper-catalyzed intramolecular cyclization of N-propargyl-adenine: synthesis of purine-fused tricyclics.

    PubMed

    Li, Ren-Long; Liang, Lei; Xie, Ming-Sheng; Qu, Gui-Rong; Niu, Hong-Ying; Guo, Hai-Ming

    2014-04-18

    A novel protocol to construct fluorescent purine-fused tricyclic products via intramolecular cyclization of N-propargyl-adenine has been developed. With CuBr as the catalyst, a series of purine-fused tricyclic products were obtained in good to excellent yields (19 examples, 75-89% yields). When R2 was a hydrogen atom in N-propargyl-adenines, the reactions only afforded the endocyclic double bond products. When R2 was an aryl group, the electron-donating groups favored the endocyclic double bond products, while the electron-withdrawing groups favored the exocyclic double bond products. PMID:24678722

  20. Effects of soluble flavin on heterogeneous electron transfer between surface-exposed bacterial cytochromes and iron oxides

    NASA Astrophysics Data System (ADS)

    Wang, Zheming; Shi, Zhi; Shi, Liang; White, Gaye F.; Richardson, David J.; Clarke, Thomas A.; Fredrickson, Jim K.; Zachara, John M.

    2015-08-01

    Dissimilatory iron-reducing bacteria can utilize insoluble Fe(Mn)-oxides as a terminal electron acceptor under anaerobic conditions. For Shewanella species specifically, evidence suggests that iron reduction is associated with the secretion of flavin mononucleotide (FMN) and riboflavin. However, the exact mechanism of flavin involvement is unclear; while some indicate that flavins mediate electron transfer (Marsili et al., 2008), others point to flavin serving as co-factors to outer membrane proteins (Okamoto et al., 2013). In this work, we used methyl viologen (MVrad +)-encapsulated, porin-cytochrome complex (MtrCAB) embedded liposomes (MELs) as a synthetic model of the Shewanella outer membrane to investigate the proposed mediating behavior of microbially produced flavins. The reduction kinetics of goethite, hematite and lepidocrocite (200 μM) by MELs ([MVrad +] ∼ 40 μM and MtrABC ⩽ 1 nM) were determined in the presence FMN at pH 7.0 in N2 atmosphere by monitoring the concentrations of MVrad + and FMN through their characteristic UV-visible absorption spectra. Experiments were performed where (i) FMN and Fe(III)-oxide were mixed and then reacted with the reduced MELs and (ii) FMN was reacted with the reduced MELs followed by addition of Fe(III)-oxide. The redox reactions proceeded in two steps: a fast step that was completed in a few seconds, and a slower one lasting over 400 s. For all three Fe(III)-oxides, the initial reaction rate in the presence of a low concentration of FMN (⩽1 μM) was at least a factor of five faster than those with MELs alone, and orders of magnitude faster than those by FMNH2, suggesting that FMN may serve as a co-factor that enhances electron transfer from outer-membrane c-cytochromes to Fe(III)-oxides. The rate and extent of the initial reaction followed the order of lepidocrocite > hematite > goethite, the same as their reduction potentials, implying thermodynamic control on reaction rate. For LEP, with the highest reduction

  1. Distribution of seawater fluorescence and dissolved flavins in the Almeria-Oran front (Alboran Sea, western Mediterranean Sea)

    NASA Astrophysics Data System (ADS)

    Momzikoff, A.; Dallot, S.; Gondry, G.

    1994-08-01

    Seawater fluorescence in the blue region of the spectrum (excitation at 360 nm) due to fluorescent dissolved organic matter (FDOM), and dissolved flavins were investigated in the Almeria-Oran geostrophic front (western Mediterranean) in the 0-200 m layer. Seawater fluorescence increased with depth from a minimum in the jet divergence, increasing towards the oligotrophic waters located outside the jet zone, and reaching a maximum in the right side of the jet, a convergence zone. Comparisons with other recorded parameters suggested both physical and biological factors were involved in its distribution along the transect. Photodegradation due to light-penetration and seawater enrichment with FDOM due to biological activity appeared as driving factors of fluorescence distribution. Fluorescence increase along the secondary circulation of the jet was attributed to the combined effects of aging of a bloom (where it was suggested that both auto- and heterotrophic populations were involved) and photodegradation. FDOM of deeper waters (found in the divergence zone) was inferred to be less photodegradable than that generated in the productive layers (the convergence zone). From these data fluorescence in the oligotrophic sites was deduced to originate from prevaling biological activity. Three flavins were investigated: riboflavin and its photoproducts (lumichrome and lumiflavin). The vertical distribution of flavins was marked by a stratification into two layers of enhanced concentrations. The upper one was found to coincide with the upper chlorophyll layer (DCM or DCM1), the lower one with the lower chlorophyll layer (DCM2, where it occurred) and/or with the base of the halocline. From these depth coincidences both auto- and heterotrophic populations were inferred to be sources of flavins although their respective contributions were hard to determine. As for fluorescence, an increase of flavins was found in the jet zone. However significant differences were found between the

  2. Predicting DNA Methylation State of CpG Dinucleotide Using Genome Topological Features and Deep Networks

    NASA Astrophysics Data System (ADS)

    Wang, Yiheng; Liu, Tong; Xu, Dong; Shi, Huidong; Zhang, Chaoyang; Mo, Yin-Yuan; Wang, Zheng

    2016-01-01

    The hypo- or hyper-methylation of the human genome is one of the epigenetic features of leukemia. However, experimental approaches have only determined the methylation state of a small portion of the human genome. We developed deep learning based (stacked denoising autoencoders, or SdAs) software named “DeepMethyl” to predict the methylation state of DNA CpG dinucleotides using features inferred from three-dimensional genome topology (based on Hi-C) and DNA sequence patterns. We used the experimental data from immortalised myelogenous leukemia (K562) and healthy lymphoblastoid (GM12878) cell lines to train the learning models and assess prediction performance. We have tested various SdA architectures with different configurations of hidden layer(s) and amount of pre-training data and compared the performance of deep networks relative to support vector machines (SVMs). Using the methylation states of sequentially neighboring regions as one of the learning features, an SdA achieved a blind test accuracy of 89.7% for GM12878 and 88.6% for K562. When the methylation states of sequentially neighboring regions are unknown, the accuracies are 84.82% for GM12878 and 72.01% for K562. We also analyzed the contribution of genome topological features inferred from Hi-C. DeepMethyl can be accessed at http://dna.cs.usm.edu/deepmethyl/.

  3. Predicting DNA Methylation State of CpG Dinucleotide Using Genome Topological Features and Deep Networks

    PubMed Central

    Wang, Yiheng; Liu, Tong; Xu, Dong; Shi, Huidong; Zhang, Chaoyang; Mo, Yin-Yuan; Wang, Zheng

    2016-01-01

    The hypo- or hyper-methylation of the human genome is one of the epigenetic features of leukemia. However, experimental approaches have only determined the methylation state of a small portion of the human genome. We developed deep learning based (stacked denoising autoencoders, or SdAs) software named “DeepMethyl” to predict the methylation state of DNA CpG dinucleotides using features inferred from three-dimensional genome topology (based on Hi-C) and DNA sequence patterns. We used the experimental data from immortalised myelogenous leukemia (K562) and healthy lymphoblastoid (GM12878) cell lines to train the learning models and assess prediction performance. We have tested various SdA architectures with different configurations of hidden layer(s) and amount of pre-training data and compared the performance of deep networks relative to support vector machines (SVMs). Using the methylation states of sequentially neighboring regions as one of the learning features, an SdA achieved a blind test accuracy of 89.7% for GM12878 and 88.6% for K562. When the methylation states of sequentially neighboring regions are unknown, the accuracies are 84.82% for GM12878 and 72.01% for K562. We also analyzed the contribution of genome topological features inferred from Hi-C. DeepMethyl can be accessed at http://dna.cs.usm.edu/deepmethyl/. PMID:26797014

  4. Structural and Functional Analysis of DDX41: a bispecific immune receptor for DNA and cyclic dinucleotide

    PubMed Central

    Omura, Hiroki; Oikawa, Daisuke; Nakane, Takanori; Kato, Megumi; Ishii, Ryohei; Ishitani, Ryuichiro; Tokunaga, Fuminori; Nureki, Osamu

    2016-01-01

    In the innate immune system, pattern recognition receptors (PRRs) specifically recognize ligands derived from bacteria or viruses, to trigger the responsible downstream pathways. DEAD box protein 41 (DDX41) is an intracellular PRR that triggers the downstream pathway involving the adapter STING, the kinase TBK1, and the transcription factor IRF3, to activate the type I interferon response. DDX41 is unique in that it recognizes two different ligands; i.e., double-stranded DNA (dsDNA) and cyclic dinucleotides (CDN), via its DEAD domain. However, the structural basis for the ligand recognition by the DDX41 DEAD domain has remained elusive. Here, we report two crystal structures of the DDX41 DEAD domain in apo forms, at 1.5 and 2.2 Å resolutions. A comparison of the two crystal structures revealed the flexibility in the ATP binding site, suggesting its formation upon ATP binding. Structure-guided functional analyses in vitro and in vivo demonstrated the overlapped binding surface for dsDNA and CDN, which is distinct from the ATP-binding site. We propose that the structural rearrangement of the ATP binding site is crucial for the release of ADP, enabling the fast turnover of DDX41 for the dsDNA/CDN-induced STING activation pathway. PMID:27721487

  5. bis-Molybdopterin Guanine Dinucleotide Is Required for Persistence of Mycobacterium tuberculosis in Guinea Pigs

    PubMed Central

    Williams, Monique J.; Shanley, Crystal A.; Zilavy, Andrew; Peixoto, Blas; Manca, Claudia; Kaplan, Gilla; Orme, Ian M.; Mizrahi, Valerie

    2014-01-01

    Mycobacterium tuberculosis is able to synthesize molybdopterin cofactor (MoCo), which is utilized by numerous enzymes that catalyze redox reactions in carbon, nitrogen, and sulfur metabolism. In bacteria, MoCo is further modified through the activity of a guanylyltransferase, MobA, which converts MoCo to bis-molybdopterin guanine dinucleotide (bis-MGD), a form of the cofactor that is required by the dimethylsulfoxide (DMSO) reductase family of enzymes, which includes the nitrate reductase NarGHI. In this study, the functionality of the mobA homolog in M. tuberculosis was confirmed by demonstrating the loss of assimilatory and respiratory nitrate reductase activity in a mobA deletion mutant. This mutant displayed no survival defects in human monocytes or mouse lungs but failed to persist in the lungs of guinea pigs. These results implicate one or more bis-MGD-dependent enzymes in the persistence of M. tuberculosis in guinea pig lungs and underscore the applicability of this animal model for assessing the role of molybdoenzymes in this pathogen. PMID:25404027

  6. bis-Molybdopterin guanine dinucleotide is required for persistence of Mycobacterium tuberculosis in guinea pigs.

    PubMed

    Williams, Monique J; Shanley, Crystal A; Zilavy, Andrew; Peixoto, Blas; Manca, Claudia; Kaplan, Gilla; Orme, Ian M; Mizrahi, Valerie; Kana, Bavesh D

    2015-02-01

    Mycobacterium tuberculosis is able to synthesize molybdopterin cofactor (MoCo), which is utilized by numerous enzymes that catalyze redox reactions in carbon, nitrogen, and sulfur metabolism. In bacteria, MoCo is further modified through the activity of a guanylyltransferase, MobA, which converts MoCo to bis-molybdopterin guanine dinucleotide (bis-MGD), a form of the cofactor that is required by the dimethylsulfoxide (DMSO) reductase family of enzymes, which includes the nitrate reductase NarGHI. In this study, the functionality of the mobA homolog in M. tuberculosis was confirmed by demonstrating the loss of assimilatory and respiratory nitrate reductase activity in a mobA deletion mutant. This mutant displayed no survival defects in human monocytes or mouse lungs but failed to persist in the lungs of guinea pigs. These results implicate one or more bis-MGD-dependent enzymes in the persistence of M. tuberculosis in guinea pig lungs and underscore the applicability of this animal model for assessing the role of molybdoenzymes in this pathogen.

  7. Measurement of liver adenine nucleotides and S-adenosyl amino acids by one-step high-performance liquid chromatography.

    PubMed

    Gourdeau, H; Lavoie, R; Grose, J H; Bélanger, L

    1986-10-01

    A reverse-phase isocratic HPLC method is described for direct simultaneous assay of ATP, ADP, AMP, S-adenosylmethionine, S-adenosylhomocysteine, S-adenosylethionine, and other adenine derivatives in liver microbiopsies. The procedure was tested in conditions which alter the hepatic content of adenine nucleotides and sulfur-adenosyl amino acids in humans, rats, and guinea pigs.

  8. A flavin-dependent halogenase catalyzes the chlorination step in the biosynthesis of Dictyostelium differentiation-inducing factor 1

    PubMed Central

    Neumann, Christopher S.; Walsh, Christopher T.; Kay, Robert R.

    2010-01-01

    Differentiation-inducing factor 1 (DIF-1) is a polyketide-derived morphogen which drives stalk cell formation in the developmental cycle of Dictyostelium discoideum. Previous experiments demonstrated that the biosynthetic pathway proceeds via dichlorination of the precursor molecule THPH, but the enzyme responsible for this transformation has eluded characterization. Our recent studies on prokaryotic flavin-dependent halogenases and insights from the sequenced Dd genome led us to a candidate gene for this transformation. In this work, we present in vivo and in vitro evidence that chlA from Dd encodes a flavin-dependent halogenase capable of catalyzing both chlorinations in the biosynthesis of DIF-1. The results provide in vitro characterization of a eukaryotic oxygen-dependent halogenase and demonstrate a broad reach in biology for this molecular tailoring strategy, notably its involvement in the differentiation program of a social amoeba. PMID:20231486

  9. Phosphorus-31 NMR visibility and characterization of rat liver mitochondrial matrix adenine nucleotides

    SciTech Connect

    Hutson, S.M.; Berkich, D.; Williams, G.D.; LaNoue, K.F.; Briggs, R.W. )

    1989-05-16

    Compartmentation and NMR visibility of mitochondrial adenine nucleotides were quantitated in isolated rat liver mitochondria respiring on succinate and glutamate in vitro at 8 and 25{degree}C. Intra- and extramitochondrial nucleotides were discriminated by adding the chelator trans-1,2-diaminocyclohexane-N,N,N{prime},N{prime}-tetraacetic acid (CDTA). T{sub 1} values of about 0.2-0.3 s for magnesium-bound matrix nucleotides were determined. Adenine nucleotide T{sub 1} values were influenced by the ionic environment; only magnesium-free ATP T{sub 1}'s were affected by temperature. Intra- and extramitochondrial adenine nucleotide ratios were varied in ATP-loaded mitochondria with added ATP and phosphate using the mitochondrial inhibitors oligomycin and carboxyatractyloside, and adenine nucleotides were quantitated by using NMR and enzymatic analysis. There was good agreement between matrix ATP concentrations (magnesium-bound ATP) calculated by using NMR and standard biochemical techniques. Although matrix ADP could be detected by NMR, it was difficult to quantitate accurately by NMR. The data indicate that mitochondrial ATP is NMR-visible in isolated mitochondria in vitro.

  10. Controlling two-phase self-assembly of an adenine derivative on HOPG via kinetic effects.

    PubMed

    Wang, Can; Jana, Pritam Kumar; Zhang, Haiming; Mu, Zhongcheng; Kehr, Gerald; Blömker, Tobias; Erker, Gerhard; Fuchs, Harald; Heuer, Andreas; Chi, Lifeng

    2014-08-21

    Large-area self-assembled structures of a nucleobase adenine derivative were successfully realized through vacuum deposition. STM images reveal two types of structures, which could be regulated by substrate temperature and the evaporation rate, indicating the relevance of kinetic effects. The results are supported by computer simulations.

  11. The effect of activated charcoal on adenine-induced chronic renal failure in rats.

    PubMed

    Ali, Badreldin H; Alza'abi, Mohamed; Ramkumar, Aishwarya; Al-Lawati, Intisar; Waly, Mostafa I; Beegam, Sumaya; Nemmar, Abderrahim; Brand, Susanne; Schupp, Nicole

    2014-03-01

    Activated charcoal (AC) is a sorbent that has been shown to remove urinary toxins like urea and indoxyl sulfate. Here, the influence of AC on kidney function of rats with experimental chronic renal failure (CRF) is investigated. CRF was induced in rats by feeding adenine (0.75%) for four weeks. As an intervention, AC was added to the feed at concentrations of 10%, 15% or 20%. Adenine treatment impaired kidney function: it lowered creatinine clearance and increased plasma concentrations of creatinine, urea, neutrophil gelatinase-associated lipocalin and vanin-1. Furthermore, it raised plasma concentrations of the uremic toxins indoxyl sulfate, phosphate and uric acid. Renal morphology was severely damaged and histopathological markers of inflammation and fibrosis were especially increased. In renal homogenates, antioxidant indices, including superoxide dismutase and catalase activity, total antioxidant capacity and reduced glutathione were adversely affected. Most of these changes were significantly ameliorated by dietary administration of AC at a concentration of 20%, while effects induced by lower doses of dietary AC on adenine nephrotoxicity were not statistically significant. The results suggest that charcoal is a useful sorbent agent in dietary adenine-induced CRF in rats and that its usability as a nephroprotective agent in human kidney disease should be studied.

  12. Effects of adenine arabinoside on lymphocytes infected with Epstein-Barr virus.

    PubMed Central

    Benz, W C; Siegel, P J; Baer, J

    1978-01-01

    Low concentrations of adenine arabinoside inhibited growth of two Epstein-Barr virus producer cell lines in culture, while not significantly affecting a nonproducer cell line and a B-cell-negative line. These observations were extended to include freshly infected cells. Mitogen-stimulated human umbilical cord blood lymphocytes were unaffected by the drug at concentration levels that inhibited [3H]thymidine incorporation into the DNA of Epstein-Barr virus-stimulated cells. DNA synthesis in Epstein-Barr virus-superinfected Raji cells was also adversely affected by adenine arabinoside. However, these same low concentrations of adenine arabinoside in the triphosphate form produced less effect on DNA synthesis in nuclear systems and DNA polymerase assays than on growth or DNA synthesis in whole cells. Therefore the effects reported here of low concentrations of the drug on whole cells may be only in part related to DNA polymerase inhibition. The work reported here suggests that adenine arabinoside has multiple sites of action in infected cells. PMID:212577

  13. Ameliorative Effect of Chrysin on Adenine-Induced Chronic Kidney Disease in Rats

    PubMed Central

    Ali, Badreldin H.; Adham, Sirin A.; Al Za’abi, Mohammed; Waly, Mostafa I.; Yasin, Javed; Nemmar, Abderrahim; Schupp, Nicole

    2015-01-01

    Chrysin (5, 7- dihydroxyflavone) is a flavonoid with several pharmacological properties that include antioxidant, anti-inflammatory and antiapoptotic activities. in this work, we investigated some effects of three graded oral doses of chrysin (10, 50 and 250 mg/kg) on kidney structure and function in rats with experimental chronic renal disease (CKD) induced by adenine (0.25% w/w in feed for 35 days), which is known to involve inflammation and oxidative stress. Using several indices in plasma, urine and kidney homogenates, adenine was found to impair kidney function as it lowered creatinine clearance and increased plasma concentrations of creatinine, urea, neutrophil gelatinase-associated lipocalin and N-Acetyl-beta-D-glucosaminidase activity. Furthermore, it raised plasma concentrations of the uremic toxin indoxyl sulfate, some inflammatory cytokines and urinary albumin concentration. Renal morphology was severely damaged and histopathological markers of inflammation and fibrosis were especially increased. In renal homogenates, antioxidant indices, including superoxide dismutase and catalase activities, total antioxidant capacity and reduced glutathione were all adversely affected. Most of these adenine – induced actions were moderately and dose -dependently mitigated by chrysin, especially at the highest dose. Chrysin did not cause any overt adverse effect on the treated rats. The results suggest that different doses of chrysin produce variable salutary effects against adenine-induced CKD in rats, and that, pending further pharmacological and toxicological studies, its usability as a possible ameliorative agent in human CKD should be considered. PMID:25909514

  14. Macrophage Trafficking as Key Mediator of Adenine-Induced Kidney Injury

    PubMed Central

    Braga, Tárcio Teodoro; Felizardo, Raphael José Ferreira; Andrade-Oliveira, Vinícius; Hiyane, Meire Ioshie; da Silva, João Santana; Câmara, Niels Olsen Saraiva

    2014-01-01

    Macrophages play a special role in the onset of several diseases, including acute and chronic kidney injuries. In this sense, tubule interstitial nephritis (TIN) represents an underestimated insult, which can be triggered by different stimuli and, in the absence of a proper regulation, can lead to fibrosis deposition. Based on this perception, we evaluated the participation of macrophage recruitment in the development of TIN. Initially, we provided adenine-enriched food to WT and searched for macrophage presence and action in the kidney. Also, a group of animals were depleted of macrophages with the clodronate liposome while receiving adenine-enriched diet. We collected blood and renal tissue from these animals and renal function, inflammation, and fibrosis were evaluated. We observed higher expression of chemokines in the kidneys of adenine-fed mice and a substantial protection when macrophages were depleted. Then, we specifically investigated the role of some key chemokines, CCR5 and CCL3, in this TIN experimental model. Interestingly, CCR5 KO and CCL3 KO animals showed less renal dysfunction and a decreased proinflammatory profile. Furthermore, in those animals, there was less profibrotic signaling. In conclusion, we can suggest that macrophage infiltration is important for the onset of renal injury in the adenine-induced TIN. PMID:25132730

  15. Structural and quantum chemical studies of 8-aryl-sulfanyl adenine class Hsp90 inhibitors.

    PubMed

    Immormino, Robert M; Kang, Yanlong; Chiosis, Gabriela; Gewirth, Daniel T

    2006-08-10

    Hsp90 chaperones play a critical role in modulating the activity of many cell signaling proteins and are an attractive target for anti-cancer therapeutics. We report here the structures of the water soluble 8-aryl-sulfanyl adenine class Hsp90 inhibitors, 1 (PU-H71) and 2 (PU-H64), in complex with the N-terminal domain of human Hsp90alpha. The conformation of 1 when bound to Hsp90 differs from previously reported 8-aryl adenine Hsp90 inhibitors including 3 (PU24FCl). While the binding mode for 3 places the 2'-halide of the 8-aryl group on top of the adenine ring, for 1 and 2, we show that the 2'-halide is rotated approximately 180 degrees away. This difference explains the opposing trends in Hsp90 inhibitory activity for the 2'-halo derivatives of the 3',4',5'-trimethoxy series where Cl > Br > I compared to the 4',5'-methylenedioxy series where I > Br > Cl. We also present quantum chemical calculations of 2 and its analogues that illuminate their basis for Hsp90 inhibition. The calculated conformation of 2 agreed well with the crystallographically observed conformations of 1 and 2. The predictive nature of the calculations has allowed the exploration of additional derivatives based on the 8-aryl adenine scaffold.

  16. SERS, XPS, and DFT Study of Adenine Adsorption on Silver and Gold Surfaces.

    PubMed

    Pagliai, Marco; Caporali, Stefano; Muniz-Miranda, Maurizio; Pratesi, Giovanni; Schettino, Vincenzo

    2012-01-19

    The adsorption of adenine on silver and gold surfaces has been investigated combining density functional theory calculations with surface-enhanced Raman scattering and angle-resolved X-ray photoelectron spectroscopy measurements, obtaining useful insight into the orientation and interaction of the nucleobase with the metal surfaces.

  17. Studies of Mg2+/Ca2+ complexes of naturally occurring dinucleotides: potentiometric titrations, NMR, and molecular dynamics.

    PubMed

    Stern, Noa; Major, Dan Thomas; Gottlieb, Hugo Emilio; Weizman, Daniel; Sayer, Alon Haim; Blum, Eliav; Fischer, Bilha

    2012-08-01

    Dinucleotides (Np(n)N'; N and N' are A, U, G, or C, n = 2-7) are naturally occurring physiologically active compounds. Despite the interest in dinucleotides, the composition of their complexes with metal ions as well as their conformations and species distribution in living systems are understudied. Therefore, we investigated a series of Mg(2+) and Ca(2+) complexes of Np(n)N's. Potentiometric titrations indicated that a longer dinucleotide polyphosphate (N is A or G, n = 3-5) linker yields more stable complexes (e.g., log K of 2.70, 3.27, and 3.73 for Ap(n)A-Mg(2+), n = 3, 4, 5, respectively). The base (A or G) or ion (Mg(2+) or Ca(2+)) has a minor effect on K(M)(ML) values. In a physiological medium, the longer Ap(n)As (n = 4, 5) are predicted to occur mostly as the Mg(2+)/Ca(2+) complexes. (31)P NMR monitored titrations of Np(n)N's with Mg(2+)/Ca(2+) ions showed that the middle phosphates of the dinucleotides coordinate with Mg(2+)/Ca(2+). Multidimensional potential of mean force (PMF) molecular dynamics (MD) simulations suggest that Ap(2)A and Ap(4)A coordinate Mg(2+) and Ca(2+) ions in both inner-sphere and outer-sphere modes. The PMF MD simulations additionally provide a detailed picture of the possible coordination sites, as well as the cation binding process. Moreover, both NMR and MD simulations showed that the conformation of the nucleoside moieties in Np(n)N'-Mg(2+)/Ca(2+) complexes remains the same as that of free mononucleotides.

  18. A nomenclature for the mammalian flavin-containing monooxygenase gene family based on amino acid sequence identities.

    PubMed

    Lawton, M P; Cashman, J R; Cresteil, T; Dolphin, C T; Elfarra, A A; Hines, R N; Hodgson, E; Kimura, T; Ozols, J; Phillips, I R

    1994-01-01

    A nomenclature based on comparisons of amino acid sequences is proposed for the members of the mammalian flavin-containing monooxygenase (FMO) gene family. This nomenclature is based on evidence of a single gene family composed of five genes. The percentage identities of the amino acid sequences of the five known forms of mammalian FMO are between 52 and 57% in rabbit and between 50 and 58% across species lines. The identities of all orthologs are greater than 82%. There is no evidence for multiple, highly related forms of the enzyme or for more than one mammalian FMO gene family. In the proposed system, the mammalian flavin-containing monooxygenase gene family is designated as "FMO" and the individual genes are distinguished by an Arabic numeral. The FMOs known as the "liver" and "lung" enzymes become FMO1 and FMO2, and the more recently described forms of the enzymes become FMO3, FMO4, and FMO5. Human FMO gene designations, FMO1 and FMO3, remain unchanged, but the gene designated FMO2 becomes FMO4. Following convention, the genes and cDNA designations will be italicized and the mRNA and protein designations will be nonitalicized. The purpose of the proposed nomenclature is to provide for the unambiguous identification of orthologous forms of mammalian FMOs, regardless of the species or tissue in question. The proposed classification considers only members of the mammalian flavin-containing monooxygenase gene family and has no bearing on the generally accepted definition of a multisubstrate flavin-containing monooxygenase.

  19. The superoxide-generating oxidase of leucocytes. NADPH-dependent reduction of flavin and cytochrome b in solubilized preparations.

    PubMed Central

    Cross, A R; Parkinson, J F; Jones, O T

    1984-01-01

    An NADPH-dependent O2.- -generating oxidase was solubilized from phorbol 12-myristate 13-acetate-activated pig neutrophils by using a mixture of detergents. Recovery of oxidase was approx. 40%. The extract contained cytochrome b-245 (331 pmol/mg of protein) and FAD (421 pmol/mg of protein); approx. 30% of each was reduced within 60s when NADPH was added to anaerobic incubations. Three different additives, quinacrine, p-chloromercuribenzoate and cetyltrimethylammonium bromide, strongly inhibited O2.- generation; they also inhibited the reduction by NADPH of cytochrome b at the same low concentrations. In the presence of p-chloromercuribenzoate cytochrome b reduction was strongly inhibited and flavin reduction was less inhibited. A detergent extract prepared from non-stimulated neutrophils also contained flavin and cytochrome b, but its rate of O2.- production was less than 1% of that from activated cells; its initial rate of cytochrome b and flavin reduction was low, although the state of reduction at equilibrium was similar to that of extracts of activated cells. Even in the non-activated cell extract the reduction of flavin and cytochrome was made fast and complete when Methyl Viologen was added to the anaerobic incubations. The oxidase was temperature-sensitive, with a sharp maximum at 25 degrees C; temperatures above this caused loss of O2.- generation, and this coincided with loss of the characteristic cytochrome b spectrum, indicate of denaturation of the cytochrome. The cytochrome b formed a complex with butyl isocyanide (close to 100% binding at 10mM); butyl isocyanide also inhibited the oxidase activity of stimulated whole neutrophils (22.5% inhibition at 10mM). Photoreduced FMN stimulated O2 uptake by the oxidase. The results support a scheme of electron transport within the oxidase complex involving NADPH, FAD, cytochrome b-245 and O2 in that sequence. PMID:6497852

  20. Multinuclear NMR studies of the flavodoxin from Anabaena 7120:. beta. -sheet structure and the flavin mononucleotide binding site

    SciTech Connect

    Stockman, B.J.

    1989-01-01

    A concerted approach to primary {sup 1}H, {sup 13}C, and {sup 15}N nuclear magnetic resonance assignments in proteins was developed. The method requires enrichment of the protein with {sup 13}C and {sup 15}N. The technique relies on the comparison of data sets from NMR experiments that correlate various nuclei: {sup 13}C({sup 13}C) double quantum correlations, {sup 1}H({sup 13}C) and {sup 1}H({sup 15}N) single bond correlations, and {sup 1}H({sup 13}C) and {sup 1}H({sup 15}N) multiple bond correlations. Comparison of data sets increases the number of resonances that can be assigned and improves assignment confidence. By combined use of these and conventional NMR techniques, sequential assignments were made for the {beta}-sheet and flavin mononucleotide (FMN) binding site residues in flavodoxin from Anabaena 7120. The {beta}-sheet structure was found to be similar to that seen in the crystal structure of Anacystis nidulans flavodoxin. In the FMN binding site, a total of 69 NOEs were identified: eight between protons of FMN, 36 between protons of binding site residues, and 25 between protons of FMN and protein. These constraints were used to determine the localized solution structure of the flavin binding site. The electronic environment and conformation of the protein-bound isoalloxazine ring were investigated by determining chemical shifts and coupling constants for the ring atoms. The carbonyl edge of the flavin ring was found to be slightly polarized by hydrogen bonding to the protein. The xylene ring was found to be nonplanar. The C{sup 6}-N{sup 5} region of the flavin appears to be solvent accessible.

  1. Mechanistic studies on the flavin-dependent N⁶-lysine monooxygenase MbsG reveal an unusual control for catalysis.

    PubMed

    Robinson, Reeder M; Rodriguez, Pedro J; Sobrado, Pablo

    2014-05-15

    The mechanism of Mycobacterium smegmatis G (MbsG), a flavin-dependent l-lysine monooxygenase, was investigated under steady-state and rapid reaction conditions using primary and solvent kinetic isotope effects, substrate analogs, pH and solvent viscosity effects as mechanistic probes. The results suggest that l-lysine binds before NAD(P)H, which leads to a decrease in the rate constant for flavin reduction. l-lysine binding has no effect on the rate of flavin oxidation, which occurs in a one-step process without the observation of a C4a-hydroperoxyflavin intermediate. Similar effects were determined with several substrate analogs. Flavin oxidation is pH independent while the kcat/Km and kred/KD pH profiles for NAD(P)H exhibit single pKa values of ∼6.0, with increasing activity as the pH decreases. At lower pH, the enzyme becomes more uncoupled, producing more hydrogen peroxide and superoxide. Hydride transfer is partially rate-limiting at neutral pH and becomes more rate-limiting at low pH. An inverse solvent viscosity effect on kcat/Km for NAD(P)H was observed at neutral pH whereas a normal solvent viscosity effect was observed at lower pH. Together, the results indicate a unique mechanism where a rate-limiting and pH-sensitive conformational change occurs in the reductive half-reaction, which affects the efficiency of lysine hydroxylation.

  2. Arg279 is the key regulator of coenzyme selectivity in the flavin-dependent ornithine monooxygenase SidA.

    PubMed

    Robinson, Reeder; Franceschini, Stefano; Fedkenheuer, Michael; Rodriguez, Pedro J; Ellerbrock, Jacob; Romero, Elvira; Echandi, Maria Paulina; Martin Del Campo, Julia S; Sobrado, Pablo

    2014-04-01

    Siderophore A (SidA) is a flavin-dependent monooxygenase that catalyzes the NAD(P)H- and oxygen-dependent hydroxylation of ornithine in the biosynthesis of siderophores in Aspergillus fumigatus and is essential for virulence. SidA can utilize both NADPH or NADH for activity; however, the enzyme is selective for NADPH. Structural analysis shows that R279 interacts with the 2'-phosphate of NADPH. To probe the role of electrostatic interactions in coenzyme selectivity, R279 was mutated to both an alanine and a glutamate. The mutant proteins were active but highly uncoupled, oxidizing NADPH and producing hydrogen peroxide instead of hydroxylated ornithine. For wtSidA, the catalytic efficiency was 6-fold higher with NADPH as compared to NADH. For the R279A mutant the catalytic efficiency was the same with both coenyzmes, while for the R279E mutant the catalytic efficiency was 5-fold higher with NADH. The effects are mainly due to an increase in the KD values, as no major changes on the kcat or flavin reduction values were observed. Thus, the absence of a positive charge leads to no coenzyme selectivity while introduction of a negative charge leads to preference for NADH. Flavin fluorescence studies suggest altered interaction between the flavin and NADP⁺ in the mutant enzymes. The effects are caused by different binding modes of the coenzyme upon removal of the positive charge at position 279, as no major conformational changes were observed in the structure for R279A. The results indicate that the positive charge at position 279 is critical for tight binding of NADPH and efficient hydroxylation.

  3. Cyclic dinucleotides bind the C-linker of HCN4 to control channel cAMP responsiveness.

    PubMed

    Lolicato, Marco; Bucchi, Annalisa; Arrigoni, Cristina; Zucca, Stefano; Nardini, Marco; Schroeder, Indra; Simmons, Katie; Aquila, Marco; DiFrancesco, Dario; Bolognesi, Martino; Schwede, Frank; Kashin, Dmitry; Fishwick, Colin W G; Johnson, A Peter; Thiel, Gerhard; Moroni, Anna

    2014-06-01

    cAMP mediates autonomic regulation of heart rate by means of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which underlie the pacemaker current If. cAMP binding to the C-terminal cyclic nucleotide binding domain enhances HCN open probability through a conformational change that reaches the pore via the C-linker. Using structural and functional analysis, we identified a binding pocket in the C-linker of HCN4. Cyclic dinucleotides, an emerging class of second messengers in mammals, bind the C-linker pocket (CLP) and antagonize cAMP regulation of the channel. Accordingly, cyclic dinucleotides prevent cAMP regulation of If in sinoatrial node myocytes, reducing heart rate by 30%. Occupancy of the CLP hence constitutes an efficient mechanism to hinder β-adrenergic stimulation on If. Our results highlight the regulative role of the C-linker and identify a potential drug target in HCN4. Furthermore, these data extend the signaling scope of cyclic dinucleotides in mammals beyond their first reported role in innate immune system. PMID:24776929

  4. Administration of α-Galactosylceramide Improves Adenine-Induced Renal Injury

    PubMed Central

    Aguiar, Cristhiane Favero; Naffah-de-Souza, Cristiane; Castoldi, Angela; Corrêa-Costa, Matheus; Braga, Tárcio T; Naka, Érika L; Amano, Mariane T; Abate, Débora T R S; Hiyane, Meire I; Cenedeze, Marcos A; Filho, Alvaro Pacheco e Silva; Câmara, Niels O S

    2015-01-01

    Natural killer T (NKT) cells are a subset of lymphocytes that reacts to glycolipids presented by CD1d. Invariant NKT cells (iNKT) correspond to >90% of the total population of NKTs and reacts to α-galactosylceramide (αGalCer). αGalCer promotes a complex mixture of Th1 and Th2 cytokines, as interferon (IFN)-γ and interleukin (IL)-4. NKT cells and IFN-γ are known to participate in some models of renal diseases, but further studies are still necessary to elucidate their mechanisms. The aim of our study was to analyze the participation of iNKT cells in an experimental model of tubule-interstitial nephritis. We used 8-wk-old C57BL/6j, Jα18KO and IFN-γKO mice. They were fed a 0.25% adenine diet for 10 d. Both adenine-fed wild-type (WT) and Jα18KO mice exhibited renal dysfunction, but adenine-fed Jα18KO mice presented higher expression of kidney injury molecule-1 (KIM-1), tumor necrosis factor (TNF)-α and type I collagen. To analyze the role of activated iNKT cells in our model, we administered αGalCer in WT mice during adenine ingestion. After αGalCer injection, we observed a significant reduction in serum creatinine, proinflammatory cytokines and renal fibrosis. However, this improvement in renal function was not observed in IFN-γKO mice after αGalCer treatment and adenine feeding, illustrating that this cytokine plays a role in our model. Our findings may suggest that IFN-γ production is one of the factors contributing to improved renal function after αGalCer administration. PMID:26101952

  5. ON THE INTERACTION OF ADENINE WITH IONIZING RADIATION: MECHANISTICAL STUDIES AND ASTROBIOLOGICAL IMPLICATIONS

    SciTech Connect

    Evans, Nicholas L.; Ullrich, Susanne; Bennett, Chris J.; Kaiser, Ralf I.

    2011-04-01

    The molecular inventory available on the prebiotic Earth was likely derived from both terrestrial and extraterrestrial sources. A complete description of which extraterrestrial molecules may have seeded early Earth is therefore necessary to fully understand the prebiotic evolution which led to life. Galactic cosmic rays (GCRs) are expected to cause both the formation and destruction of important biomolecules-including nucleic acid bases such as adenine-in the interstellar medium within the ices condensed on interstellar grains. The interstellar ultraviolet (UV) component is expected to photochemically degrade gas-phase adenine on a short timescale of only several years. However, the destruction rate is expected to be significantly reduced when adenine is shielded in dense molecular clouds or even within the ices of interstellar grains. Here, biomolecule destruction by the energetic charged particle component of the GCR becomes important as it is not fully attenuated. Presented here are results on the destruction rate of the nucleobase adenine in the solid state at 10 K by energetic electrons, as generated in the track of cosmic ray particles as they penetrate ices. When both UV and energetic charged particle destructive processes are taken into account, the half-life of adenine within dense interstellar clouds is found to be {approx}6 Myr, which is on the order of a star-forming molecular cloud. We also discuss chemical reaction pathways within the ices to explain the production of observed species, including the formation of nitriles (R-C{identical_to}N), epoxides (C-O-C), and carbonyl functions (R-C=O).

  6. Bound Flavin-Cytochrome Model of Extracellular Electron Transfer in Shewanella oneidensis: Analysis by Free Energy Molecular Dynamics Simulations.

    PubMed

    Hong, Gongyi; Pachter, Ruth

    2016-06-30

    Flavins are known to enhance extracellular electron transfer (EET) in Shewanella oneidensis MR-1 bacteria, which reduce electron acceptors through outer-membrane (OM) cytochromes c. Free-shuttle and bound-redox cofactor mechanisms were proposed to explain this enhancement, but recent electrochemical reports favor a flavin-bound model, proposing two one-electron reductions of flavin, namely, oxidized (Ox) to semiquinone (Sq) and semiquinone to hydroquinone (Hq), at anodic and cathodic conditions, respectively. In this work, to provide a mechanistic understanding of riboflavin (RF) binding at the multiheme OM cytochrome OmcA, we explored binding configurations at hemes 2, 5, 7, and 10. Subsequently, on the basis of molecular dynamics (MD) simulations, binding free energies and redox potential shifts upon RF binding for the Ox/Sq and Sq/Hq reductions were analyzed. Our results demonstrated an upshift in the Ox/Sq and a downshift in the Sq/Hq redox potentials, consistent with a bound RF-OmcA model. Furthermore, binding free energy MD simulations indicated an RF binding preference at heme 7. MD simulations of the OmcA-MtrC complex interfacing at hemes 5 revealed a small interprotein redox potential difference with an electron transfer rate of 10(7)-10(8)/s. PMID:27266856

  7. Convenient microtiter plate-based, oxygen-independent activity assays for flavin-dependent oxidoreductases based on different redox dyes.

    PubMed

    Brugger, Dagmar; Krondorfer, Iris; Zahma, Kawah; Stoisser, Thomas; Bolivar, Juan M; Nidetzky, Bernd; Peterbauer, Clemens K; Haltrich, Dietmar

    2014-04-01

    Flavin-dependent oxidoreductases are increasingly recognized as important biocatalysts for various industrial applications. In order to identify novel activities and to improve these enzymes in engineering approaches, suitable screening methods are necessary. We developed novel microtiter-plate-based assays for flavin-dependent oxidases and dehydrogenases using redox dyes as electron acceptors for these enzymes. 2,6-dichlorophenol-indophenol, methylene green, and thionine show absorption changes between their oxidized and reduced forms in the visible range, making it easy to judge visually changes in activity. A sample set of enzymes containing both flavoprotein oxidases and dehydrogenases - pyranose 2-oxidase, pyranose dehydrogenase, cellobiose dehydrogenase, D-amino acid oxidase, and L-lactate oxidase - was selected. Assays for these enzymes are based on a direct enzymatic reduction of the redox dyes and not on the coupled detection of a reaction product as in the frequently used assays based on hydrogen peroxide formation. The different flavoproteins show low Michaelis constants with these electron acceptor substrates, and therefore these dyes need to be added in only low concentrations to assure substrate saturation. In conclusion, these electron acceptors are useful in selective, reliable and cheap MTP-based screening assays for a range of flavin-dependent oxidoreductases, and offer a robust method for library screening, which could find applications in enzyme engineering programs. PMID:24376171

  8. Agonists of Toll-like receptor 9 containing synthetic dinucleotide motifs.

    PubMed

    Yu, Dong; Putta, Mallikarjuna R; Bhagat, Lakshmi; Li, Yukui; Zhu, Fugang; Wang, Daqing; Tang, Jimmy X; Kandimalla, Ekambar R; Agrawal, Sudhir

    2007-12-13

    Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs activate Toll-like receptor 9 (TLR9). Our previous studies have shown that ODNs containing two 5'-ends are more immunostimulatory than those with one 5'-end. In the present study, to understand the role of functional groups in TLR9 recognition and subsequent immune response, we substituted C or G of a CpG dinucleotide with 5-OH-dC, 5-propyne-dC, furano-dT, 1-(2'-deoxy-beta- d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine, dF, 4-thio-dU, N(3)-Me-dC, N (4)-Et-dC, Psi-iso-dC, and arabinoC or 7-deaza-dG, 7-deaza-8-aza-dG, 9-deaza-dG, N(1)-Me-dG, N(2)-Me-dG, 6-Thio-dG, dI, 8-OMe-dG, 8-O-allyl-dG, and arabinoG in ODN containing two 5'-ends. Agonists of TLR9 containing cytosine or guanine modification showed activity in HEK293 cells expressing TLR9, mouse spleen, and human cell-based assays and in vivo in mice. The results presented here provide insight into which specific chemical modifications at C or G of the CpG motif are recognized by TLR9 and the ability to modulate immune responses substituting natural C or G in immune modulatory oligonucleotides. PMID:17988082

  9. Rationale, progress and development of vaccines utilizing STING-activating cyclic dinucleotide adjuvants

    PubMed Central

    Kanne, David B.; Leong, Meredith L.

    2013-01-01

    A principal barrier to the development of effective vaccines is the availability of adjuvants and formulations that can elicit both effector and long-lived memory CD4 and CD8 T cells. Cellular immunity is the presumptive immune correlate of protection against intracellular pathogens: a group composed of bacteria, viruses and protozoans that is responsible for a staggering level of morbidity and mortality on a global scale. T-cell immunity is also correlated with clinical benefit in cancer, and the development of therapeutic strategies to harness the immune system to treat diverse malignancies is currently undergoing a renaissance. Cyclic dinucleotides (CDNs) are ubiquitous small molecule second messengers synthesized by bacteria that regulate diverse processes and are a relatively new class of adjuvants that have been shown to increase vaccine potency. CDNs activate innate immunity by directly binding the endoplasmic reticulum-resident receptor STING (stimulator of interferon genes), activating a signaling pathway that induces the expression of interferon-β (IFN-β) and also nuclear factor-κB (NF-κB) dependent inflammatory cytokines. The STING signaling pathway has emerged as a central Toll-like receptor (TLR) independent mediator of host innate defense in response to sensing cytosolic nucleic acids, either through direct binding of CDNs secreted by bacteria, or, as shown recently, through binding of a structurally distinct CDN produced by a host cell receptor in response to binding cytosolic double-stranded (ds)DNA. Although this relatively new class of adjuvants has to date only been evaluated in mice, newly available CDN-STING cocrystal structures will likely intensify efforts in this field towards further development and evaluation in human trials both in preventive vaccine and immunotherapy settings. PMID:24757520

  10. Interaction of Biologically Active Flavins inside Bile Salt Aggregates: Molecular Level Investigation.

    PubMed

    Maity, Banibrata; Ahmed, Sayeed Ashique; Seth, Debabrata

    2016-09-22

    In this work we have studied the photophysics of biologically active flavin molecule lumichrome (LCM) in different bile-salt aggregates. With alteration of the functional groups of the bile salts, the photophysics of confined fluorophore is largely affected and shows difference in their spectral behavior. This study also reveals the selective prototropic species of LCM present in bile salt aggregates. In the presence of the bile salt aggregates, LCM molecule shows excitation and emission wavelength-dependent emission properties, indicating switch over of the structural change of different prototropic form of the LCM molecule. The observation of higher rotational relaxation time in NaDC aggregates compared to NaTC aggregates clearly reflects that NaDC aggregates are more rigid due to its greater hydrophobicity and large in size, which is capable to bind the guest molecule more into their nanoconfined medium. Moreover, due to less acidic nature, NaDC aggregates have more ability to accept hydrogen bond from the LCM molecule and show the selective formation of isoalloxazine N10 anion (A1 monoanionic form) of LCM. PMID:27557394

  11. The non-enzymatic reduction of azo dyes by flavin and nicotinamide cofactors under varying conditions.

    PubMed

    Morrison, Jessica M; John, Gilbert H

    2013-10-01

    Azo dyes are ubiquitous in products and often become environmental pollutants due to their anthropogenic nature. Azoreductases are enzymes which are present within many bacteria and are capable of breaking down the azo dyes via reduction of the azo bond. Often, though, carcinogenic aromatic amines are formed as metabolites and are of concern to humans. Azoreductases function via an oxidation-reduction reaction and require cofactors (a nicotinamide cofactor and sometimes a flavin cofactor) to perform their function. Non-enzymatic reduction of azo dyes in the absence of an azoreductase enzyme has been suggested in previous studies, but has never been studied in detail in terms of varying cofactor combinations, different oxygen states or pHs, nor has the enzymatic reduction been compared to azoreduction in terms of dye reduction or metabolites produced, which was the aim of this study. Reduction of azo dyes by different cofactor combinations was found to occur under both aerobic and anaerobic conditions and under physiologically-relevant pHs to produce the same metabolites as an azoreductase. Our results show that, in some cases, the non-enzymatic reduction by the cofactors was found to be equal to that seen with the azoreductase, suggesting that all dye reduction in these cases is due to the cofactors themselves. This study details the importance of the use of a cofactor-only control when studying azoreductase enzymes.

  12. Flavin containing monooxygenase 3 exerts broad effects on glucose and lipid metabolism and atherosclerosis.

    PubMed

    Shih, Diana M; Wang, Zeneng; Lee, Richard; Meng, Yonghong; Che, Nam; Charugundla, Sarada; Qi, Hannah; Wu, Judy; Pan, Calvin; Brown, J Mark; Vallim, Thomas; Bennett, Brian J; Graham, Mark; Hazen, Stanley L; Lusis, Aldons J

    2015-01-01

    We performed silencing and overexpression studies of flavin containing monooxygenase (FMO) 3 in hyperlipidemic mouse models to examine its effects on trimethylamine N-oxide (TMAO) levels and atherosclerosis. Knockdown of hepatic FMO3 in LDL receptor knockout mice using an antisense oligonucleotide resulted in decreased circulating TMAO levels and atherosclerosis. Surprisingly, we also observed significant decreases in hepatic lipids and in levels of plasma lipids, ketone bodies, glucose, and insulin. FMO3 overexpression in transgenic mice, on the other hand, increased hepatic and plasma lipids. Global gene expression analyses suggested that these effects of FMO3 on lipogenesis and gluconeogenesis may be mediated through the PPARα and Kruppel-like factor 15 pathways. In vivo and in vitro results were consistent with the concept that the effects were mediated directly by FMO3 rather than trimethylamine/TMAO; in particular, overexpression of FMO3 in the human hepatoma cell line, Hep3B, resulted in significantly increased glucose secretion and lipogenesis. Our results indicate a major role for FMO3 in modulating glucose and lipid homeostasis in vivo, and they suggest that pharmacologic inhibition of FMO3 to reduce TMAO levels would be confounded by metabolic interactions. PMID:25378658

  13. How can EPR spectroscopy help to unravel molecular mechanisms of flavin-dependent photoreceptors?

    PubMed Central

    Nohr, Daniel; Rodriguez, Ryan; Weber, Stefan; Schleicher, Erik

    2015-01-01

    Electron paramagnetic resonance (EPR) spectroscopy is a well-established spectroscopic method for the examination of paramagnetic molecules. Proteins can contain paramagnetic moieties in form of stable cofactors, transiently formed intermediates, or spin labels artificially introduced to cysteine sites. The focus of this review is to evaluate potential scopes of application of EPR to the emerging field of optogenetics. The main objective for EPR spectroscopy in this context is to unravel the complex mechanisms of light-active proteins, from their primary photoreaction to downstream signal transduction. An overview of recent results from the family of flavin-containing, blue-light dependent photoreceptors is given. In detail, mechanistic similarities and differences are condensed from the three classes of flavoproteins, the cryptochromes, LOV (Light-oxygen-voltage), and BLUF (blue-light using FAD) domains. Additionally, a concept that includes spin-labeled proteins and examination using modern pulsed EPR is introduced, which allows for a precise mapping of light-induced conformational changes. PMID:26389123

  14. Oxygen-dependent photochemistry and photophysics of "miniSOG," a protein-encased flavin.

    PubMed

    Pimenta, Frederico M; Jensen, Rasmus L; Breitenbach, Thomas; Etzerodt, Michael; Ogilby, Peter R

    2013-01-01

    Selected photochemical and photophysical parameters of flavin mononucleotide (FMN) have been examined under conditions in which FMN is (1) solvated in a buffered aqueous solution, and (2) encased in a protein likewise solvated in a buffered aqueous solution. The latter was achieved using the so-called "mini Singlet Oxygen Generator" (miniSOG), an FMN-containing flavoprotein engineered from Arabidopsis thaliana phototropin 2. Although FMN is a reasonably good singlet oxygen photosensitizer in bulk water (ϕΔ = 0.65 ± 0.04), enclosing FMN in this protein facilitates photoinitiated electron-transfer reactions (Type-I chemistry) at the expense of photosensitized singlet oxygen production (Type-II chemistry) and results in a comparatively poor yield of singlet oxygen (ϕΔ = 0.030 ± 0.002). This observation on the effect of the local environment surrounding FMN is supported by a host of spectroscopic and chemical trapping experiments. The results of this study not only elucidate the behavior of miniSOG but also provide useful information for the further development of well-characterized chromophores suitable for use as intracellular sensitizers in mechanistic studies of reactive oxygen species.

  15. Localization of genes encoding three distinct flavin-containing monooxygenases to human chromosome 1q

    SciTech Connect

    Shephard, E.A.; Fox, M.F.; Povey, S. ); Dolphin, C.T.; Phillips, I.R.; Smith, R. )

    1993-04-01

    The authors have used the polymerase chain reaction to map the gene encoding human flavin-containing monooxygenase (FMO) form II (N. Lomri, Q. Gu, and J. R. Cashman, 1992, Proc. Natl. Acad. Sci. USA 89: 1685--1689) to chromosome 1. They propose the designation FMO3 for this gene as it is the third FMO gene to be mapped. The two other human FMO genes identified to date, FMO1 and FMO2, are also located on chromosome 1 (C. Dolphin, E. A. Shephard, S. Povey, C. N. A. Palmer, D. M. Ziegler, R. Ayesh, R. L. Smith, and 1. R. Phillips, 1991, J. Biol. Chem. 266: 12379--12385; C. Dolphin, E. A. Shephard, S. F. Povey, R. L. Smith, and I. R. Phillips, 1992, Biochem. J. 286: 261--267). The localization of FMO1, FMO2, and FMO3 has been refined to the long arm of chromosome 1. Analysis of human metaphase chromosomes by in situ hybridization confirmed the mapping of FMO1 and localized this gene more precisely to 1 q23-q25. 28 refs., 3 figs., 2 tabs.

  16. Mechanism of N-hydroxylation catalyzed by flavin-dependent monooxygenases.

    PubMed

    Badieyan, Somayesadat; Bach, Robert D; Sobrado, Pablo

    2015-02-20

    Aspergillus fumigatus siderophore (SidA), a member of class B flavin-dependent monooxygenases, was selected as a model system to investigate the hydroxylation mechanism of heteroatom-containing molecules by this group of enzymes. SidA selectively hydroxylates ornithine to produce N(5)-hydroxyornithine. However, SidA is also able to hydroxylate lysine with lower efficiency. In this study, the hydroxylation mechanism and substrate selectivity of SidA were systematically studied using DFT calculations. The data show that the hydroxylation reaction is initiated by homolytic cleavage of the O-O bond in the C(4a)-hydroperoxyflavin intermediate, resulting in the formation of an internal hydrogen-bonded hydroxyl radical (HO(•)). As the HO(•) moves to the ornithine N(5) atom, it rotates and donates a hydrogen atom to form the C(4a)-hydroxyflavin. Oxygen atom transfer yields an aminoxide, which is subsequently converted to hydroxylamine via water-mediated proton shuttling, with the water molecule originating from dehydration of the C(4a)-hydroxyflavin. The selectivity of SidA for ornithine is predicted to be the result of the lower energy barrier for oxidation of ornithine relative to that of lysine (16 vs 24 kcal/mol, respectively), which is due to the weaker stabilizing hydrogen bond between the incipient HO(•) and O3' of the ribose ring of NADP(+) in the transition state for lysine.

  17. Redox linked flavin sites in extracellular decaheme proteins involved in microbe-mineral electron transfer.

    DOE PAGES

    Edwards, Marcus J.; White, Gaye F.; Norman, Michael; Tome-Fernandez, Alice; Ainsworth, Emma; Shi, Liang; Fredrickson, Jim K.; Zachara, John M.; Butt, Julea N.; Richardson, David J.; et al

    2015-07-01

    Extracellular microbe-mineral electron transfer is a major driving force for the oxidation of organic carbon in many subsurface environments. Extracellular multi-heme cytochromes of the Shewenella genus play a major role in this process but the mechanism of electron exchange at the interface between cytochrome and acceptor is widely debated. The 1.8 Å x-ray crystal structure of the decaheme MtrC revealed a highly conserved CX₈C disulfide that, when substituted for AX₈A, severely compromised the ability of S. oneidensis to grow under aerobic conditions. Reductive cleavage of the disulfide in the presence of flavin mononucleotide (FMN) resulted in the reversible formation ofmore » a stable flavocytochrome. Similar results were also observed with other decaheme cytochromes, OmcA, MtrF and UndA. The data suggest that these decaheme cytochromes can transition between highly reactive flavocytochromes or less reactive cytochromes, and that this transition is controlled by a redox active disulfide that responds to the presence of oxygen.« less

  18. Redox linked flavin sites in extracellular decaheme proteins involved in microbe-mineral electron transfer.

    SciTech Connect

    Edwards, Marcus J.; White, Gaye F.; Norman, Michael; Tome-Fernandez, Alice; Ainsworth, Emma; Shi, Liang; Fredrickson, Jim K.; Zachara, John M.; Butt, Julea N.; Richardson, David J.; Clarke, Thomas A.

    2015-07-01

    Extracellular microbe-mineral electron transfer is a major driving force for the oxidation of organic carbon in many subsurface environments. Extracellular multi-heme cytochromes of the Shewenella genus play a major role in this process but the mechanism of electron exchange at the interface between cytochrome and acceptor is widely debated. The 1.8 Å x-ray crystal structure of the decaheme MtrC revealed a highly conserved CX₈C disulfide that, when substituted for AX₈A, severely compromised the ability of S. oneidensis to grow under aerobic conditions. Reductive cleavage of the disulfide in the presence of flavin mononucleotide (FMN) resulted in the reversible formation of a stable flavocytochrome. Similar results were also observed with other decaheme cytochromes, OmcA, MtrF and UndA. The data suggest that these decaheme cytochromes can transition between highly reactive flavocytochromes or less reactive cytochromes, and that this transition is controlled by a redox active disulfide that responds to the presence of oxygen.

  19. C. elegans flavin-containing monooxygenase-4 is essential for osmoregulation in hypotonic stress

    PubMed Central

    Hirani, Nisha; Westenberg, Marcel; Seed, Paul T.; Petalcorin, Mark I. R.; Dolphin, Colin T.

    2016-01-01

    ABSTRACT Studies in Caenorhabditis elegans have revealed osmoregulatory systems engaged when worms experience hypertonic conditions, but less is known about measures employed when faced with hypotonic stress. Inactivation of fmo-4, which encodes flavin-containing monooxygenase-4, results in dramatic hypoosmotic hypersensitivity; worms are unable to prevent overwhelming water influx and swell rapidly, finally rupturing due to high internal hydrostatic pressure. fmo-4 is expressed prominently in hypodermis, duct and pore cells but is excluded from the excretory cell. Thus, FMO-4 plays a crucial osmoregulatory role by promoting clearance of excess water that enters during hypotonicity, perhaps by synthesizing an osmolyte that acts to establish an osmotic gradient from excretory cell to duct and pore cells. C. elegans FMO-4 contains a C-terminal extension conserved in all nematode FMO-4s. The coincidently numbered human FMO4 also contains an extended C-terminus with features similar to those of FMO-4. Although these shared sequence characteristics suggest potential orthology, human FMO4 was unable to rescue the fmo-4 osmoregulatory defect. Intriguingly, however, mammalian FMO4 is expressed predominantly in the kidney – an appropriate site if it too is, or once was, involved in osmoregulation. PMID:27010030

  20. Performance of Protein-Ligand Force Fields for the Flavodoxin-Flavin Mononucleotide System.

    PubMed

    Robertson, Michael J; Tirado-Rives, Julian; Jorgensen, William L

    2016-08-01

    The ability to accurately perform molecular dynamics and free energy perturbation calculations for protein-ligand systems is of broad interest to the biophysical and pharmaceutical sciences. In this work, several popular force fields are evaluated for reproducing experimental properties of the flavodoxin/flavin mononucleotide system. Calculated (3)J couplings from molecular dynamics simulations probing φ and χ1 dihedral angles are compared to over 1000 experimental measurements. Free energy perturbation calculations were also executed between different protein mutants for comparison with experimental data for relative free energies of binding. Newer versions of popular protein force fields reproduced (3)J backbone and side chain couplings with good accuracy, with RMSD values near or below one hertz in most cases. OPLS-AA/M paired with CM5 charges for the ligand performed particularly well, both for the (3)J couplings and FEP results, with a mean unsigned error for relative free energies of binding of 0.36 kcal/mol. PMID:27441982

  1. Mammalian flavin-containing monooxygenases: structure/function, genetic polymorphisms and role in drug metabolism

    PubMed Central

    Krueger, Sharon K.; Williams, David E.

    2005-01-01

    Flavin-containing monooxygenase (FMO) oxygenates drugs and xenobiotics containing a “soft-nucleophile”, usually nitrogen or sulfur. FMO, like cytochrome P450 (CYP), is a monooxygenase, utilizing the reducing equivalents of NADPH to reduce 1 atom of molecular oxygen to water, while the other atom is used to oxidize the substrate. FMO and CYP also exhibit similar tissue and cellular location, molecular weight, substrate specificity, and exist as multiple enzymes under developmental control. The human FMO functional gene family is much smaller (5 families each with a single member) than CYP. FMO does not require a reductase to transfer electrons from NADPH and the catalytic cycle of the 2 monooxygenases is strikingly different. Another distinction is the lack of induction of FMOs by xenobiotics. In general, CYP is the major contributor to oxidative xenobiotic metabolism. However, FMO activity may be of significance in a number of cases and should not be overlooked. FMO and CYP have overlapping substrate specificities, but often yield distinct metabolites with potentially significant toxicological/pharmacological consequences. The physiological function(s) of FMO are poorly understood. Three of the 5 expressed human FMO genes, FMO1, FMO2 and FMO3, exhibit genetic polymorphisms. The most studied of these is FMO3 (adult human liver) in which mutant alleles contribute to the disease known as trimethylaminuria. The consequences of these FMO genetic polymorphisms in drug metabolism and human health are areas of research requiring further exploration. PMID:15922018

  2. Redox Linked Flavin Sites in Extracellular Decaheme Proteins Involved in Microbe-Mineral Electron Transfer.

    PubMed Central

    Edwards, Marcus J.; White, Gaye F.; Norman, Michael; Tome-Fernandez, Alice; Ainsworth, Emma; Shi, Liang; Fredrickson, Jim K.; Zachara, John M.; Butt, Julea N.; Richardson, David J.; Clarke, Thomas A.

    2015-01-01

    Extracellular microbe-mineral electron transfer is a major driving force for the oxidation of organic carbon in many subsurface environments. Extracellular multi-heme cytochromes of the Shewenella genus play a major role in this process but the mechanism of electron exchange at the interface between cytochrome and acceptor is widely debated. The 1.8 Å x-ray crystal structure of the decaheme MtrC revealed a highly conserved CX8C disulfide that, when substituted for AX8A, severely compromised the ability of S. oneidensis to grow under aerobic conditions. Reductive cleavage of the disulfide in the presence of flavin mononucleotide (FMN) resulted in the reversible formation of a stable flavocytochrome. Similar results were also observed with other decaheme cytochromes, OmcA, MtrF and UndA. The data suggest that these decaheme cytochromes can transition between highly reactive flavocytochromes or less reactive cytochromes, and that this transition is controlled by a redox active disulfide that responds to the presence of oxygen. PMID:26126857

  3. How can EPR spectroscopy help to unravel molecular mechanisms of flavin-dependent photoreceptors?

    PubMed

    Nohr, Daniel; Rodriguez, Ryan; Weber, Stefan; Schleicher, Erik

    2015-01-01

    Electron paramagnetic resonance (EPR) spectroscopy is a well-established spectroscopic method for the examination of paramagnetic molecules. Proteins can contain paramagnetic moieties in form of stable cofactors, transiently formed intermediates, or spin labels artificially introduced to cysteine sites. The focus of this review is to evaluate potential scopes of application of EPR to the emerging field of optogenetics. The main objective for EPR spectroscopy in this context is to unravel the complex mechanisms of light-active proteins, from their primary photoreaction to downstream signal transduction. An overview of recent results from the family of flavin-containing, blue-light dependent photoreceptors is given. In detail, mechanistic similarities and differences are condensed from the three classes of flavoproteins, the cryptochromes, LOV (Light-oxygen-voltage), and BLUF (blue-light using FAD) domains. Additionally, a concept that includes spin-labeled proteins and examination using modern pulsed EPR is introduced, which allows for a precise mapping of light-induced conformational changes.

  4. iRSpot-PseDNC: identify recombination spots with pseudo dinucleotide composition.

    PubMed

    Chen, Wei; Feng, Peng-Mian; Lin, Hao; Chou, Kuo-Chen

    2013-04-01

    Meiotic recombination is an important biological process. As a main driving force of evolution, recombination provides natural new combinations of genetic variations. Rather than randomly occurring across a genome, meiotic recombination takes place in some genomic regions (the so-called 'hotspots') with higher frequencies, and in the other regions (the so-called 'coldspots') with lower frequencies. Therefore, the information of the hotspots and coldspots would provide useful insights for in-depth studying of the mechanism of recombination and the genome evolution process as well. So far, the recombination regions have been mainly determined by experiments, which are both expensive and time-consuming. With the avalanche of genome sequences generated in the postgenomic age, it is highly desired to develop automated methods for rapidly and effectively identifying the recombination regions. In this study, a predictor, called 'iRSpot-PseDNC', was developed for identifying the recombination hotspots and coldspots. In the new predictor, the samples of DNA sequences are formulated by a novel feature vector, the so-called 'pseudo dinucleotide composition' (PseDNC), into which six local DNA structural properties, i.e. three angular parameters (twist, tilt and roll) and three translational parameters (shift, slide and rise), are incorporated. It was observed by the rigorous jackknife test that the overall success rate achieved by iRSpot-PseDNC was >82% in identifying recombination spots in Saccharomyces cerevisiae, indicating the new predictor is promising or at least may become a complementary tool to the existing methods in this area. Although the benchmark data set used to train and test the current method was from S. cerevisiae, the basic approaches can also be extended to deal with all the other genomes. Particularly, it has not escaped our notice that the PseDNC approach can be also used to study many other DNA-related problems. As a user-friendly web-server, i

  5. BII stability and base step flexibility of N6-adenine methylated GATC motifs.

    PubMed

    Karolak, Aleksandra; van der Vaart, Arjan

    2015-01-01

    The effect of N6-adenine methylation on the flexibility and shape of palindromic GATC sequences has been investigated by molecular dynamics simulations. Variations in DNA backbone geometry were observed, which were dependent on the degree of methylation and the identity of the bases. While the effect was small, more frequent BI to BII conversions were observed in the GA step of hemimethylated DNA. The increased BII population of the hemimethylated system positively correlated with increased stacking interactions between methylated adenine and guanine, while stacking interactions decreased at the TC step for the fully methylated strand. The flexibility of the AT and TC steps was marginally affected by methylation, in a fashion that was correlated with stacking interactions. The facilitated BI to BII conversion in hemimethylated strands might be of importance for SeqA selectivity and binding. PMID:26004863

  6. Role of vacuum ultraviolet (VUV) radiation in abiogenic synthesis of adenine nucleotides

    NASA Astrophysics Data System (ADS)

    Kuzicheva, E. A.; Simakov, M. B.; Mal'Ko, I. L.; Dodonova, N. Ya.; Gontareva, N. B.

    With the use of high performance liquid chromatography the products of abiogenic synthesis of adenine nucleotides in solid films were indentified and estimated quantitatively. The main products of photosynthesis appeared to be adenosine and deoxyadenosine monophosphates. Maximal yield of these products in case of adenosine has been 0.36 for 5'AMP, 0.41% for 2'(3')AMP, 0.20 for 2'3'cAMP in case of deoxyadenosine 0.13% for 5'dAMP, 0.15% for 3'dAMP, 0.24% for 3'5'cdAMP. The destruction of initial adenosine and deoxyadenosine by the end of the experiment was 10 and 15%, respectively. By the increasing of irradiation dose, 5'AMP and 5'dAMP synthesized in the cource of VUV photolysis were destructed up to adenine, its yield being 15% in both cases.

  7. Theoretical Study of Tautomerization Reactions for the Ground and First Excited Electronic States of Adenine

    NASA Technical Reports Server (NTRS)

    Salter, Latasha M.; Chaban, Galina M.; Kwak, Dochan (Technical Monitor)

    2002-01-01

    Geometrical structures and energetic properties for different tautomers of adenine are calculated in this study, using multi-configurational wave functions. Both the ground and the lowest singlet excited state potential energy surfaces are studied. Four tautomeric forms are considered, and their energetic order is found to be different on the ground and the excited state potential energy surfaces. Minimum energy reaction paths are obtained for hydrogen atom transfer (tautomerization) reactions in the ground and the lowest excited electronic states. It is found that the barrier heights and the shapes of the reaction paths are different for the ground and the excited electronic states, suggesting that the probability of such tautomerization reaction is higher on the excited state potential energy surface. This tautomerization process should become possible in the presence of water or other polar solvent molecules and should play an important role in the photochemistry of adenine.

  8. First prebiotic generation of a ribonucleotide from adenine, D-ribose and trimetaphosphate.

    PubMed

    Baccolini, Graziano; Boga, Carla; Micheletti, Gabriele

    2011-03-28

    Adenosine monophosphate isomers are obtained by self-assembling of adenine, D-ribose and trimetaphosphate in aqueous solution in good yields. This generation of a ribonucleotide from its three molecular components occurs in a one-pot reaction at room temperature for about 30-40 days and with high chemio-, regio-, and stereo-selectivity. Similar results are obtained with guanine. A mechanism is also proposed. PMID:21305098

  9. Protection of Chinese herbs against adenine-induced chronic renal failure in rats.

    PubMed

    Tong, Yanqing; Han, Bing; Guo, Hongyang; Liu, Yanru

    2010-01-01

    The aim of the study is to evaluate the efficacy of Chinese herbs (Angelica sinensis, Ligusticum wallichii, Salvia miltiorrhiza, Rhizoma dioscoreae, Rhodiola crenilata, Astragalus membranaceus and Angelica sinensis) on adenine-induced chronic renal failure in rats. 30 age-matched male Wistar rats were divided into three groups. Rats in group A (n = 10), B (n = 10) and C (n = 10) were fed a standard laboratory chow and allowed tap water ad libitum. In group B and C, renal failure was induced by the administration of a diet containing 0.75% adenine for 28 days which began at day 0. Rats in group C were given Chinese herbs (40 ml/kg with drug concentration 1.75 g/ml) beginning at day 0. Urine albumin, blood urea nitrogen (BUN) and creatinine were determined at days 0, 14 and 28. At day 28, the animals were killed and their kidneys removed for light microscope evaluation. Body weight in Group B decreased more significantly than that in Group C (p = 0.032) at day 28. The rats in group B demonstrated more severe proteinuria and higher Serum creatinine and BUN levels than group C at day 14 and day 28 (P < 0.05, 0.01). All rats given adenine developed marked structural renal damage involving the tubule and interstitium. The values were much less severe in group C than those in group B. In adenine-induced chronic renal failure rats, the protective effects of these Chinese herbs were of a significant nature. Our results do support the notion that these Chinese herbs are useful in deferring the advance of chronic renal failure. We recommend Chinese herbs as a beneficial treatment for pre-end stage chronic renal failure.

  10. Linear free energy relationships demonstrate a catalytic role for the flavin mononucleotide coenzyme of the type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase.

    PubMed

    Thibodeaux, Christopher J; Chang, Wei-chen; Liu, Hung-wen

    2010-07-28

    The type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) catalyzes the reversible isomerization of the two ubiquitous isoprene units, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are required to initiate the biosynthesis of all isoprenoid compounds found in nature. The overall chemical transformation catalyzed by IDI-2 involves a net 1,3-proton addition/elimination reaction. Surprisingly, IDI-2 requires a reduced flavin mononucleotide (FMN) coenzyme to carry out this redox neutral isomerization. The exact function of FMN in catalysis has not yet been clearly defined. To provide mechanistic insight into the role of the reduced flavin in IDI-2 catalysis, several FMN analogues with altered electronic properties were chemoenzymatically prepared, and their effects on the kinetic properties of the IDI-2 catalyzed reaction were investigated. Linear free energy relationships (LFERs) between the electronic properties of the flavin and the steady state kinetic parameters of the IDI-2 catalyzed reaction were observed. The LFER studies are complemented with kinetic isotope effect studies and kinetic characterization of an active site mutant enzyme (Q154N). Cumulatively, the data presented in this work (and in other studies) suggest that the reduced FMN coenzyme of IDI-2 functions as an acid/base catalyst, with the N5 atom of the flavin likely playing a critical role in the deprotonation of IPP en route to DMAPP formation. Several potential chemical mechanisms involving the reduced flavin as an acid/base catalyst are presented and discussed.

  11. Two Novel Flavin-Containing Monooxygenases Involved in Biosynthesis of Aliphatic Glucosinolates.

    PubMed

    Kong, Wenwen; Li, Jing; Yu, Qingyue; Cang, Wei; Xu, Rui; Wang, Yang; Ji, Wei

    2016-01-01

    Glucosinolates, a class of secondary metabolites from cruciferous plants, are derived from amino acids and have diverse biological activities, such as in biotic defense, depending on their side chain modification. The first structural modification step in the synthesis of aliphatic (methionine-derived) glucosinolates-S-oxygenation of methylthioalkyl glucosinolates to methylsulfinylalkyl glucosinolates-was found to be catalyzed by five flavin-containing monooxygenases (FMOs), FMOGS-OX1-5. Here, we report two additional FMOGS-OX enzymes, FMOGS-OX6, and FMOGS-OX7, encoded by At1g12130 and At1g12160, respectively. The overexpression of both FMOGS-OX6 and FMOGS-OX7 decreased the ratio of methylthioalkyl glucosinolates to the sum of methylthioalkyl and methylsulfinylalkyl glucosinolates, suggesting that the introduction of the two genes converted methylthioalkyl glucosinolates into methylsulfinylalkyl glucosinolates. Analysis of expression pattern revealed that the spatial expression of the two genes is quite similar and partially overlapped with the other FMOGS-OX genes, which are primarily expressed in vascular tissue. We further analyzed the responsive expression pattern of all the seven FMOGS-OX genes to exogenous treatment with abscisic acid, 1-aminocyclopropane-1-carboxylic acid (ACC), jasmonic acid (JA), salicylic acid, indole-3-acetic acid (IAA), and low and high temperatures. Although these genes showed same tendency toward the changing stimulus, the sensitivity of each gene was quite different. The variety in spatial expression among the FMOGS-OX genes while responding to environmental stimulus indicated a complex and finely tuned regulation of glucosinolates modifications. Identification of these two novel FMOGS-OX enzymes will enhance the understanding of glucosinolates modifications and the importance of evolution of these duplicated genes. PMID:27621741

  12. Two Novel Flavin-Containing Monooxygenases Involved in Biosynthesis of Aliphatic Glucosinolates

    PubMed Central

    Kong, Wenwen; Li, Jing; Yu, Qingyue; Cang, Wei; Xu, Rui; Wang, Yang; Ji, Wei

    2016-01-01

    Glucosinolates, a class of secondary metabolites from cruciferous plants, are derived from amino acids and have diverse biological activities, such as in biotic defense, depending on their side chain modification. The first structural modification step in the synthesis of aliphatic (methionine-derived) glucosinolates—S-oxygenation of methylthioalkyl glucosinolates to methylsulfinylalkyl glucosinolates—was found to be catalyzed by five flavin-containing monooxygenases (FMOs), FMOGS-OX1-5. Here, we report two additional FMOGS-OX enzymes, FMOGS-OX6, and FMOGS-OX7, encoded by At1g12130 and At1g12160, respectively. The overexpression of both FMOGS-OX6 and FMOGS-OX7 decreased the ratio of methylthioalkyl glucosinolates to the sum of methylthioalkyl and methylsulfinylalkyl glucosinolates, suggesting that the introduction of the two genes converted methylthioalkyl glucosinolates into methylsulfinylalkyl glucosinolates. Analysis of expression pattern revealed that the spatial expression of the two genes is quite similar and partially overlapped with the other FMOGS-OX genes, which are primarily expressed in vascular tissue. We further analyzed the responsive expression pattern of all the seven FMOGS-OX genes to exogenous treatment with abscisic acid, 1-aminocyclopropane-1-carboxylic acid (ACC), jasmonic acid (JA), salicylic acid, indole-3-acetic acid (IAA), and low and high temperatures. Although these genes showed same tendency toward the changing stimulus, the sensitivity of each gene was quite different. The variety in spatial expression among the FMOGS-OX genes while responding to environmental stimulus indicated a complex and finely tuned regulation of glucosinolates modifications. Identification of these two novel FMOGS-OX enzymes will enhance the understanding of glucosinolates modifications and the importance of evolution of these duplicated genes.

  13. Flavin-Containing Monooxygenase S-Oxygenation of a Series of Thioureas and Thiones

    PubMed Central

    Henderson, Marilyn C.; Siddens, Lisbeth K.; Krueger, Sharon K.; Stevens, J. Fred; Kedzie, Karen; Fang, Ken; Heidelbaugh, Todd; Nguyen, Phong; Chow, Ken; Garst, Michael; Gil, Daniel; Williams, David E.

    2014-01-01

    Mammalian flavin-containing monooxygenase (FMO) is active towards many drugs with a heteroatom having the properties of a soft nucleophile. Thiocarbamides and thiones are S-oxygenated to the sulfenic acid which can either react with glutathione and initiate a redox-cycle or be oxygenated a second time to the unstable sulfinic acid. In this study, we utilized LC-MS/MS to demonstrate that the oxygenation by hFMO of the thioureas under test terminated at the sulfenic acid. With thiones, hFMO catalyzed the second reaction and the sulfinic acid rapidly lost sulfite to form the corresponding imidazole. Thioureas are often pulmonary toxicants in mammals and, as previously reported by our laboratory, are excellent substrates for hFMO2. This isoform is expressed at high levels in the lung of most mammals, including non-human primates. Genotyping to date indicates that individuals of African (up to 49%) or Hispanic (2–7%) ancestry have at least one allele for functional hFMO2 in lung, but not Caucasians nor Asians. In this study the major metabolite formed by hFMO2 with thioureas from Allergan, Inc. was the sulfenic acid that reacted with glutathione. The majority of thiones were poor substrates for hFMO3, the major form in adult human liver. However, hFMO1, the major isoform expressed in infant and neonatal liver and adult kidney and intestine, readily S-oxygenated thiones under test, with Kms ranging from 7–160 μM and turnover numbers of 30–40 min−1. The product formed was identified by LC-MS/MS as the imidazole. The activities of the mouse and human FMO1 and FMO3 orthologs were in good agreement with the exception of some thiones for which activity was much greater with hFMO1 than mFMO1. PMID:24727368

  14. Flavin-containing monooxygenase S-oxygenation of a series of thioureas and thiones.

    PubMed

    Henderson, Marilyn C; Siddens, Lisbeth K; Krueger, Sharon K; Stevens, J Fred; Kedzie, Karen; Fang, Wenkui K; Heidelbaugh, Todd; Nguyen, Phong; Chow, Ken; Garst, Michael; Gil, Daniel; Williams, David E

    2014-07-15

    Mammalian flavin-containing monooxygenase (FMO) is active towards many drugs with a heteroatom having the properties of a soft nucleophile. Thiocarbamides and thiones are S-oxygenated to the sulfenic acid which can either react with glutathione and initiate a redox-cycle or be oxygenated a second time to the unstable sulfinic acid. In this study, we utilized LC-MS/MS to demonstrate that the oxygenation by hFMO of the thioureas under test terminated at the sulfenic acid. With thiones, hFMO catalyzed the second reaction and the sulfinic acid rapidly lost sulfite to form the corresponding imidazole. Thioureas are often pulmonary toxicants in mammals and, as previously reported by our laboratory, are excellent substrates for hFMO2. This isoform is expressed at high levels in the lung of most mammals, including non-human primates. Genotyping to date indicates that individuals of African (up to 49%) or Hispanic (2-7%) ancestry have at least one allele for functional hFMO2 in lung, but not Caucasians nor Asians. In this study the major metabolite formed by hFMO2 with thioureas from Allergan, Inc. was the sulfenic acid that reacted with glutathione. The majority of thiones were poor substrates for hFMO3, the major form in adult human liver. However, hFMO1, the major isoform expressed in infant and neonatal liver and adult kidney and intestine, readily S-oxygenated thiones under test, with Kms ranging from 7 to 160 μM and turnover numbers of 30-40 min(-1). The product formed was identified by LC-MS/MS as the imidazole. The activities of the mouse and human FMO1 and FMO3 orthologs were in good agreement with the exception of some thiones for which activity was much greater with hFMO1 than mFMO1. PMID:24727368

  15. Flavin-Dependent Monooxygenases as a Detoxification Mechanism in Insects: New Insights from the Arctiids (Lepidoptera)

    PubMed Central

    Langel, Dorothee; Heckel, David G.; Mohagheghi, Hoda; Petschenka, Georg; Ober, Dietrich

    2010-01-01

    Insects experience a wide array of chemical pressures from plant allelochemicals and pesticides and have developed several effective counterstrategies to cope with such toxins. Among these, cytochrome P450 monooxygenases are crucial in plant-insect interactions. Flavin-dependent monooxygenases (FMOs) seem not to play a central role in xenobiotic detoxification in insects, in contrast to mammals. However, the previously identified senecionine N-oxygenase of the arctiid moth Tyria jacobaeae (Lepidoptera) indicates that FMOs have been recruited during the adaptation of this insect to plants that accumulate toxic pyrrolizidine alkaloids. Identification of related FMO-like sequences of various arctiids and other Lepidoptera and their combination with expressed sequence tag (EST) data and sequences emerging from the Bombyx mori genome project show that FMOs in Lepidoptera form a gene family with three members (FMO1 to FMO3). Phylogenetic analyses suggest that FMO3 is only distantly related to lepidopteran FMO1 and FMO2 that originated from a more recent gene duplication event. Within the FMO1 gene cluster, an additional gene duplication early in the arctiid lineage provided the basis for the evolution of the highly specific biochemical, physiological, and behavioral adaptations of these butterflies to pyrrolizidine-alkaloid-producing plants. The genes encoding pyrrolizidine-alkaloid-N-oxygenizing enzymes (PNOs) are transcribed in the fat body and the head of the larvae. An N-terminal signal peptide mediates the transport of the soluble proteins into the hemolymph where PNOs efficiently convert pro-toxic pyrrolizidine alkaloids into their non-toxic N-oxide derivatives. Heterologous expression of a PNO of the generalist arctiid Grammia geneura produced an N-oxygenizing enzyme that shows noticeably expanded substrate specificity compared with the related enzyme of the specialist Tyria jacobaeae. The data about the evolution of FMOs within lepidopteran insects and the

  16. Metabolism of ketoconazole and deacetylated ketoconazole by rat hepatic microsomes and flavin-containing monooxygenases.

    PubMed

    Rodriguez, R J; Acosta, D

    1997-06-01

    Ketoconazole (KT) has been reported to cause hepatotoxicity, which is probably not mediated through an immunoallergic mechanism. Although KT is extensively metabolized by hepatic microsomal enzymes, the nature, route of formation, and toxicity of suspected metabolites are largely unknown. Recent reports indicate that N-deacetyl ketoconazole (DAK) is a major initial metabolite in mice, which, like lipophilic 4-alkylpiperazines, is susceptible to successive oxidative attacks on the N-1 position producing ring-opened dialdehydes. The rate of formation of DAK from hepatic rat microsomal incubations of KT was determined by HPLC. The rate of disappearance for KT was almost equal to the rate of DAK formation: 5.96 and 5.88 microM/hr, respectively. Also, the potential bioactivation of DAK was evaluated by measuring substrate activity of DAK with purified pig liver flavin-containing monooxygenase (FMO) and rat liver microsomes. Activity was measured by following DAK-dependent oxygen uptake polarographically at 37 degrees C in pyrophosphate buffer (pH 8.8) containing the glucose-6-phosphate NADPH-generating system. The K(M)'s of DAK were 34.6 and 77.4 microM for the purified FMO and rat microsomal FMO, respectively. Lastly, DAK was found to be metabolized by an NADPH-dependent rat liver microsomal monooxygenases at pH 8.8 to two metabolites as determined by HPLC. Heat inactivation of rat liver microsomal FMO abolished the formation of these metabolites from DAK. SKF-525A and anti-rat NADPH cytochrome P450 reductase did not inhibit this reaction. These results suggest that deacetylation of KT yields a major product, DAK, for further metabolism by microsomal monooxygenases that seem to be FMO-related.

  17. Flavin-containing monooxygenase-mediated metabolism of N-deacetyl ketoconazole by rat hepatic microsomes.

    PubMed

    Rodriguez, R J; Proteau, P J; Marquez, B L; Hetherington, C L; Buckholz, C J; O'Connell, K L

    1999-08-01

    Although ketoconazole is extensively metabolized by hepatic microsomal enzymes, the route of formation and toxicity of suspected metabolites are largely unknown. Reports indicate that N-deacetyl ketoconazole (DAK) is a major initial metabolite in mice. DAK may be susceptible to successive oxidative attacks on the N-1 position by flavin-containing monooxygenases (FMO) producing potentially toxic metabolites. Previous laboratory findings have demonstrated that postnatal rat hepatic microsomes metabolize DAK by NADPH-dependent monooxygenases to two metabolites as determined by HPLC. Our current investigation evaluated DAK's metabolism in adult male and female rats and identified metabolites that may be responsible for ketoconazole's hepatotoxicity. DAK was extensively metabolized by rat liver microsomal monooxygenases at pH 8.8 in pyrophosphate buffer containing the glucose 6-phosphate NADPH-generating system to three metabolites as determined by HPLC. The initial metabolite of DAK was a secondary hydroxylamine, N-deacetyl-N-hydroxyketoconazole, which was confirmed by liquid chromatography/mass spectrometry and NMR spectroscopy. Extensive metabolism of DAK occurred at pH 8.8 in pyrophosphate buffer (female 29% and male 53% at 0.25 h; female 55% and male 57% at 0.5 h; and female 62% and male 66% at 1.0 h). Significantly less metabolism of DAK occurred at pH 7.4 in phosphate buffer (female 11%, male 17% at 0.25 h; female 20%, male 31% at 0.5 h; and female 27%, male 37% at 1 h). Heat inactivation of microsomal-FMO abolished the formation of these metabolites from DAK. SKF-525A did not inhibit this reaction. These results suggest that DAK appears to be extensively metabolized by adult FMO-mediated monooxygenation.

  18. Isoform specificity of N-deacetyl ketoconazole by human and rabbit flavin-containing monooxygenases.

    PubMed

    Rodriguez, R J; Miranda, C L

    2000-09-01

    N-Deacetyl ketoconazole (DAK) is the major metabolite of orally administered ketoconazole. This major metabolite has been demonstrated to be further metabolized predominately by the flavin-containing monooxygenases (FMOs) to the secondary hydroxylamine, N-deacetyl-N-hydroxyketoconazole (N-hydroxy-DAK) by adult and postnatal rat hepatic microsomes. Our current investigation evaluated the FMO isoform specificity of DAK in a pyrophosphate buffer (pH 8.8) containing the glucose 6-phosphate NADPH-generating system. cDNA-expressed human FMOs (FMO1, FMO3, and FMO5) and cDNA-expressed rabbit FMOs (FMO1, FMO2, FMO3, and FMO5) were used to assess the metabolism of DAK to its subsequent FMO-mediated metabolites by HPLC analysis. Human and rabbit cDNA-expressed FMO3 resulted in extensive metabolism of DAK in 1 h (71.2 and 64.5%, respectively) to N-hydroxy-DAK (48.2 and 47.7%, respectively) and two other metabolites, metabolite 1 (11.7 and 7.8%, respectively) and metabolite 3 (10.5 and 10.0%, respectively). Previous studies suggest that metabolite 1 is the nitrone formed after successive FMO-mediated metabolism of N-hydroxy-DAK. Moreover, these studies display similar metabolic profiles seen with adult and postnatal rat hepatic microsomes. The human and rabbit FMO1 metabolized DAK predominately to the N-hydroxy-DAK in 1 h (36.2 and 25.3%, respectively) with minimal metabolism to the other metabolites (

  19. Flavin and iron-sulfur containing ferredoxin-linked glutamate synthase from spinach leaves.

    PubMed

    Hirasawa, M; Tamura, G

    1984-04-01

    Ferredoxin-dependent glutamate synthase (native enzyme) [EC 1.4.7.1] of spinach has been purified to homogeneity in the presence of 2-oxoglutarate and sodium chloride and the properties of the enzyme have been studied. The molecular weight of the enzyme was estimated to be 140,000 by gel filtration. Subunit analysis by SDS-gel electrophoresis yielded a single protein band whose molecular weight was about 170,000. This purified enzyme showed a flavo-protein-like absorption spectrum having maxima at 279 and 438 nm with shoulders at 415 and 460 nm and a broad band around 360 nm. Fluorometric data indicated the presence of 2 mol of flavin per mol of the enzyme. Preliminary paper chromatography results indicated the presence of FAD and FMN in the purified enzyme. The enzyme also contained 4 mol of acid-labile sulfide and 4 g-atoms iron per mol of enzyme. In the absence of 2-oxoglutarate and/or sodium chloride, the purified enzyme was separated by either DE-52 cellulose chromatography or gel filtration with Ultrogel AcA 34 into two molecular forms (modified enzymes) with considerable inactivation. When reduced methyl viologen plus ferredoxin was used as the electron donor, the purified (native) enzyme showed high ferredoxin-dependent activity with a specific activity of 100 units/mg protein. Methyl viologen-dependent activity was negligible in the absence of ferredoxin. Kinetic properties and results of ESR studies were described. The results indicate that ferredoxin-linked glutamate synthase of spinach leaves is an iron-sulfur flavoprotein.

  20. Flavin-containing monooxygenase S-oxygenation of a series of thioureas and thiones

    SciTech Connect

    Henderson, Marilyn C.; Siddens, Lisbeth K.; Krueger, Sharon K.; Stevens, J. Fred; Kedzie, Karen; Fang, Wenkui K.; Heidelbaugh, Todd; Nguyen, Phong; Chow, Ken; Garst, Michael; Gil, Daniel; Williams, David E.

    2014-07-15

    Mammalian flavin-containing monooxygenase (FMO) is active towards many drugs with a heteroatom having the properties of a soft nucleophile. Thiocarbamides and thiones are S-oxygenated to the sulfenic acid which can either react with glutathione and initiate a redox-cycle or be oxygenated a second time to the unstable sulfinic acid. In this study, we utilized LC–MS/MS to demonstrate that the oxygenation by hFMO of the thioureas under test terminated at the sulfenic acid. With thiones, hFMO catalyzed the second reaction and the sulfinic acid rapidly lost sulfite to form the corresponding imidazole. Thioureas are often pulmonary toxicants in mammals and, as previously reported by our laboratory, are excellent substrates for hFMO2. This isoform is expressed at high levels in the lung of most mammals, including non-human primates. Genotyping to date indicates that individuals of African (up to 49%) or Hispanic (2–7%) ancestry have at least one allele for functional hFMO2 in lung, but not Caucasians nor Asians. In this study the major metabolite formed by hFMO2 with thioureas from Allergan, Inc. was the sulfenic acid that reacted with glutathione. The majority of thiones were poor substrates for hFMO3, the major form in adult human liver. However, hFMO1, the major isoform expressed in infant and neonatal liver and adult kidney and intestine, readily S-oxygenated thiones under test, with K{sub m}s ranging from 7 to 160 μM and turnover numbers of 30–40 min{sup −1}. The product formed was identified by LC–MS/MS as the imidazole. The activities of the mouse and human FMO1 and FMO3 orthologs were in good agreement with the exception of some thiones for which activity was much greater with hFMO1 than mFMO1.

  1. Novel Variants of the Human Flavin-Containing Monooxygenase 3 (FMO3) Gene Associated with Trimethylaminuria

    PubMed Central

    Motika, Meike S.; Zhang, Jun; Zheng, Xueying; Riedler, Kiersten; Cashman, John R.

    2009-01-01

    The disorder trimethylaminuria (TMAu) often manifests itself in a body odor for individuals affected. TMAu is due to decreased metabolism of dietary-derived trimethylamine (TMA). In a healthy individual, 95% or more of TMA is converted by the flavin-containing monooxygenase 3 (FMO3, EC 1.14.13.8) to non-odorous trimethylamine N-oxide (TMA N-oxide). Several single nucleotide polymorphisms (SNPs) of the FMO3 gene have been described and result in an enzyme with decreased or abolished functional activity for TMA N-oxygenation thus leading to TMAu. Herein, we report two novel mutations observed from phenotyping and genotyping two self-reporting individuals. Sequence analysis of the exon regions of the FMO3 gene of a young woman with severe TMAu revealed heterozygous mutations at positions 187 (V187A), 158 (E158K), 308 (E308G), and 305 (E305X). Familial genetic analysis showed that the E158K/V187A/E308G derived from the same allele from the mother, and the E305X was derived from the father. FMO3 variants V187A and V187A/E158K were characterized for oxygenation of several common FMO3 substrates (i.e., 5- and 8-DPT, mercaptoimidazole (MMI), TMA, and sulindac sulfide) and for its thermal stability. Our findings show that with the combination of V187A/E158K mutations in FMO3, the enzyme activity is severely affected and possibly contributes to the TMAu observed. In another study, genotyping analysis of a 17 year old female revealed a mutation that caused a frame shift after K415 and resulted in a protein variant with only 486 amino acid residues that was associated with severe TMAu. PMID:19321370

  2. Two Novel Flavin-Containing Monooxygenases Involved in Biosynthesis of Aliphatic Glucosinolates

    PubMed Central

    Kong, Wenwen; Li, Jing; Yu, Qingyue; Cang, Wei; Xu, Rui; Wang, Yang; Ji, Wei

    2016-01-01

    Glucosinolates, a class of secondary metabolites from cruciferous plants, are derived from amino acids and have diverse biological activities, such as in biotic defense, depending on their side chain modification. The first structural modification step in the synthesis of aliphatic (methionine-derived) glucosinolates—S-oxygenation of methylthioalkyl glucosinolates to methylsulfinylalkyl glucosinolates—was found to be catalyzed by five flavin-containing monooxygenases (FMOs), FMOGS-OX1-5. Here, we report two additional FMOGS-OX enzymes, FMOGS-OX6, and FMOGS-OX7, encoded by At1g12130 and At1g12160, respectively. The overexpression of both FMOGS-OX6 and FMOGS-OX7 decreased the ratio of methylthioalkyl glucosinolates to the sum of methylthioalkyl and methylsulfinylalkyl glucosinolates, suggesting that the introduction of the two genes converted methylthioalkyl glucosinolates into methylsulfinylalkyl glucosinolates. Analysis of expression pattern revealed that the spatial expression of the two genes is quite similar and partially overlapped with the other FMOGS-OX genes, which are primarily expressed in vascular tissue. We further analyzed the responsive expression pattern of all the seven FMOGS-OX genes to exogenous treatment with abscisic acid, 1-aminocyclopropane-1-carboxylic acid (ACC), jasmonic acid (JA), salicylic acid, indole-3-acetic acid (IAA), and low and high temperatures. Although these genes showed same tendency toward the changing stimulus, the sensitivity of each gene was quite different. The variety in spatial expression among the FMOGS-OX genes while responding to environmental stimulus indicated a complex and finely tuned regulation of glucosinolates modifications. Identification of these two novel FMOGS-OX enzymes will enhance the understanding of glucosinolates modifications and the importance of evolution of these duplicated genes. PMID:27621741

  3. Contribution of flavin covalent linkage with histidine 99 to the reaction catalyzed by choline oxidase.

    PubMed

    Quaye, Osbourne; Cowins, Sharonda; Gadda, Giovanni

    2009-06-19

    The FAD-dependent choline oxidase has a flavin cofactor covalently attached to the protein via histidine 99 through an 8alpha-N(3)-histidyl linkage. The enzyme catalyzes the four-electron oxidation of choline to glycine betaine, forming betaine aldehyde as an enzyme-bound intermediate. The variant form of choline oxidase in which the histidine residue has been replaced with asparagine was used to investigate the contribution of the 8alpha-N(3)-histidyl linkage of FAD to the protein toward the reaction catalyzed by the enzyme. Decreases of 10-fold and 30-fold in the k(cat)/K(m) and k(cat) values were observed as compared with wild-type choline oxidase at pH 10 and 25 degrees C, with no significant effect on k(cat)/K(O) using choline as substrate. Both the k(cat)/K(m) and k(cat) values increased with increasing pH to limiting values at high pH consistent with the participation of an unprotonated group in the reductive half-reaction and the overall turnover of the enzyme. The pH independence of both (D)(k(cat)/K(m)) and (D)k(cat), with average values of 9.2 +/- 3.3 and 7.4 +/- 0.5, respectively, is consistent with absence of external forward and reverse commitments to catalysis, and the chemical step of CH bond cleavage being rate-limiting for both the reductive half-reaction and the overall enzyme turnover. The temperature dependence of the (D)k(red) values suggests disruption of the preorganization in the asparagine variant enzyme. Altogether, the data presented in this study are consistent with the FAD-histidyl covalent linkage being important for the optimal positioning of the hydride ion donor and acceptor in the tunneling reaction catalyzed by choline oxidase.

  4. Selective self-assembly of adenine-silver nanoparticles forms rings resembling the size of cells

    PubMed Central

    Choi, Sungmoon; Park, Soonyoung; Yang, Seon-Ah; Jeong, Yujin; Yu, Junhua

    2015-01-01

    Self-assembly has played critical roles in the construction of functional nanomaterials. However, the structure of the macroscale multicomponent materials built by the self-assembly of nanoscale building blocks is hard to predict due to multiple intermolecular interactions of great complexity. Evaporation of solvents is usually an important approach to induce kinetically stable assemblies of building blocks with a large-scale specific arrangement. During such a deweting process, we tried to monitor the possible interactions between silver nanoparticles and nucleobases at a larger scale by epifluorescence microscopy, thanks to the doping of silver nanoparticles with luminescent silver nanodots. ssDNA oligomer-stabilized silver nanoparticles and adenine self-assemble to form ring-like compartments similar to the size of modern cells. However, the silver ions only dismantle the self-assembly of adenine. The rings are thermodynamically stable as the drying process only enrich the nanoparticles-nucleobase mixture to a concentration that activates the self-assembly. The permeable membrane-like edge of the ring is composed of adenine filaments glued together by silver nanoparticles. Interestingly, chemicals are partially confined and accumulated inside the ring, suggesting that this might be used as a microreactor to speed up chemical reactions during a dewetting process. PMID:26643504

  5. Ethanol-induced activation of adenine nucleotide turnover. Evidence for a role of acetate

    SciTech Connect

    Puig, J.G.; Fox, I.H.

    1984-09-01

    Consumption of alcohol causes hyperuricemia by decreasing urate excretion and increasing its production. Our previous studies indicate that ethanol administration increases uric acid production by increasing ATP degradation to uric acid precursors. To test the hypothesis that ethanol-induced increased urate production results from acetate metabolism and enhanced adenosine triphosphate turnover, we gave intravenous sodium acetate, sodium chloride and ethanol (0.1 mmol/kg per min for 1 h) to five normal subjects. Acetate plasma levels increased from 0.04 +/- 0.01 mM (mean +/- SE) to peak values of 0.35 +/- 0.07 mM and to 0.08 +/- 0.01 mM during acetate and ethanol infusions, respectively. Urinary oxypurines increased to 223 +/- 13% and 316 +/- 44% of the base-line values during acetate and ethanol infusions, respectively. Urinary radioactivity from the adenine nucleotide pool labeled with (8-14C) adenine increased to 171 +/- 27% and to 128 +/- 8% of the base-line values after acetate and ethanol infusions. These data indicate that both ethanol and acetate increase purine nucleotide degradation by enhancing the turnover of the adenine nucleotide pool. They support the hypothesis that acetate metabolism contributes to the increased production of urate associated with ethanol intake.

  6. Stability Constants of Mixed Ligand Complexes of Nickel(II) with Adenine and Some Amino Acids

    PubMed Central

    Türkel, Naciye

    2015-01-01

    Nickel is one of the essential trace elements found in biological systems. It is mostly found in nickel-based enzymes as an essential cofactor. It forms coordination complexes with amino acids within enzymes. Nickel is also present in nucleic acids, though its function in DNA or RNA is still not clearly understood. In this study, complex formation tendencies of Ni(II) with adenine and certain L-amino acids such as aspartic acid, glutamic acid, asparagine, leucine, phenylalanine, and tryptophan were investigated in an aqueous medium. Potentiometric equilibrium measurements showed that both binary and ternary complexes of Ni(II) form with adenine and the above-mentioned L-amino acids. Ternary complexes of Ni(II)-adenine-L-amino acids are formed by stepwise mechanisms. Relative stabilities of the ternary complexes are compared with those of the corresponding binary complexes in terms of Δlog10⁡K, log10⁡X, and % RS values. It was shown that the most stable ternary complex is Ni(II):Ade:L-Asn while the weakest one is Ni(II):Ade:L-Phe in aqueous solution used in this research. In addition, results of this research clearly show that various binary and ternary type Ni(II) complexes are formed in different concentrations as a function of pH in aqueous solution. PMID:26843852

  7. Chemical evolution: The mechanism of the formation of adenine under prebiotic conditions

    PubMed Central

    Roy, Debjani; Najafian, Katayoun; von Ragué Schleyer, Paul

    2007-01-01

    Fundamental building blocks of life have been detected extraterrestrially, even in interstellar space, and are known to form nonenzymatically. Thus, the HCN pentamer, adenine (a base present in DNA and RNA), was first isolated in abiogenic experiments from an aqueous solution of ammonia and HCN in 1960. Although many variations of the reaction conditions giving adenine have been reported since then, the mechanistic details remain unexplored. Our predictions are based on extensive computations of sequences of reaction steps along several possible mechanistic routes. H2O- or NH3-catalyzed pathways are more favorable than uncatalyzed neutral or anionic alternatives, and they may well have been the major source of adenine on primitive earth. Our report provides a more detailed understanding of some of the chemical processes involved in chemical evolution, and a partial answer to the fundamental question of molecular biogenesis. Our investigation should trigger similar explorations of the detailed mechanisms of the abiotic formation of the remaining nucleic acid bases and other biologically relevant molecules. PMID:17951429

  8. Identification and characterization of a novel plastidic adenine nucleotide uniporter from Solanum tuberosum.

    PubMed

    Leroch, Michaela; Kirchberger, Simon; Haferkamp, Ilka; Wahl, Markus; Neuhaus, H Ekkehard; Tjaden, Joachim

    2005-05-01

    Homologs of BT1 (the Brittle1 protein) are found to be phylogenetically related to the mitochondrial carrier family and appear to occur in both mono- and dicotyledonous plants. Whereas BT1 from cereals is probably involved in the transport of ADP-glucose, which is essential for starch metabolism in endosperm plastids, BT1 from a noncereal plant, Solanum tuberosum (StBT1), catalyzes an adenine nucleotide uniport when functionally integrated into the bacterial cytoplasmic membrane. Import studies into intact Escherichia coli cells harboring StBT1 revealed a narrow substrate spectrum with similar affinities for AMP, ADP, and ATP of about 300-400 mum. Transiently expressed StBT1-green fluorescent protein fusion protein in tobacco leaf protoplasts showed a plastidic localization of the StBT1. In vitro synthesized radioactively labeled StBT1 was targeted to the envelope membranes of isolated spinach chloroplasts. Furthermore, we showed by real time reverse transcription-PCR a ubiquitous expression pattern of the StBT1 in autotrophic and heterotrophic potato tissues. We therefore propose that StBT1 is a plastidic adenine nucleotide uniporter used to provide the cytosol and other compartments with adenine nucleotides exclusively synthesized inside plastids.

  9. Selective self-assembly of adenine-silver nanoparticles forms rings resembling the size of cells.

    PubMed

    Choi, Sungmoon; Park, Soonyoung; Yang, Seon-Ah; Jeong, Yujin; Yu, Junhua

    2015-12-08

    Self-assembly has played critical roles in the construction of functional nanomaterials. However, the structure of the macroscale multicomponent materials built by the self-assembly of nanoscale building blocks is hard to predict due to multiple intermolecular interactions of great complexity. Evaporation of solvents is usually an important approach to induce kinetically stable assemblies of building blocks with a large-scale specific arrangement. During such a deweting process, we tried to monitor the possible interactions between silver nanoparticles and nucleobases at a larger scale by epifluorescence microscopy, thanks to the doping of silver nanoparticles with luminescent silver nanodots. ssDNA oligomer-stabilized silver nanoparticles and adenine self-assemble to form ring-like compartments similar to the size of modern cells. However, the silver ions only dismantle the self-assembly of adenine. The rings are thermodynamically stable as the drying process only enrich the nanoparticles-nucleobase mixture to a concentration that activates the self-assembly. The permeable membrane-like edge of the ring is composed of adenine filaments glued together by silver nanoparticles. Interestingly, chemicals are partially confined and accumulated inside the ring, suggesting that this might be used as a microreactor to speed up chemical reactions during a dewetting process.

  10. Adenine Synthesis in a Model Prebiotic Reaction: Connecting Origin of Life Chemistry with Biology

    PubMed Central

    2011-01-01

    Many high school laboratory experiments demonstrate concepts related to biological evolution, but few exist that allow students to investigate life’s chemical origins. This series of laboratory experiments has been developed to allow students to explore and appreciate the deep connection that exists between prebiotic chemistry, chemical evolution, and contemporary biological systems. In the first experiment of the series, students synthesize adenine, one of the purine nucleobases of DNA and RNA, from plausibly prebiotic precursor molecules. Students compare their product to authentic standards using thin-layer chromatography. The second and third experiments of the series allow students to extract DNA from a familiar organism, the strawberry, and hydrolyze it, releasing adenine, which they can then compare to the previously chemically-synthesized adenine. A fourth, optional experiment is included where the technique of thin-layer chromatography is introduced and chromatographic skills are developed for use in the other three experiments that comprise this series. Concepts relating to organic and analytical chemistry, as well as biochemistry and DNA structure, are incorporated throughout, allowing this series of laboratory experiments to be easily inserted into existing laboratory courses and to reinforce concepts already included in any high school chemistry or biology curriculum. PMID:22075932

  11. Monitoring potential molecular interactions of adenine with other amino acids using Raman spectroscopy and DFT modeling.

    PubMed

    Singh, Shweta; Donfack, P; Srivastava, Sunil K; Singh, Dheeraj K; Materny, A; Asthana, B P; Mishra, P C

    2015-01-01

    We report on the modes of inter-molecular interaction between adenine (Ade) and the amino acids: glycine (Gly), lysine (Lys) and arginine (Arg) using Raman spectroscopy of binary mixtures of adenine and each of the three amino acids at varying molar ratios in the spectral region 1550-550 cm(-1). We focused our attention on certain specific changes in the Raman bands of adenine arising due to its interaction with the amino acids. While the changes are less apparent in the Ade/Gly system, in the Ade/Lys or Ade/Arg systems, significant changes are observed, particularly in the Ade Raman bands that involve the amino group moiety and the N7 and N1 atoms of the purine ring. The ν(N1-C6), ν(N1-C2), δ(C8-H) and δ(N7-C8-N9) vibrations at 1486, 1332, 1253 and 948 cm(-1) show spectral changes on varying the Ade to amino acid molar ratio, the extent of variation being different for the three amino acids. This observation suggests a specific interaction mode between Ade and Lys or Arg, which is due to the hydrogen bonding. The measured spectral changes provide a clear indication that the interaction of Ade depends strongly on the structures of the amino acids, especially their side chains. Density functional theory (DFT) calculations were carried out to elucidate the most probable interaction modes of Ade with the different amino acids.

  12. Structure-wise discrimination of adenine and guanine by proteins on the basis of their nonbonded interactions.

    PubMed

    Usha, S; Selvaraj, S

    2015-01-01

    We have analyzed the nonbonded interactions of the structurally similar moieties, adenine and guanine forming complexes with proteins. The results comprise (a) the amino acid-ligand atom preferences, (b) solvent accessibility of ligand atoms before and after complex formation with proteins, and (c) preferred amino acid residue atoms involved in the interactions. We have observed that the amino acid preferences involved in the hydrogen bonding interactions vary for adenine and guanine. The structural variation between the purine atoms is clearly reflected by their burial tendency in the solvent environment. Correlation of the mean amino acid preference values show the variation that exists between adenine and guanine preferences of all the amino acid residues. All our observations provide evidence for the discriminating nature of the proteins in recognizing adenine and guanine. PMID:25245205

  13. Structure-wise discrimination of adenine and guanine by proteins on the basis of their nonbonded interactions.

    PubMed

    Usha, S; Selvaraj, S

    2015-01-01

    We have analyzed the nonbonded interactions of the structurally similar moieties, adenine and guanine forming complexes with proteins. The results comprise (a) the amino acid-ligand atom preferences, (b) solvent accessibility of ligand atoms before and after complex formation with proteins, and (c) preferred amino acid residue atoms involved in the interactions. We have observed that the amino acid preferences involved in the hydrogen bonding interactions vary for adenine and guanine. The structural variation between the purine atoms is clearly reflected by their burial tendency in the solvent environment. Correlation of the mean amino acid preference values show the variation that exists between adenine and guanine preferences of all the amino acid residues. All our observations provide evidence for the discriminating nature of the proteins in recognizing adenine and guanine.

  14. Structure of STING bound to cyclic di-GMP reveals the mechanism of cyclic dinucleotide recognition by the immune system.

    PubMed

    Shu, Chang; Yi, Guanghui; Watts, Tylan; Kao, C Cheng; Li, Pingwei

    2012-06-24

    STING (stimulator of interferon genes) is an innate immune sensor of cyclic dinucleotides that regulates the induction of type I interferons. STING's C-terminal domain forms a V-shaped dimer and binds a cyclic diguanylate monophosphate (c-di-GMP) at the dimer interface by both direct and solvent-mediated hydrogen bonds. Guanines of c-di-GMP stack against the phenolic rings of a conserved tyrosine, and mutations at the c-di-GMP binding surface reduce nucleotide binding and affect signaling.

  15. Kinetic and mechanistic analysis of dinucleotide and oligonucleotide formation from the 5'-phosphorimidazolide of adenosine on Na(+)-montmorillonite

    NASA Technical Reports Server (NTRS)

    Kawamura, K.; Ferris, J. P.

    1994-01-01

    The rate constants for the condensation reaction of the 5'-phosphorimidazolide of adenosine (ImpA) to form dinucleotides and oligonucleotides have been measured in the presence of Na(+)-volclay (a Na(+)-montmorillonite) in pH 8 aqueous solution at 25 degrees C. The rates of the reaction of ImpA with an excess of adenosine 5'-monophosphoramidate (NH2pA), P1,P2-diadenosine 5',5'-pyrophosphate (A5'ppA), or adenosine 5'-monophosphate (5'-AMP or pA) in the presence of the montmorillonite to form NH2pA3'pA, A5'ppA3'pA, and pA3'pA, respectively, were measured. Only 3',5'-linked products were observed. The magnitude of the rate constants decrease in the order NH2pA3'pA > A5'-ppA3'pA > pA3'pA. The binding of ImpA to montmorillonite was measured, and the adsorption isotherm was determined. The binding of ImpA to montmorillonite and the formation of higher oligonucleotides is not observed in the absence of salts. Mg2+ enhances binding and oligonucleotide formation more than Ca2+ and Na+. The rate constants for the oligonucleotide formation were determined from the reaction products formed from 10 to 40 mM ImpA in the presence of Na(+)-montmorillonite using the computer program SIMFIT. The magnitudes of the rate constants for the formation of oligonucleotides increased in the order 2-mer < 3-mer < 4-mer ... 7-mer. The rate constants for dinucleotide and trinucleotide formation are more than 1000 times larger than those measured in the absence of montmorillonite. The rate constants for the formation of dinucleotide, trinucleotide, and tetranucleotide are 41,2.6, and 3.7 times larger than those for the formation of oligo(G)s with a poly(C) template. The hydrolysis of ImpA was accelerated 35 times in the presence of the montmorillonite. The catalytic ability of montmorillonite to form dinucleotides and oligonucleotides is quantitatively evaluated and possible pathways for oligo(A) formation are proposed.

  16. Intracellular coenzymes as natural biomarkers for metabolic activities and mitochondrial anomalies.

    PubMed

    Heikal, Ahmed A

    2010-04-01

    Mitochondria play a pivotal role in energy metabolism, programmed cell death and oxidative stress. Mutated mitochondrial DNA in diseased cells compromises the structure of key enzyme complexes and, therefore, mitochondrial function, which leads to a myriad of health-related conditions such as cancer, neurodegenerative diseases, diabetes and aging. Early detection of mitochondrial and metabolic anomalies is an essential step towards effective diagnoses and therapeutic intervention. Reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) play important roles in a wide range of cellular oxidation-reduction reactions. Importantly, NADH and FAD are naturally fluorescent, which allows noninvasive imaging of metabolic activities of living cells and tissues. Furthermore, NADH and FAD autofluorescence, which can be excited using distinct wavelengths for complementary imaging methods and is sensitive to protein binding and local environment. This article highlights recent developments concerning intracellular NADH and FAD as potential biomarkers for metabolic and mitochondrial activities.

  17. Combined Endoscopic Optical Coherence Tomography and Laser Induced Fluorescence

    NASA Astrophysics Data System (ADS)

    Barton, Jennifer K.; Tumlinson, Alexandre R.; Utzinger, Urs

    Optical coherence tomography (OCT) and laser-induced fluorescence (LIF) are promising modalities for tissue characterization in human patients and animal models. OCT detects coherently backscattered light, whereas LIF detects fluorescence emission of endogenous biochemicals, such as reduced nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), collagen, and fluorescent proteins, or exogenous substances such as cyanine dyes. Given the complementary mechanisms of contrast for OCT and LIF, the combination of the two modalities could potentially provide more sensitive and specific detection of disease than either modality alone. Sample probes for both OCT and LIF can be implemented using small diameter optical fibers, suggesting a particular synergy for endoscopic applications. In this chapter, the mechanisms of contrast and diagnostic capability for both OCT and LIF are briefly examined. Evidence of complementary capability is described. Example published combined OCT-LIF systems are reviewed, one successful commercial instrument is discussed, and example applications are provided.

  18. Gold nanorods as photothermal agents and autofluorescence enhancer to track cell death during plasmonic photothermal therapy

    NASA Astrophysics Data System (ADS)

    Kannadorai, Ravi Kumar; Chiew, Geraldine Giap Ying; Luo, Kathy Qian; Liu, Quan

    2015-07-01

    The transverse and longitudinal plasmon resonance in gold nanorods can be exploited to localize the photothermal therapy and influence the fluorescence to monitor the treatment outcome at the same time. While the longitudinal plasmon peak contributes to the photothermal effect, the transverse peak can enhance fluorescence. After cells take in PEGylated nanorods through endocytosis, autofluorescence from endogenous fluorophores such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in the mitochondria is enhanced two times, which is a good indicator of the respiratory status of the cell. When cells are illuminated continuously with near infrared laser, the temperature reaches the hyperthermic region within the first four minutes, which demonstrates the efficiency of gold nanorods in photothermal therapy. The cell viability test and autofluorescence intensity show good correlation indicating the progress of cell death over time.

  19. Nonlinear optical molecular imaging enables metabolic redox sensing in tissue-engineered constructs

    NASA Astrophysics Data System (ADS)

    Chen, Leng-Chun; Lloyd, William R.; Wilson, Robert H.; Kuo, Shiuhyang; Marcelo, Cynthia L.; Feinberg, Stephen E.; Mycek, Mary-Ann

    2011-07-01

    Tissue-engineered constructs require noninvasive monitoring of cellular viability prior to implantation. In a preclinical study on human Ex Vivo Produced Oral Mucosa Equivalent (EVPOME) constructs, nonlinear optical molecular imaging was employed to extract morphological and functional information from intact constructs. Multiphoton excitation fluorescence images were acquired using endogenous fluorescence from cellular nicotinamide adenine dinucleotide phosphate [NAD(P)H] and flavin adenine dinucleotide (FAD). The images were analyzed to report quantitatively on tissue structure and metabolism (redox ratio). Both thickness variations over time and cell distribution variations with depth were identified, while changes in redox were quantified. Our results show that nonlinear optical molecular imaging has the potential to visualize and quantitatively monitor the growth and viability of a tissue-engineered construct over time.

  20. Integrated detection of intrinsic fluorophores in live microbial cells using an array of thin film amorphous silicon photodetectors.

    PubMed

    Jóskowiak, A; Stasio, N; Chu, V; Prazeres, D M F; Conde, J P

    2012-01-01

    Two-dimensional fluorescence spectroscopy (2D FS) provides a non-invasive means to assess cell condition without the introduction of changes to the cell environment. The method relies on the measurement of the excitation-emission fluorescence intensity matrix of key intrinsic fluorophores, like aromatic amino acids, enzyme cofactors, and vitamins. Commonly used detection systems are complex, with multiple bandpass filters, and are hard to miniaturize. Here, an amorphous silicon photodetector array system integrated with amorphous silicon-carbon alloy filters designed to detect three key fluorophores - tryptophan (Trp), reduced nicotine adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) - is demonstrated. These intrinsic fluorophores were detected in pure solutions and also in suspended yeast cells. The array system was used to monitor changes in intrinsic fluorophore concentration when a yeast cell solution was subject to a thermal shock stress. PMID:22565094

  1. Sensing cell metabolism by time-resolved autofluorescence.

    PubMed

    Wu, Yicong; Zheng, Wei; Qu, Jianan Y

    2006-11-01

    We built a time-resolved confocal fluorescence spectroscopy system equipped with the multichannel time-correlated single-photon-counting technique. The instrument provides a unique approach to study the fluorescence sensing of cell metabolism via analysis of the wavelength- and time-resolved intracellular autofluorescence. The experiments on monolayered cell cultures show that with UV excitation at 365 nm the time-resolved autofluorescence decays, dominated by free-bound reduced nicotinamide adenine dinucleotide signals, are sensitive indicators for cell metabolism. However, the sensitivity decreases with the increase of excitation wavelength possibly due to the interference from free-bound flavin adenine dinucleotide fluorescence. The results demonstrate that time-resolved autofluorescence can be potentially used as an important contrast mechanism to detect epithelial precancer. PMID:17041655

  2. Sensing cell metabolism by time-resolved autofluorescence

    NASA Astrophysics Data System (ADS)

    Wu, Yicong; Zheng, Wei; Qu, Jianan Y.

    2006-11-01

    We built a time-resolved confocal fluorescence spectroscopy system equipped with the multichannel time-correlated single-photon-counting technique. The instrument provides a unique approach to study the fluorescence sensing of cell metabolism via analysis of the wavelength- and time-resolved intracellular autofluorescence. The experiments on monolayered cell cultures show that with UV excitation at 365 nm the time-resolved autofluorescence decays, dominated by free-bound reduced nicotinamide adenine dinucleotide signals, are sensitive indicators for cell metabolism. However, the sensitivity decreases with the increase of excitation wavelength possibly due to the interference from free-bound flavin adenine dinucleotide fluorescence. The results demonstrate that time-resolved autofluorescence can be potentially used as an important contrast mechanism to detect epithelial precancer.

  3. Redox Reactions of Reduced Flavin Mononucleotide (FMN), Riboflavin (RBF), and Anthraquinone-2,6-disulfonate (AQDS) with Ferrihydrite and Lepidocrocite

    SciTech Connect

    Shi, Zhi; Zachara, John M.; Shi, Liang; Wang, Zheming; Moore, Dean A.; Kennedy, David W.; Fredrickson, Jim K.

    2012-09-17

    Flavins are secreted by the dissimilatory iron-reducing bacterium Shewanella and can function as endogenous electron transfer mediators (ETM). In order to assess the potential importance of flavins in Fe(III) bioreduction, we investigated the redox reaction kinetics of reduced flavins (FMNH2 and RBFH2) with ferrihydrite and lepidocrocite. The organic reductants rapidly reduced and dissolved ferrihydrite and lepidocrocite in the pH range 4-8. The rate constant k for 2-line ferrihydrite reductive dissolution by FMNH2 was 87.5 ± 3.5 M-1∙s-1 at pH 7.0 in batch reactors, and the k was similar for RBFH2. For lepidocrocite, the k was 500 ± 61 M-1∙s-1 for FMNH2, and 236 ± 22 M-1∙s-1 for RBFH2. The surface area normalized initial reaction rates (ra) were between 0.08 and 77 μmoles∙m-2∙s-1 for various conditions in stopped-flow experiments. Initial rates (ro) were first-order with respect to Fe(III) oxide concentration, and ra increased with decreasing pH. Poorly crystalline 2-line ferrihydrite yielded the highest ra, followed by more crystalline 6-line ferrihydrite, and crystalline lepidocrocite. Compared to a previous whole-cell study with Shewanella oneidensis strain MR-1, our findings suggest that ETM reduction by the Mtr pathway coupled to lactate oxidation are rate limiting, rather than heterogeneous electron transfer to the Fe(III) oxide.

  4. A flavin-mononucleotide-binding site in Hansenula anomala nicked flavocytochrome b2, requiring the association of two domains.

    PubMed

    Gervais, M; Labeyrie, F; Risler, Y; Vergnes, O

    1980-10-01

    Previous experiments in our laboratory with Saccharomyces cervisiae flavocytochrom b2 indicated that both fragments alpha and beta of the enzyme after cleavage by yeast proteases are required to form the flavin site. More detailed experiments have not been carried out on the nicked Hansenula anomala enzyme obtained by tryptic cleavage. A method has been devised that gives a quantitative separation in 4 M urea of beta, and alpha with its heme still bound. The characteristics of the various species: isolated alpha and beta and mixed alpha + beta were studied in 4 M urea and after elimination of this reagent by dialysis in the presence of FMN and 2-mercaptoethanol. Several methods, including heme spectroscopy, tryptophan fluorescence, sedimentation studies, and titration of bound flavin, were used. The results indicate that isolated alpha and beta have a folded globular structure after renaturation. The flavin binding to the alpha + beta mixture was important (50-100%) with recovery of the flavodehydrogenase activity. In contrast, binding was not detectable (< 0.5%, Kf > 10 mM) for isolated alpha and beta. As far as mononucleotide binding is concerned, such a cooperative requirement for two folding domains has never been reported in other enzymes. The present results are discussed together with others obtained in our laboratory which demonstrate that, as deduced from their sensitivity to trypsin, the structure of S. cerevisiae and H. anomala flavocytochrome b2 protomers is triglobular 'n-x-beta' (n and x combined within alpha). The tetramer assembly, which remains intact as a nicked enzyme (alpha beta)4 after the first trypsin cleavage, is broken down following a second cleavage of the chain into four cytochrome cores (n) and a functional T-flavodehydrogenase entity, a tetramer of the type (x beta)4. PMID:7439181

  5. Adenine photodimerization in deoxyadenylate sequences: elucidation of the mechanism through structural studies of a major d(ApA) photoproduct.

    PubMed Central

    Kumar, S; Joshi, P C; Sharma, N D; Bose, S N; Jeremy, R; Davies, H; Takeda, N; McCloskey, J A

    1991-01-01

    The mechanism of the photodimerization of adjacent adenine bases on the same strand of DNA has been elucidated by determining the structure of one of the two major photoproducts that are formed by UV irradiation of the deoxydinucleoside monophosphate d(ApA). The photoproduct, denoted d(ApA)*, corresponds to a species of adenine photodimer first described by Pörschke (Pörschke, D. (1973) J.Am.Chem.Soc. 95, 8440-8446). From a detailed examination of its chemical and spectroscopic properties, including comparisons with the model compound N-cyano-N1-(1-methylimidazol-5-yl)formamidine, it is deduced that d(ApA)* contains a deoxyadenosine unit covalently linked through its C(8) position to C(4) of an imidazole N(1) deoxyribonucleoside moiety bearing an N-cyanoformamidino substituent at C(5). On treatment with acid, d(ApA)* is degraded with high specificity to 8-(5-amino-imidazol-4-yl)adenine whose identity has been confirmed by independent chemical synthesis. It is concluded that the primary event in adenine photodimerization entails photoaddition of the N(7)-C(8) double bond of the 5'-adenine across the C(6) and C(5) positions of the 3'-adenine. The azetidine species thus generated acts as a common precursor to both types of d(ApA) photoproduct which are formed from it by competing modes of azetidine ring fission. PMID:2057348

  6. Movement and Metabolism of Kinetin-14C and of Adenine-14C in Coleus Petiole Segments of Increasing Age 1

    PubMed Central

    Veen, Henk; Jacobs, William P.

    1969-01-01

    To see if polar movement was typical of growth-regulators other than auxins, the movement of adenine-8-14C and of kinetin-8-14C was studied in segments cut from petioles of increasing age. No polarity was found. In time-course experiments lasting 24 hr, kinetin showed a progressive increase of radioactivity in receiver blocks, while adenine showed a maximum at 8 hr with a decline thereafter. More kinetin moved through older segments than through younger ones. There was no difference in net loss as far as the position of the donor block is concerned. However, the loss of radioactivity from adenine donor blocks was much higher than the loss of radioactivity from kinetin donor blocks. The radioactivity in receiver blocks after 24 hr treatment with kinetin-14C was still with kinetin, judging by location on chromatograms. By the same criterion, adenine and a smaller amount of some other compound were in receiver blocks after a 6 hr transport with adenine-14C in the donors. By contrast, more zones of radioactivity were extracted from petiole segments to which kinetin or adenine had been added. For both purine derivatives the original compound represented no more than 20% of the total radioactivity extracted from the tissue after a transport period of 24 hr. PMID:16657203

  7. Photoactivation of the flavin cofactor in Xenopus laevis (6–4) photolyase: Observation of a transient tyrosyl radical by time-resolved electron paramagnetic resonance

    PubMed Central

    Weber, Stefan; Kay, Christopher W. M.; Mögling, Heike; Möbius, Klaus; Hitomi, Kenichi; Todo, Takeshi

    2002-01-01

    The light-induced electron transfer reaction of flavin cofactor photoactivation in Xenopus laevis (6–4) photolyase has been studied by continuous-wave and time-resolved electron paramagnetic resonance spectroscopy. When the photoactivation is initiated from the fully oxidized form of the flavin, a neutral flavin radical is observed as a long-lived paramagnetic intermediate of two consecutive single-electron reductions under participation of redox-active amino acid residues. By time-resolved electron paramagnetic resonance, a spin-polarized transient radical-pair signal was detected that shows remarkable differences to the signals observed in the related cyclobutane pyrimidine dimer photolyase enzyme. In (6–4) photolyase, a neutral tyrosine radical has been identified as the final electron donor, on the basis of the characteristic line width, hyperfine splitting pattern, and resonance magnetic field position of the tyrosine resonances of the transient radical pair. PMID:11805294

  8. UU/UA dinucleotide frequency reduction in coding regions results in increased mRNA stability and protein expression.

    PubMed

    Al-Saif, Maher; Khabar, Khalid S A

    2012-05-01

    UU and UA dinucleotides are rare in mammalian genes and may offer natural selection against endoribonuclease-mediated mRNA decay. This study hypothesized that reducing UU and UA (UW) dinucleotides in the mRNA-coding sequence, including the codons and the dicodon boundaries, may promote resistance to mRNA decay, thereby increasing protein production. Indeed, protein expression from UW-reduced coding regions of enhanced green fluorescent protein (EGFP), luciferase, interferon-α, and hepatitis B surface antigen (HBsAg) was higher when compared to the wild-type protein expression. The steady-state level of UW-reduced EGFP mRNA was higher and the mRNA half-life was also longer. Ectopic expression of the endoribonuclease, RNase L, did not reduce the wild type or UW-reduced mRNA. A mutant form of the mRNA decay-promoting protein, tristetraprolin (TTP/ZFP36), which has a point mutation in the zinc-finger domain (C124R), was used. The wild-type EGFP mRNA but not the UW-reduced mRNA responded to the dominant negative action of the C124R ZFP36/TTP mutant. The results indicate the efficacy of the described rational approach to formulate a general scheme for boosting recombinant protein production in mammalian cells.

  9. Undetectable levels of N6-methyl adenine in mouse DNA: Cloning and analysis of PRED28, a gene coding for a putative mammalian DNA adenine methyltransferase.

    PubMed

    Ratel, David; Ravanat, Jean-Luc; Charles, Marie-Pierre; Platet, Nadine; Breuillaud, Lionel; Lunardi, Joël; Berger, François; Wion, Didier

    2006-05-29

    Three methylated bases, 5-methylcytosine, N4-methylcytosine and N6-methyladenine (m6A), can be found in DNA. However, to date, only 5-methylcytosine has been detected in mammalian genomes. To reinvestigate the presence of m6A in mammalian DNA, we used a highly sensitive method capable of detecting one N6-methyldeoxyadenosine per million nucleosides. Our results suggest that the total mouse genome contains, if any, less than 10(3) m6A. Experiments were next performed on PRED28, a putative mammalian N6-DNA methyltransferase. The murine PRED28 encodes two alternatively spliced RNA. However, although recombinant PRED28 proteins are found in the nucleus, no evidence for an adenine-methyltransferase activity was detected. PMID:16684535

  10. Trp(359) regulates flavin thermodynamics and coenzyme selectivity in Mycobacterium tuberculosis FprA.

    PubMed

    Neeli, Rajasekhar; Sabri, Muna; McLean, Kirsty J; Dunford, Adrian J; Scrutton, Nigel S; Leys, David; Munro, Andrew W

    2008-05-01

    Mtb (Mycobacterium tuberculosis) FprA (flavoprotein reductase A) is an NAD(P)H-dependent FAD-binding reductase that is structurally related to mammalian adrenodoxin reductase, and which supports the catalytic function of Mtb cytochrome P450s. Trp(359), proximal to the FAD, was investigated in light of its potential role in controlling coenzyme interactions, as observed for similarly located aromatic residues in diflavin reductases. Phylogenetic analysis indicated that a tryptophan residue corresponding to Trp(359) is conserved across FprA-type enzymes and in adrenodoxin reductases. W359A/H mutants of Mtb FprA were generated, expressed and the proteins characterized to define the role of Trp(359). W359A/H mutants exhibited perturbed UV-visible absorption/fluorescence properties. The FAD semiquinone formed in wild-type NADPH-reduced FprA was destabilized in the W359A/H mutants, which also had more positive FAD midpoint reduction potentials (-168/-181 mV respectively, versus the standard hydrogen electrode, compared with -230 mV for wild-type FprA). The W359A/H mutants had lower ferricyanide reductase k(cat) and NAD(P)H K(m) values, but this led to improvements in catalytic efficiency (k(cat)/K(m)) with NADH as reducing coenzyme (9.6/18.8 muM(-1).min(-1) respectively, compared with 5.7 muM(-1).min(-1) for wild-type FprA). Stopped-flow spectroscopy revealed NAD(P)H-dependent FAD reduction as rate-limiting in steady-state catalysis, and to be retarded in mutants (e.g. limiting rate constants for NADH-dependent FAD reduction were 25.4 s(-1) for wild-type FprA and 4.8 s(-1)/13.4 s(-1) for W359A/H mutants). Diminished mutant FAD content (particularly in W359H FprA) highlighted the importance of Trp(359) for flavin stability. The results demonstrate that the conserved Trp(359) is critical in regulating FprA FAD binding, thermodynamic properties, catalytic efficiency and coenzyme selectivity.

  11. MNDO-MOCIC evaluation of the uracil force field: Application to the interpretation of flavin vibrational spectra

    NASA Astrophysics Data System (ADS)

    Bowman, W. David; Spiro, Thomas G.

    1980-12-01

    A normal mode analysis has been carried out on the in-plane vibrations of uracil and three isotopic molecules, N,N-dideutero, C,C-dideutero, and perdeutero uracil, using a molecular orbital calculation (MNDO) to constrain the ratios of off-diagonal to diagonal elements in the compliance matrix (MOCIC). The 81 observed frequencies are calculated with a mean error of 9.7 cm-1. The force constants and normal mode patterns are discussed. The characteristic up-shift of the 1236 cm-1 mode upon N,N deuteration is reproduced by the calculation, and is ascribable to a change in mode composition from predominantly C=O deformation to predominantly C-N stretching, in agreement with the pattern observed for other cyclic imides. The analogous mode in flavin is variable in different flavoproteins and has been suggested to be sensitive to hydrogen bonding at flavin N3. This effect of hydrogen bonding is modeled in the present calculation by increasing the N-H deformation force constant. A similar perturbation of the C=O deformation constants shows that the 1236 cm-1 mode frequency may also be somewhat sensitive to hydrogen bonding at C4=0, but not at C2=0.

  12. Crystallographic Evidence of Drastic Conformational Changes in the Active Site of a Flavin-Dependent N-Hydroxylase

    PubMed Central

    2015-01-01

    The soil actinomycete Kutzneria sp. 744 produces a class of highly decorated hexadepsipeptides, which represent a new chemical scaffold that has both antimicrobial and antifungal properties. These natural products, known as kutznerides, are created via nonribosomal peptide synthesis using various derivatized amino acids. The piperazic acid moiety contained in the kutzneride scaffold, which is vital for its antibiotic activity, has been shown to derive from the hydroxylated product of l-ornithine, l-N5-hydroxyornithine. The production of this hydroxylated species is catalyzed by the action of an FAD- and NAD(P)H-dependent N-hydroxylase known as KtzI. We have been able to structurally characterize KtzI in several states along its catalytic trajectory, and by pairing these snapshots with the biochemical and structural data already available for this enzyme class, we propose a structurally based reaction mechanism that includes novel conformational changes of both the protein backbone and the flavin cofactor. Further, we were able to recapitulate these conformational changes in the protein crystal, displaying their chemical competence. Our series of structures, with corroborating biochemical and spectroscopic data collected by us and others, affords mechanistic insight into this relatively new class of flavin-dependent hydroxylases and adds another layer to the complexity of flavoenzymes. PMID:25184411

  13. Reaction of organic nitrate esters and S-nitrosothiols with reduced flavins: a possible mechanism of bioactivation.

    PubMed

    Wong, P S; Fukuto, J M

    1999-04-01

    Organic nitrate esters, such as glyceryl trinitrate and isosorbide dinitrate, are a class of compounds used to treat a variety of vascular ailments. Their effectiveness relies on their ability to be bioactivated to nitric oxide (NO) which, in turn, relaxes vascular smooth muscle. Although there have been many biological studies that indicate that NO can be formed from organic nitrate esters in a biological environment, the chemical mechanism by which this occurs has yet to be established. Previous studies have implicated both flavins and thiols in organic nitrate ester bioactivation. Thus, we examined the chemical interactions of flavins and thiols with organic nitrate esters as a means of determining the role these species may play in NO production. Based on these studies we concluded that a reasonable chemical mechanism for organic nitrate ester bioactivation involves reduction to the organic nitrite ester followed by conversion to a nitrosothiol. The release of NO from nitrosothiols can occur via a variety of processes including reaction with dihydroflavins and NADH. PMID:10232931

  14. Is the flavin-deficient red blood cell common in Maremma, Italy, an important defense against malaria in this area?

    PubMed Central

    Anderson, B. B.; Scattoni, M.; Perry, G. M.; Galvan, P.; Giuberti, M.; Buonocore, G.; Vullo, C.

    1994-01-01

    There is a high prevalence of a familial flavin-deficient red blood cell in Ferrara province in the Po delta in northern Italy, believed to have been selected for by malaria which was endemic from the 12th century. In the present study, activities of FAD-dependent red-cell glutathione reductase (EGR) in the Grosseto area of Maremma on the west coast of Italy where malaria was endemic from 300 B.C. are compared both with activities in the Ferrara area and with activities where there was no history of endemic malaria--in the Florence area and in London in people of Anglo-Saxon origin. EGR activities were similar in Grosseto and Ferrara and were significantly lower than in Florence and London. As previously found in Ferrara, low EGR activity in Grosseto was shown to be unrelated to low dietary riboflavin intake. These findings in Grosseto, suggesting selection by malaria, are particularly interesting because, unlike the situation in Ferrara and most other malarial areas, the prevalence of thalassemia and glucose-6-phosphate dehydrogenase deficiency is very low, and they do not appear to have been selected for in Maremma. It is possible that a flavin-deficient red cell, known to inhibit growth of the malaria parasite, was an important protecting factor in the population of this area over the centuries. PMID:7977361

  15. Fragmentation of the adenine and guanine molecules induced by electron collisions

    NASA Astrophysics Data System (ADS)

    Minaev, B. F.; Shafranyosh, M. I.; Svida, Yu. Yu; Sukhoviya, M. I.; Shafranyosh, I. I.; Baryshnikov, G. V.; Minaeva, V. A.

    2014-05-01

    Secondary electron emission is the most important stage in the mechanism of radiation damage to DNA biopolymers induced by primary ionizing radiation. These secondary electrons ejected by the primary electron impacts can produce further ionizations, initiating an avalanche effect, leading to genome damage through the energy transfer from the primary objects to sensitive biomolecular targets, such as nitrogenous bases, saccharides, and other DNA and peptide components. In this work, the formation of positive and negative ions of purine bases of nucleic acids (adenine and guanine molecules) under the impact of slow electrons (from 0.1 till 200 eV) is studied by the crossed electron and molecular beams technique. The method used makes it possible to measure the molecular beam intensity and determine the total cross-sections for the formation of positive and negative ions of the studied molecules, their energy dependences, and absolute values. It is found that the maximum cross section for formation of the adenine and guanine positive ions is reached at about 90 eV energy of the electron beam and their absolute values are equal to 2.8 × 10-15 and 3.2 × 10-15 cm2, respectively. The total cross section for formation of the negative ions is 6.1 × 10-18 and 7.6 × 10-18 cm2 at the energy of 1.1 eV for adenine and guanine, respectively. The absolute cross-section values for the molecular ions are measured and the cross-sections of dissociative ionization are determined. Quantum chemical calculations are performed for the studied molecules, ions and fragments for interpretation of the crossed beams experiments.

  16. Fragmentation of the adenine and guanine molecules induced by electron collisions

    SciTech Connect

    Minaev, B. F. E-mail: boris@theochem.kth.se; Shafranyosh, M. I.; Svida, Yu. Yu; Sukhoviya, M. I.; Shafranyosh, I. I.; Baryshnikov, G. V.; Minaeva, V. A.

    2014-05-07

    Secondary electron emission is the most important stage in the mechanism of radiation damage to DNA biopolymers induced by primary ionizing radiation. These secondary electrons ejected by the primary electron impacts can produce further ionizations, initiating an avalanche effect, leading to genome damage through the energy transfer from the primary objects to sensitive biomolecular targets, such as nitrogenous bases, saccharides, and other DNA and peptide components. In this work, the formation of positive and negative ions of purine bases of nucleic acids (adenine and guanine molecules) under the impact of slow electrons (from 0.1 till 200 eV) is studied by the crossed electron and molecular beams technique. The method used makes it possible to measure the molecular beam intensity and determine the total cross-sections for the formation of positive and negative ions of the studied molecules, their energy dependences, and absolute values. It is found that the maximum cross section for formation of the adenine and guanine positive ions is reached at about 90 eV energy of the electron beam and their absolute values are equal to 2.8 × 10{sup −15} and 3.2 × 10{sup −15} cm{sup 2}, respectively. The total cross section for formation of the negative ions is 6.1 × 10{sup −18} and 7.6 × 10{sup −18} cm{sup 2} at the energy of 1.1 eV for adenine and guanine, respectively. The absolute cross-section values for the molecular ions are measured and the cross-sections of dissociative ionization are determined. Quantum chemical calculations are performed for the studied molecules, ions and fragments for interpretation of the crossed beams experiments.

  17. NF-κB activation mediates crystal translocation and interstitial inflammation in adenine overload nephropathy.

    PubMed

    Okabe, Cristiene; Borges, Raquel Lerner; de Almeida, Danilo Candido; Fanelli, Camilla; Barlette, Grasiela Pedreira; Machado, Flavia Gomes; Arias, Simone Costa Alarcon; Malheiros, Denise Maria Avancini Costa; Camara, Niels Olsen Saraiva; Zatz, Roberto; Fujihara, Clarice Kazue

    2013-07-15

    Adenine overload promotes intratubular crystal precipitation and interstitial nephritis. We showed recently that these abnormalities are strongly attenuated in mice knockout for Toll-like receptors-2, -4, MyD88, ASC, or caspase-1. We now investigated whether NF-κB activation also plays a pathogenic role in this model. Adult male Munich-Wistar rats were distributed among three groups: C (n = 17), receiving standard chow; ADE (n = 17), given adenine in the chow at 0.7% for 1 wk and 0.5% for 2 wk; and ADE + pyrrolidine dithiocarbamate (PDTC; n = 14), receiving adenine as above and the NF-κB inhibitor PDTC (120 mg·kg⁻¹·day⁻¹ in the drinking water). After 3 wk, widespread crystal deposition was seen in tubular lumina and in the renal interstitium, along with granuloma formation, collagen accumulation, intense tubulointerstitial proliferation, and increased interstitial expression of inflammatory mediators. Part of the crystals were segregated from tubular lumina by a newly formed cell layer and, at more advanced stages, appeared to be extruded to the interstitium. p65 nuclear translocation and IKK-α increased abundance indicated activation of the NF-κB system. PDTC treatment prevented p65 migration and normalized IKK-α, limited crystal shift to the interstitium, and strongly attenuated interstitial fibrosis/inflammation. These findings indicate that the complex inflammatory phenomena associated with this model depend, at least in part, on NF-κB activation, and suggest that the NF-κB system may become a therapeutic target in the treatment of chronic kidney disease.

  18. The effect of pi-stacking, h-bonding, and electrostatic interactions on the ionization energies of nucleic acid bases: adenine-adenine, thymine-thymine and adenine-thymine dimers

    SciTech Connect

    Bravaya, Ksenia B.; Kostko, Oleg; Ahmed, Musahid; Krylov, Anna I.

    2009-09-02

    A combined theoretical and experimental study of the ionized dimers of thymine and adenine, TT, AA, and AT, is presented. Adiabatic and vertical ionization energies(IEs) for monomers and dimers as well as thresholds for the appearance of the protonated species are reported and analyzed. Non-covalent interactions stronglyaffect the observed IEs. The magnitude and the nature of the effect is different for different isomers of the dimers. The computations reveal that for TT, the largestchanges in vertical IEs (0.4 eV) occur in asymmetric h-bonded and symmetric pi- stacked isomers, whereas in the lowest-energy symmetric h-bonded dimer the shiftin IEs is much smaller (0.1 eV). The origin of the shift and the character of the ionized states is different in asymmetric h-bonded and symmetric stacked isomers. Inthe former, the initial hole is localized on one of the fragments, and the shift is due to the electrostatic stabilization of the positive charge of the ionized fragment by thedipole moment of the neutral fragment. In the latter, the hole is delocalized, and the change in IE is proportional to the overlap of the fragments' MOs. The shifts in AAare much smaller due to a less effcient overlap and a smaller dipole moment. The ionization of the h-bonded dimers results in barrierless (or nearly barrierless) protontransfer, whereas the pi-stacked dimers relax to structures with the hole stabilized by the delocalization or electrostatic interactions.

  19. The structure, stability, H-bonding pattern, and electrostatic potential of adenine tetrads

    NASA Astrophysics Data System (ADS)

    Gu, Jiande; Leszczynski, Jerzy

    2001-03-01

    Two conformations of the adenine tetrad were investigated at the HF and B3LYP/6-311G(d,p) levels of theory. Both conformations are predicted to be stable only in the nonplanar form. They adopt the bowl type structure. Since the planar form offers better geometry for stacking with the adjacent G-tetrad, both planar forms are expected to be important in the formation of the tetraplexes. Based on electrostatic potential map the positive electrostatic potential in the central area of both conformations is expected to reinforce the stacking between the A-tetrads and the G-tetrads in the tetraplexes.

  20. Strong coupling between adenine nucleobases in DNA single strands revealed by circular dichroism using synchrotron radiation

    NASA Astrophysics Data System (ADS)

    Kadhane, Umesh; Holm, Anne I. S.; Hoffmann, Søren Vrønning; Nielsen, Steen Brøndsted

    2008-02-01

    Circular dichroism (CD) experiments on DNA single strands (dAn) at the ASTRID synchrotron radiation facility reveal that eight adenine (A) bases electronically couple upon 190nm excitation. After n=8 , the CD signal increases linearly with n with a slope equal to the sum of the coupling terms. Nearest neighbor interactions account for only 24% of the CD signal whereas electronic communication is limited to nearest neighbors for two other exciton bands observed at 218 and 251nm (i.e., dimer excited states). Electronic coupling between bases in DNA is important for nonradiative deexcitation of electronically excited states since the hazardous energy is spread over a larger spatial region.

  1. Synthesis, spectroscopic, structural and thermal characterizations of vanadyl(IV) adenine complex prospective as antidiabetic drug agent

    NASA Astrophysics Data System (ADS)

    El-Megharbel, Samy M.; Hamza, Reham Z.; Refat, Moamen S.

    2015-01-01

    The vanadyl(IV) adenine complex; [VO(Adn)2]ṡSO4; was synthesized and characterized. The molar conductivity of this complex was measured in DMSO solution that showed an electrolyte nature. Spectroscopic investigation of the green solid complex studied here indicate that the adenine acts as a bidentate ligand, coordinated to vanadyl(IV) ions through the nitrogen atoms N7 and nitrogen atom of amino group. Thus, from the results presented the vanadyl(IV) complex has square pyramid geometry. Further characterizations using thermal analyses and scanning electron techniques was useful. The aim of this paper was to introduce a new drug model for the diabetic complications by synthesized a novel mononuclear vanadyl(IV) adenine complex to mimic insulin action and reducing blood sugar level. The antidiabetic ability of this complex was investigated in STZ-induced diabetic mice. The results suggested that VO(IV)/adenine complex has antidiabetic activity, it improved the lipid profile, it improved liver and kidney functions, also it ameliorated insulin hormone and blood glucose levels. The vanadyl(IV) complex possesses an antioxidant activity and this was clear through studying SOD, CAT, MDA, GSH and methionine synthase. The current results support the therapeutic potentiality of vanadyl(IV)/adenine complex for the management and treatment of diabetes.

  2. Simultaneous Determination of Adenine and Guanine Using Cadmium Selenide Quantum Dots-Graphene Oxide Nanocomposite Modified Electrode.

    PubMed

    Kalaivani, Arumugam; Narayanan, Sangilimuthu Sriman

    2015-06-01

    A novel electrochemical sensor was fabricated by immobilizing Cadmium Selenide Quantum Dots (CdSe QDs)-Graphene Oxide (GO) nanocomposite on a paraffin wax impregnated graphite electrode (PIGE) and was used for the simultaneous determination of adenine and guanine. The CdSe QDs-GO nanocomposite was prepared by ultrasonication and was characterized with spectroscopic and microscopic techniques. The nanocomposite modified electrode was characterized by cyclic voltammetry (CV). The modified electrode showed excellent electrocatalytic activity towards the oxidative determination of adenine and guanine with a good peak separation of 0.31 V. This may be due to the high surface area and fast electron transfer kinetics of the nanocomposite. The modified electrode exhibited wide linear ranges from 0.167 μM to 245 μM for Guanine and 0.083 μM to 291 μM for Adenine with detection limits of 0.055 μM Guanine and 0.028 μM of Adenine (S/N = 3) respectively. Further, the modified electrode was used for the quantitative determination of adenine and guanine in herring sperm DNA with satisfactory results. The modified electrode showed acceptable selectivity, reproducibility and stability under optimal conditions. PMID:26369099

  3. The isolation and characterisation of a new type of dimeric adenine photoproduct in UV-irradiated deoxyadenylates.

    PubMed Central

    Kumar, S; Sharma, N D; Davies, R J; Phillipson, D W; McCloskey, J A

    1987-01-01

    A new type of dimeric adenine photoproduct has been isolated from d(ApA) irradiated at 254 nm in neutral aqueous solution. It is formed in comparable amounts to another, quite distinct, adenine photoproduct first described by Pörschke (J. Am. Chem. Soc. (1973), 95, 8440-8446). Results from high resolution mass spectrometry and 1H NMR indicate that the new photoproduct comprises a mixture of two stereoisomers whose formation involves covalent coupling of the adenine bases in d(ApA) and concomitant incorporation of the elements of one molecule of water. The photoproduct is degraded specifically by acid to 4,6-diamino-5-guanidinopyrimidine (DGPY) whose identity has been confirmed by independent chemical synthesis. Formation of the new photoproduct in UV-irradiated d(pA)2 and poly(dA), but not poly(rA), has been demonstrated by assaying their acid hydrolysates for the presence of DGPY. The properties of the photoproduct are consistent with it being generated by the hydrolytic fission of an azetidine photoadduct in which the N(7) and C(8) atoms of the 5'-adenine in d(ApA) are linked respectively to the C(6) and C(5) positions of the 3'-adenine. PMID:3822822

  4. Metabolic fate of 14C-labelled nicotinamide and adenine in germinating propagules of the mangrove Bruguiera gymnorrhiza.

    PubMed

    Yin, Yuling; Watanabe, Shin; Ashihara, Hiroshi

    2012-01-01

    We studied the metabolic fate of [carbonyl-14C]nicotinamide and [8-(14)C]adenine in segments taken from young and developing leaves, stem, hypocotyls, and roots of a shoot-root type emerging propagule of the mangrove plant Bruguiera gymnorrhiza. Thin-layer chromatography was used together with a bioimaging analyser system. During 4 h of incubation, incorporation of radioactivity from [carbonyl-14C]nicotinamide into NAD and trigonelline was found in all parts of the propagules; the highest incorporation rates into NAD and trigonelline were found in newly emerged stem and young leaves, respectively. Radioactivity from [8-(14)C]adenine was distributed mainly in the salvage products (adenine nucleotides and RNA), and incorporation was less in catabolites (allantoin, allantoic acid, and CO2). Adenine salvage activity was higher in young leaves and stem than in hypocotyls and roots. Over a short time, the effect of 500 mM NaCl on nicotinamide and adenine metabolism indicated that NaCl inhibits both salvage and degradation activities in roots. PMID:22888538

  5. Accurate crystal molecular dynamics simulations using particle-mesh-Ewald: RNA dinucleotides — ApU and GpC

    NASA Astrophysics Data System (ADS)

    Lee, Hsing; Darden, Thomas; Pedersen, Lee

    1995-09-01

    Long molecular dynamics (MD) simulations for two crystal RNA dinucleotides ApU (2.0 ns) and GpC (1.5 ns) were performed, starting from the crystallographic positions of all heavy atoms in the crystals. By employing the particle-mesh-Ewald algorithm [Darden et al., J. Chem. Phys. 98 (1993) 10089] to accommodate the long-range Coulomb interactions, highly accurate MD structures were obtained for both crystals. The instantaneous root-mean-square positional deviations of the heavy atoms equilibrate at approximately 0.4 Å for both systems, while the experimental and calculated temperature factors are comparable in size. These results describe the first successful crystal MD simulation of RNA molecules.

  6. EXPRESSION OF BRANCHIAL FLAVIN-CONTAINING MONOOXYGENASE IS DIRECTLY CORRELATED WITH SALINITY-INDUCED ALDICARB TOXICITY IN THE EURYHALINE FISH (ORYZIAS LATIPES). (R826109)

    EPA Science Inventory

    Abstract

    Earlier studies in our laboratory have demonstrated a reduction of flavin-containing monooxygenase (FMO) activity when salt-water adapted euryhaline fish were transferred to water of less salinity. Since FMOs have been shown to be responsible for the bioact...

  7. Tolerance to Acetaminophen Hepatotoxicity in the Mouse Model of Autoprotection is Associated with Induction of Flavin-containing Monooxygenase-3 (FMO3) in Hepatocytes

    EPA Science Inventory

    Acetaminophen (APAP) pretreatment with a low hepatotoxic dose in mice results in resistance to a second, higher dose of APAP (APAP autoprotection). Recent microarray work by our group showed a drastic induction of liver flavin containing monooxygenase-3 (Fmo3) mRNA expression in...

  8. Effects of soluble flavin on heterogeneous electron transfer between surface-exposed bacterial cytochromes and iron oxides

    SciTech Connect

    Wang, Zheming; Shi, Zhi; Shi, Liang; White, Gaye F.; Richardson, David J.; Clarke, Thomas A.; Fredrickson, Jim K.; Zachara, John M.

    2015-08-25

    Dissimilatory iron-reducing bacteria can utilize insoluble Fe(Mn)-oxides as a terminal electron acceptor under anaerobic conditions. For Shewanella species specifically, some evidence suggests that iron reduction is associated with the secretion of flavin mononucleotide (FMN) and riboflavin that are proposed to mediate electron transfer (Marsili et al., 2008). In this work, we used methyl viologen (MV•+)-encapsulated, porin-cytochrome complex (MtrCAB) embedded liposomes (MELs) as a synthetic model of the Shewanella outer membrane to investigate the proposed mediating behavior of secreted flavins. The reduction kinetics of goethite, hematite and lepidocrocite (200 µM) by MELs ([MV•+] ~ 42 µM and MtrABC ≤ 1 nM) were determined in the presence FMN at pH 7.0 in N2 atmosphere by monitoring the concentrations of MV•+ and FMN through their characteristic UV-visible absorption spectra. Experiments were performed where i) FMN and Fe(III)-oxide were mixed and then reacted with the reduced MELs and ii) FMN was reacted with the reduced MELs followed by addition of Fe(III)-oxide. The redox reactions proceeded in two steps: a fast step that was completed in a few seconds, and a slower one lasting over 400 seconds. For all three Fe(III)-oxides, the initial reaction rate in the presence of a low concentration of FMN (≤ 1 µM) was at least a factor of five faster than those with MELs alone, and orders of magnitude faster than those by FMNH2, suggesting that FMN may serve as a co-factor that enhances electron transfer from outer-membrane c-cytochromes to Fe(III)-oxides. The rate and extent of the initial reaction followed the order of lepidocrocite > hematite > goethite, the same as their reduction potentials, implying thermodynamic control on reaction rate. However, at higher FMN concentrations (> 1 µM), the reaction rates for both steps decreased and varied inversely with FMN concentration, indicating that FMN inhibited the MEL to Fe(III)-oxide electron transfer

  9. Bacteriophage adenine methyltransferase: a life cycle regulator? Modelled using Vibrio harveyi myovirus like.

    PubMed

    Bochow, S; Elliman, J; Owens, L

    2012-11-01

    The adenine methyltransferase (DAM) gene methylates GATC sequences that have been demonstrated in various bacteria to be a powerful gene regulator functioning as an epigenetic switch, particularly with virulence gene regulation. However, overproduction of DAM can lead to mutations, giving rise to variability that may be important for adaptation to environmental change. While most bacterial hosts carry a DAM gene, not all bacteriophage carry this gene. Currently, there is no literature regarding the role DAM plays in life cycle regulation of bacteriophage. Vibrio campbellii strain 642 carries the bacteriophage Vibrio harveyi myovirus like (VHML) that has been proven to increase virulence. The complete genome sequence of VHML bacteriophage revealed a putative adenine methyltransferase gene. Using VHML, a new model of phage life cycle regulation, where DAM plays a central role between the lysogenic and lytic states, will be hypothesized. In short, DAM methylates the rha antirepressor gene and once methylation is removed, homologous CI repressor protein becomes repressed and non-functional leading to the switching to the lytic cycle. Greater understanding of life cycle regulation at the genetic level can, in the future, lead to the genesis of chimeric bacteriophage with greater control over their life cycle for their safe use as probiotics within the aquaculture industry. PMID:22681538

  10. 3D Magnetically Ordered Open Supramolecular Architectures Based on Ferrimagnetic Cu/Adenine/Hydroxide Heptameric Wheels.

    PubMed

    Pérez-Aguirre, Rubén; Beobide, Garikoitz; Castillo, Oscar; de Pedro, Imanol; Luque, Antonio; Pérez-Yáñez, Sonia; Rodríguez Fernández, Jesús; Román, Pascual

    2016-08-01

    The present work provides two new examples of supramolecular metal-organic frameworks consisting of three-dimensional extended noncovalent assemblies of wheel-shaped heptanuclear [Cu7(μ-H2O)6(μ3-OH)6(μ-adeninato-κN3:κN9)6](2+) entities. The heptanuclear entity consists of a central [Cu(OH)6](4-) core connected to six additional copper(II) metal centers in a radial and planar arrangement through the hydroxides. It generates a wheel-shaped entity in which water molecules and μ-κN3:κN9 adeninato ligands bridge the peripheral copper atoms. The magnetic characterization indicates the central copper(II) center is anti-ferromagnetically coupled to external copper(II) centers, which are ferromagnetically coupled among them leading to an S = 5/2 ground state. The packing of these entities is sustained by π-π stacking interactions between the adenine nucleobases and by hydrogen bonds established among the hydroxide ligands, sulfate anions, and adenine nucleobases. The sum of both types of supramolecular interactions creates a rigid synthon that in combination with the rigidity of the heptameric entity generates an open supramolecular structure (40-50% of available space) in which additional sulfate and triethylammonium ions are located altogether with solvent molecules. These compounds represent an interesting example of materials combining both porosity and magnetic relevant features.

  11. Differentiation alters the unstable expression of adenine phosphoribosyltransferase in mouse teratocarcinoma cells.

    PubMed

    Turker, M S; Tischfield, J A; Rabinovitch, P; Stambrook, P J; Trill, J J; Smith, A C; Ogburn, C E; Martin, G M

    1986-01-01

    Three multipotent mouse teratocarcinoma stem lines, all exhibiting unstable expression for the purine salvage enzyme adenine phosphoribosyltransferase (APRT) were used for the isolation of differentiated cell lines from neoplasms developed in syngeneic mice. Two of the stem cell lines (DAP1B and DAP1C) exhibited homozygous deficiencies for APRT expression while the third stem cell line (E140) exhibited a heterozygous deficiency (Turker, M.S., Smith, A.C., and Martin, G.M.; Somat. Cell Mol. Genet.; 10:55-69; 1984). A total of 16 morphologically differentiated cell lines were established from these neoplasms; most were no longer tumorigenic. Differentiated cell lines derived from the E140-induced tumors segregated homozygous deficient mutants in a single step, consistent with their retention of the heterozygous deficient state. Differentiated homozygous deficient cell lines gave rise to phenotypic revertants at very high frequencies (10(-1) to 10(-2)). The majority of these putative revertants, however, yielded cell-free extracts with little or no detectable APRT activity. These putative revertants were capable of adenine salvage and were therefore termed APRT pseudorevertants. Since the APRT pseudorevertant phenotype was only observed in the differentiated progeny of the APRT deficient stem cell lines, we conclude that this change in the nature of the revertant phenotype was a consequence of cellular differentiation.

  12. Effect of Electronic Excitation on Hydrogen Atom Transfer (Tautomerization) Reactions for the DNA Base Adenine

    NASA Technical Reports Server (NTRS)

    Chaban, Galina M.; Salter, Latasha M.; Kwak, Dochan (Technical Monitor)

    2002-01-01

    Geometrical structures and energetic properties for four different tautomers of adenine are calculated in this study, using multi-configurational wave functions. Both the ground and the lowest single excited state potential energy surface are studied. The energetic order of the tautomers on the ground state potential surface is 9H less than 7H less than 3H less than 1H, while on the excited state surface this order is found to be different: 3H less than 1H less than 9H less than 7H. Minimum energy reaction paths are obtained for hydrogen atom transfer (9 yields 3 tautomerization) reactions in the ground and the lowest excited electronic state. It is found that the barrier heights and the shapes of the reaction paths are different for the ground and the excited electronic state, suggesting that the probability of such tautomerization reaction is higher on the excited state potential energy surface. The barrier for this reaction in the excited state may become very low in the presence of water or other polar solvent molecules, and therefore such tautomerization reaction may play an important role in the solution phase photochemistry of adenine.

  13. Development of bright fluorescent quadracyclic adenine analogues: TDDFT-calculation supported rational design

    PubMed Central

    Foller Larsen, Anders; Dumat, Blaise; Wranne, Moa S.; Lawson, Christopher P.; Preus, Søren; Bood, Mattias; Gradén, Henrik; Marcus Wilhelmsson, L.; Grøtli, Morten

    2015-01-01

    Fluorescent base analogues (FBAs) comprise a family of increasingly important molecules for the investigation of nucleic acid structure and dynamics. We recently reported the quantum chemical calculation supported development of four microenvironment sensitive analogues of the quadracyclic adenine (qA) scaffold, the qANs, with highly promising absorptive and fluorescence properties that were very well predicted by TDDFT calculations. Herein, we report on the efficient synthesis, experimental and theoretical characterization of nine novel quadracyclic adenine derivatives. The brightest derivative, 2-CNqA, displays a 13-fold increased brightness (εΦF = 4500) compared with the parent compound qA and has the additional benefit of being a virtually microenvironment-insensitive fluorophore, making it a suitable candidate for nucleic acid incorporation and use in quantitative FRET and anisotropy experiments. TDDFT calculations, conducted on the nine novel qAs a posteriori, successfully describe the relative fluorescence quantum yield and brightness of all qA derivatives. This observation suggests that the TDDFT-based rational design strategy may be employed for the development of bright fluorophores built up from a common scaffold to reduce the otherwise costly and time-consuming screening process usually required to obtain useful and bright FBAs. PMID:26227585

  14. Development of bright fluorescent quadracyclic adenine analogues: TDDFT-calculation supported rational design

    NASA Astrophysics Data System (ADS)

    Foller Larsen, Anders; Dumat, Blaise; Wranne, Moa S.; Lawson, Christopher P.; Preus, Søren; Bood, Mattias; Gradén, Henrik; Marcus Wilhelmsson, L.; Grøtli, Morten

    2015-07-01

    Fluorescent base analogues (FBAs) comprise a family of increasingly important molecules for the investigation of nucleic acid structure and dynamics. We recently reported the quantum chemical calculation supported development of four microenvironment sensitive analogues of the quadracyclic adenine (qA) scaffold, the qANs, with highly promising absorptive and fluorescence properties that were very well predicted by TDDFT calculations. Herein, we report on the efficient synthesis, experimental and theoretical characterization of nine novel quadracyclic adenine derivatives. The brightest derivative, 2-CNqA, displays a 13-fold increased brightness (ɛΦF = 4500) compared with the parent compound qA and has the additional benefit of being a virtually microenvironment-insensitive fluorophore, making it a suitable candidate for nucleic acid incorporation and use in quantitative FRET and anisotropy experiments. TDDFT calculations, conducted on the nine novel qAs a posteriori, successfully describe the relative fluorescence quantum yield and brightness of all qA derivatives. This observation suggests that the TDDFT-based rational design strategy may be employed for the development of bright fluorophores built up from a common scaffold to reduce the otherwise costly and time-consuming screening process usually required to obtain useful and bright FBAs.

  15. Probing ultrafast dynamics in adenine with mid-UV four-wave mixing spectroscopies.

    PubMed

    West, Brantley A; Womick, Jordan M; Moran, Andrew M

    2011-08-11

    Heterodyne-detected transient grating (TG) and two-dimensional photon echo (2DPE) spectroscopies are extended to the mid-UV spectral range in this investigation of photoinduced relaxation processes of adenine in aqueous solution. These experiments are the first to combine a new method for generating 25 fs laser pulses (at 263 nm) with the passive phase stability afforded by diffractive optics-based interferometry. We establish a set of conditions (e.g., laser power density, solute concentration) appropriate for the study of dynamics involving the neutral solute. Undesired solute photoionization is shown to take hold at higher peak powers of the laser pulses. Signatures of internal conversion and vibrational cooling dynamics are examined using TG measurements with signal-to-noise ratios as high as 350 at short delay times. In addition, 2DPE line shapes reveal correlations between excitation and emission frequencies in adenine, which reflect electronic and nuclear relaxation processes associated with particular tautomers. Overall, this study demonstrates the feasibility of techniques that will hold many advantages for the study of biomolecules whose lowest-energy electronic resonances are found in the mid-UV (e.g., DNA bases, amino acids).

  16. Microwave-assisted stereospecific synthesis of novel tetrahydropyran adenine isonucleosides and crystal structures determination

    NASA Astrophysics Data System (ADS)

    Silva, Fábio P. L.; Cirqueira, Marilia L.; Martins, Felipe T.; Vasconcellos, Mário L. A. A.

    2013-11-01

    We describe in this article stereospecific syntheses for new isonucleosides analogs of adenine 5-7 from tosyl derivatives 2-4 accessing by microwave irradiations (50-80%). The adenine reacts entirely at the N(9) position. Compounds 2-4 were prepared in two steps from the corresponding alcohols 1, 8 and 9 (81-92%). These tetrahydropyrans alcohols 1, 8 and 9 are achiral (Meso compounds) and were prepared in two steps with complete control of 2,4,6-cis relative configuration by Prins cyclization reaction (60-63%) preceded by the Barbier reaction between allyl bromide with benzaldehyde, 4-fluorobenzaldehyde and 2-naphthaldehyde respectively under Lewis acid conditions (96-98%). The configurations and preferential conformations of 5-7 were determined by crystal structure of 6. These novel isonucleosides 5-7 present in silico potentiality to act as GPCR ligand, kinase inhibitor and enzyme inhibitor, evaluated by Molinspiration program, consistent with the expected antiviral and anticancer bioactivities.

  17. Ultraviolet photolysis of adenine: Dissociation via the {sup 1}{pi}{sigma}{sup *} state

    SciTech Connect

    Nix, Michael G. D.; Devine, Adam L.; Cronin, Brid; Ashfold, Michael N. R.

    2007-03-28

    High resolution total kinetic energy release (TKER) spectra of the H atom fragments resulting from photodissociation of jet-cooled adenine molecules at 17 wavelengths in the range 280>{lambda}{sub phot}>214 nm are reported. TKER spectra obtained at {lambda}{sub phot}>233 nm display broad, isotropic profiles that peak at low TKER ({approx}1800 cm{sup -1}) and are largely insensitive to the choice of excitation wavelength. The bulk of these products is attributed to unintended multiphoton dissociation processes. TKER spectra recorded at {lambda}{sub phot}{<=}233 nm display additional fast structure, which is attributed to N{sub 9}-H bond fission on the {sup 1}{pi}{sigma}{sup *} potential energy surface (PES). Analysis of the kinetic energies and recoil anisotropies of the H atoms responsible for the fast structure suggests excitation to two {sup 1}{pi}{pi}{sup *} excited states (the {sup 1}L{sub a} and {sup 1}B{sub b} states) at {lambda}{sub phot}{approx}230 nm, both of which dissociate to yield H atoms together with ground state adeninyl fragments by radiationless transfer through conical intersections with the {sup 1}{pi}{sigma}{sup *} PES. Parallels with the photochemistry exhibited by other, smaller heteroaromatics (pyrrole, imidazole, phenol, etc.) are highlighted, as are inconsistencies between the present conclusions and those reached in two other recent studies of excited state adenine molecules.

  18. 3D Magnetically Ordered Open Supramolecular Architectures Based on Ferrimagnetic Cu/Adenine/Hydroxide Heptameric Wheels.

    PubMed

    Pérez-Aguirre, Rubén; Beobide, Garikoitz; Castillo, Oscar; de Pedro, Imanol; Luque, Antonio; Pérez-Yáñez, Sonia; Rodríguez Fernández, Jesús; Román, Pascual

    2016-08-01

    The present work provides two new examples of supramolecular metal-organic frameworks consisting of three-dimensional extended noncovalent assemblies of wheel-shaped heptanuclear [Cu7(μ-H2O)6(μ3-OH)6(μ-adeninato-κN3:κN9)6](2+) entities. The heptanuclear entity consists of a central [Cu(OH)6](4-) core connected to six additional copper(II) metal centers in a radial and planar arrangement through the hydroxides. It generates a wheel-shaped entity in which water molecules and μ-κN3:κN9 adeninato ligands bridge the peripheral copper atoms. The magnetic characterization indicates the central copper(II) center is anti-ferromagnetically coupled to external copper(II) centers, which are ferromagnetically coupled among them leading to an S = 5/2 ground state. The packing of these entities is sustained by π-π stacking interactions between the adenine nucleobases and by hydrogen bonds established among the hydroxide ligands, sulfate anions, and adenine nucleobases. The sum of both types of supramolecular interactions creates a rigid synthon that in combination with the rigidity of the heptameric entity generates an open supramolecular structure (40-50% of available space) in which additional sulfate and triethylammonium ions are located altogether with solvent molecules. These compounds represent an interesting example of materials combining both porosity and magnetic relevant features. PMID:27409976

  19. A van der Waals density functional study of adenine on graphene: Single molecular adsorption and overlayer binding

    SciTech Connect

    Berland, Kristian; Cooper, Valentino R; Langreth, David C.; Schroder, Prof. Elsebeth; Chakarova-Kack, Svetla

    2011-01-01

    The adsorption of an adenine molecule on graphene is studied using a first-principles van der Waals functional (vdW-DF) [Dion et al., Phys. Rev. Lett. 92, 246401 (2004)]. The cohesive energy of an ordered adenine overlayer is also estimated. For the adsorption of a single molecule, we determine the optimal binding configuration and adsorption energy by translating and rotating the molecule. The adsorption energy for a single molecule of adenine is found to be 711 meV, which is close to the calculated adsorption energy of the similar-sized naphthalene. Based on the single molecular binding configuration, we estimate the cohesive energy of a two-dimensional ordered overlayer. We find a significantly stronger binding energy for the ordered overlayer than for single-molecule adsorption.

  20. DFT Studies of the Extent of Hole Delocalization in One-electron Oxidized Adenine and Guanine base Stacks

    PubMed Central

    Kumar, Anil

    2011-01-01

    This study investigates the extent of hole delocalization in one-electron oxidized adenine (A)- and guanine (G)-stacks and shows that new IR vibrational bands are predicted that are characteristic of hole delocalization within A-stacks. The geometries of A-stack (Ai; i = 2 – 8) and G-stack (GG and GGG) in their neutral and one-electron oxidized states were optimized with the bases in a B-DNA conformation using the M06-2X/6-31G* method. The highest occupied molecular orbital (HOMO) is localized on a single adenine in A-stacks and on a single guanine in GG and GGG stacks; located at the 5′-site of the stack. On one-electron oxidation (removal of an electron from the HOMO of the neutral A- and G-stacks) a “hole” is created. Mulliken charge analysis shows that these “holes” are delocalized over 2 – 3 adenine bases in the A-stack. The calculated spin density distribution of (Ai)•+ (i = 2 – 8), also, showed delocalization of the hole predominantly on two adenine bases with some delocalization on a neighboring base. For GG and GGG radical cations, the hole was found to be localized on a single G in the stack. The calculated HFCCs of GG and GGG are in good agreement with the experiment. Further, from the vibrational frequency analysis, it was found that IR spectra of neutral and the corresponding one-electron oxidized adenine stacks are quite different. The IR spectra of (A2)•+ has intense IR peaks between 900 – 1500 cm−1 which are not present in the neutral A2 stack. The presence of (A2)•+ in the adenine stack has a characteristic intense peak at ~1100 cm−1. Thus IR and Raman spectroscopy has potential for monitoring the extent of hole delocalization in A stacks. PMID:21417208

  1. Development of a new model for the induction of chronic kidney disease via intraperitoneal adenine administration, and the effect of treatment with gum acacia thereon

    PubMed Central

    Al Za’abi, Mohammed; Al Busaidi, Mahfouda; Yasin, Javid; Schupp, Nicole; Nemmar, Abderrahim; Ali, Badreldin H

    2015-01-01

    Oral adenine (0.75% w/w in feed), is an established model for human chronic kidney disease (CKD). Gum acacia (GA) has been shown to be a nephroprotective agent in this model. Here we aimed at developing a new adenine-induced CKD model in rats via a systemic route (intraperitoneal, i.p.) and to test it with GA to obviate the possibility of a physical interaction between GA and adenine in the gut. Adenine was injected i.p. (50 or 100 mg/Kg for four weeks), and GA was given concomitantly in drinking water at a concentration of 15%, w/v. Several plasma and urinary biomarkers of oxidative stress were measured and the renal damage was assessed histopathologically. Adenine, at the two given i.p. doses, significantly reduced body weight, and increased relative kidney weight, water intake and urine output. It dose-dependently increased plasma and urinary inflammatory and oxidative stress biomarkers, and caused morphological and histological damage resembling that which has been reported with oral adenine. Concomitant treatment with GA significantly mitigated almost all the above measured indices. Administration of adenine i.p. induced CKD signs very similar to those induced by oral adenine. Therefore, this new model is quicker, more practical and accurate than the original (oral) model. GA ameliorates the CKD effects caused by adenine given i.p. suggesting that the antioxidant and anti-inflammatory properties possessed by oral GA are the main mechanism for its salutary action in adenine-induced CKD, an action that is independent of its possible interaction with adenine in the gut. PMID:25755826

  2. Development of a new model for the induction of chronic kidney disease via intraperitoneal adenine administration, and the effect of treatment with gum acacia thereon.

    PubMed

    Al Za'abi, Mohammed; Al Busaidi, Mahfouda; Yasin, Javid; Schupp, Nicole; Nemmar, Abderrahim; Ali, Badreldin H

    2015-01-01

    Oral adenine (0.75% w/w in feed), is an established model for human chronic kidney disease (CKD). Gum acacia (GA) has been shown to be a nephroprotective agent in this model. Here we aimed at developing a new adenine-induced CKD model in rats via a systemic route (intraperitoneal, i.p.) and to test it with GA to obviate the possibility of a physical interaction between GA and adenine in the gut. Adenine was injected i.p. (50 or 100 mg/Kg for four weeks), and GA was given concomitantly in drinking water at a concentration of 15%, w/v. Several plasma and urinary biomarkers of oxidative stress were measured and the renal damage was assessed histopathologically. Adenine, at the two given i.p. doses, significantly reduced body weight, and increased relative kidney weight, water intake and urine output. It dose-dependently increased plasma and urinary inflammatory and oxidative stress biomarkers, and caused morphological and histological damage resembling that which has been reported with oral adenine. Concomitant treatment with GA significantly mitigated almost all the above measured indices. Administration of adenine i.p. induced CKD signs very similar to those induced by oral adenine. Therefore, this new model is quicker, more practical and accurate than the original (oral) model. GA ameliorates the CKD effects caused by adenine given i.p. suggesting that the antioxidant and anti-inflammatory properties possessed by oral GA are the main mechanism for its salutary action in adenine-induced CKD, an action that is independent of its possible interaction with adenine in the gut.

  3. Localization of human flavin-containing monooxygenase genes FMO2 and FMO5 to chromosome 1q

    SciTech Connect

    McCombie, R.R.; Shephard, E.A.; Dolphin, C.T.

    1996-06-15

    The human flavin-containing monooxygenase (FMO) gene family comprises at least five distinct members (FMO1 to FMO5) that code for enzymes responsible for the oxidation of a wide variety of soft nucleophilic substrates, including drugs and environmental pollutants. Three of these genes (FMO1, FMO3, and FMO4) have previously been localized to human chromosome 1q, raising the possibility that the entire gene family is clustered in this chromosomal region. Analysis by polymerase chain reaction of DNA isolated from a panel of human-rodent somatic cell hybrids demonstrates that the two remaining identified members of the FMO gene family, FMO2 and FMO5, also are located on chromosome 1q. 19 refs., 1 fig., 1 tab.

  4. Identification of the Lomofungin Biosynthesis Gene Cluster and Associated Flavin-Dependent Monooxygenase Gene in Streptomyces lomondensis S015

    PubMed Central

    Zhang, Chunxiao; Sheng, Chaolan; Wang, Wei; Hu, Hongbo; Peng, Huasong; Zhang, Xuehong

    2015-01-01

    Streptomyces lomondensis S015 synthesizes the broad-spectrum phenazine antibiotic lomofungin. Whole genome sequencing of this strain revealed a genomic locus consisting of 23 open reading frames that includes the core phenazine biosynthesis gene cluster lphzGFEDCB. lomo10, encoding a putative flavin-dependent monooxygenase, was also identified in this locus. Inactivation of lomo10 by in-frame partial deletion resulted in the biosynthesis of a new phenazine metabolite, 1-carbomethoxy-6-formyl-4,9-dihydroxy-phenazine, along with the absence of lomofungin. This result suggests that lomo10 is responsible for the hydroxylation of lomofungin at its C-7 position. This is the first description of a phenazine hydroxylation gene in Streptomyces, and the results of this study lay the foundation for further investigation of phenazine metabolite biosynthesis in Streptomyces. PMID:26305803

  5. Effect of thiol compounds and flavins on mercury and organomercurial degrading enzymes in mercury resistant aquatic bacteria

    SciTech Connect

    Pahan, K.; Ray, S.; Gachhui, R.; Chaudhuri, J.; Mandal, A. )

    1990-02-01

    Plasmid-determined mercuric and organomercurial resistance in microorganisms has been studied by several workers. Mercury reductase, catalyzing the reduction of mercury depends on sulfhydryl compounds. Organomercurial lyase that catalyzes the splitting of C-Hg linkages also needs thiol compounds for its activity. Until recently, no study has been reported on thiol specificity of these enzymes from various sources. In the present study, the authors report on enzymatic volatilization of HgCl{sub 2} by fourteen Hg-resistant bacterial strains. They have also studied thiol specificity of Hg-reductases and organomercurial lyases isolated from the above bacterial species. Hg-reductase is known to have FAD-moiety which stimulates enzyme activity whereas FMN and riboflavin are ineffective in this regard. The effect of flavins, namely FAD, FMN and riboflavin, on Hg-reductase and organomercurial lyase activity is also reported here.

  6. REVERSAL BY ADENINE OF THE ETHIONINE-INDUCED LIPID ACCUMULATION IN THE ENDOPLASMIC RETICULUM OF THE RAT LIVER

    PubMed Central

    Baglio, Corrado M.; Farber, Emmanuel

    1965-01-01

    Within 3.5 to 4 hours after thionine administration, numerous small osmiophilic bodies, liposomes, appear in the endoplasmic reticulum of the liver cells. By fusion, the liposomes lead to the formation of larger collections of fat, giant liposomes. Adenine administration to ethionine-treated rats removes the liposomes from the hepatocytes and causes the transitory appearance of osmiophilic droplets in the sinusoidal space of Disse. The characteristic disaggregation of hepatic polysomes seen in the liver after ethionine administration is corrected by the injection of adenine. PMID:5885431

  7. Progesterone-adenine hybrids as bivalent inhibitors of P-glycoprotein-mediated multidrug efflux: design, synthesis, characterization and biological evaluation.

    PubMed

    Zeinyeh, Waël; Mahiout, Zahia; Radix, Sylvie; Lomberget, Thierry; Dumoulin, Axel; Barret, Roland; Grenot, Catherine; Rocheblave, Luc; Matera, Eva-Laure; Dumontet, Charles; Walchshofer, Nadia

    2012-10-01

    Bivalent ligands were designed on the basis of the described close proximity of the ATP-site and the putative steroid-binding site of P-glycoprotein (ABCB1). The syntheses of 19 progesterone-adenine hybrids are described. Their abilities to inhibit P-glycoprotein-mediated daunorubicin efflux in K562/R7 human leukemic cells overexpressing P-glycoprotein were evaluated versus progesterone. The hybrid with a hexamethylene linker chain showed the best inhibitory potency. The efficiency of these progesterone-adenine hybrids depends on two main factors: (i) the nature of the linker and (ii) its attachment point on the steroid skeleton.

  8. Autofluorescence of viable cultured mammalian cells.

    PubMed

    Aubin, J E

    1979-01-01

    The autofluorescence other than intrinsic protein emission of viable cultured mammalian cells has been investigated. The fluorescence was found to originate in discrete cytoplasmic vesicle-like regions and to be absent from the nucleus. Excitation and emission spectra of viable cells revealed at least two distinct fluorescent species. Comparison of cell spectra with spectra of known cellular metabolites suggested that most, if not all, of the fluorescence arises from intracellular nicotinamide adenine dinucleotide (NADH) and riboflavin and flavin coenzymes. Various changes in culture conditions did not affect the observed autofluorescence intensity. A multiparameter flow system (MACCS) was used to compare the fluorescence intensities of numerous cultured mammalian cells.

  9. Modeling the high-energy electronic state manifold of adenine: Calibration for nonlinear electronic spectroscopy

    SciTech Connect

    Nenov, Artur Giussani, Angelo; Segarra-Martí, Javier; Jaiswal, Vishal K.; Rivalta, Ivan; Cerullo, Giulio; Mukamel, Shaul; Garavelli, Marco E-mail: marco.garavelli@ens-lyon.fr

    2015-06-07

    Pump-probe electronic spectroscopy using femtosecond laser pulses has evolved into a standard tool for tracking ultrafast excited state dynamics. Its two-dimensional (2D) counterpart is becoming an increasingly available and promising technique for resolving many of the limitations of pump-probe caused by spectral congestion. The ability to simulate pump-probe and 2D spectra from ab initio computations would allow one to link mechanistic observables like molecular motions and the making/breaking of chemical bonds to experimental observables like excited state lifetimes and quantum yields. From a theoretical standpoint, the characterization of the electronic transitions in the visible (Vis)/ultraviolet (UV), which are excited via the interaction of a molecular system with the incoming pump/probe pulses, translates into the determination of a computationally challenging number of excited states (going over 100) even for small/medium sized systems. A protocol is therefore required to evaluate the fluctuations of spectral properties like transition energies and dipole moments as a function of the computational parameters and to estimate the effect of these fluctuations on the transient spectral appearance. In the present contribution such a protocol is presented within the framework of complete and restricted active space self-consistent field theory and its second-order perturbation theory extensions. The electronic excited states of adenine have been carefully characterized through a previously presented computational recipe [Nenov et al., Comput. Theor. Chem. 1040–1041, 295-303 (2014)]. A wise reduction of the level of theory has then been performed in order to obtain a computationally less demanding approach that is still able to reproduce the characteristic features of the reference data. Foreseeing the potentiality of 2D electronic spectroscopy to track polynucleotide ground and excited state dynamics, and in particular its expected ability to provide

  10. Modeling the high-energy electronic state manifold of adenine: Calibration for nonlinear electronic spectroscopy

    NASA Astrophysics Data System (ADS)

    Nenov, Artur; Giussani, Angelo; Segarra-Martí, Javier; Jaiswal, Vishal K.; Rivalta, Ivan; Cerullo, Giulio; Mukamel, Shaul; Garavelli, Marco

    2015-06-01

    Pump-probe electronic spectroscopy using femtosecond laser pulses has evolved into a standard tool for tracking ultrafast excited state dynamics. Its two-dimensional (2D) counterpart is becoming an increasingly available and promising technique for resolving many of the limitations of pump-probe caused by spectral congestion. The ability to simulate pump-probe and 2D spectra from ab initio computations would allow one to link mechanistic observables like molecular motions and the making/breaking of chemical bonds to experimental observables like excited state lifetimes and quantum yields. From a theoretical standpoint, the characterization of the electronic transitions in the visible (Vis)/ultraviolet (UV), which are excited via the interaction of a molecular system with the incoming pump/probe pulses, translates into the determination of a computationally challenging number of excited states (going over 100) even for small/medium sized systems. A protocol is therefore required to evaluate the fluctuations of spectral properties like transition energies and dipole moments as a function of the computational parameters and to estimate the effect of these fluctuations on the transient spectral appearance. In the present contribution such a protocol is presented within the framework of complete and restricted active space self-consistent field theory and its second-order perturbation theory extensions. The electronic excited states of adenine have been carefully characterized through a previously presented computational recipe [Nenov et al., Comput. Theor. Chem. 1040-1041, 295-303 (2014)]. A wise reduction of the level of theory has then been performed in order to obtain a computationally less demanding approach that is still able to reproduce the characteristic features of the reference data. Foreseeing the potentiality of 2D electronic spectroscopy to track polynucleotide ground and excited state dynamics, and in particular its expected ability to provide

  11. Hydroxyl radical reactions with adenine: reactant complexes, transition states, and product complexes.

    PubMed

    Cheng, Qianyi; Gu, Jiande; Compaan, Katherine R; Schaefer, Henry F

    2010-10-18

    In order to address problems such as aging, cell death, and cancer, it is important to understand the mechanisms behind reactions causing DNA damage. One specific reaction implicated in DNA oxidative damage is hydroxyl free-radical attack on adenine (A) and other nucleic acid bases. The adenine reaction has been studied experimentally, but there are few theoretical results. In the present study, adenine dehydrogenation at various sites, and the potential-energy surfaces for these reactions, are investigated theoretically. Four reactant complexes [A···OH]* have been found, with binding energies relative to A+OH* of 32.8, 11.4, 10.7, and 10.1 kcal mol(-1). These four reactant complexes lead to six transition states, which in turn lie +4.3, -5.4, (-3.7 and +0.8), and (-2.3 and +0.8) kcal mol(-1) below A+OH*, respectively. Thus the lowest lying [A···OH]* complex faces the highest local barrier to formation of the product (A-H)*+H(2)O. Between the transition states and the products lie six product complexes. Adopting the same order as the reactant complexes, the product complexes [(A-H)···H(2)O]* lie at -10.9, -22.4, (-24.2 and -18.7), and (-20.5 and -17.5) kcal mol(-1), respectively, again relative to separated A+OH*. All six A+OH* → (A-H)*+H(2)O pathways are exothermic, by -0.3, -14.7, (-17.4 and -7.8), and (-13.7 and -7.8) kcal mol(-1), respectively. The transition state for dehydrogenation at N(6) lies at the lowest energy (-5.4 kcal mol(-1) relative to A+OH*), and thus reaction is likely to occur at this site. This theoretical prediction dovetails with the observed high reactivity of OH radicals with the NH(2) group of aromatic amines. However, the high barrier (37.1 kcal mol(-1)) for reaction at the C(8) site makes C(8) dehydrogenation unlikely. This last result is consistent with experimental observation of the imidazole ring opening upon OH radical addition to C(8). In addition, TD-DFT computed electronic transitions of the N(6) product around 420 nm

  12. Chronic kidney disease induced by adenine: a suitable model of growth retardation in uremia.

    PubMed

    Claramunt, Débora; Gil-Peña, Helena; Fuente, Rocío; García-López, Enrique; Loredo, Vanessa; Hernández-Frías, Olaya; Ordoñez, Flor A; Rodríguez-Suárez, Julián; Santos, Fernando

    2015-07-01

    Growth retardation is a major manifestation of chronic kidney disease (CKD) in pediatric patients. The involvement of the various pathogenic factors is difficult to evaluate in clinical studies. Here, we present an experimental model of adenine-induced CKD for the study of growth failure. Three groups (n = 10) of weaning female rats were studied: normal diet (control), 0.5% adenine diet (AD), and normal diet pair fed with AD (PF). After 21 days, serum urea nitrogen, creatinine, parathyroid hormone (PTH), weight and length gains, femur osseous front advance as an index of longitudinal growth rate, growth plate histomorphometry, chondrocyte proliferative activity, bone structure, aorta calcifications, and kidney histology were analyzed. Results are means ± SE. AD rats developed renal failure (serum urea nitrogen: 70 ± 6 mg/dl and creatinine: 0.6 ± 0.1 mg/dl) and secondary hyperparathyroidism (PTH: 480 ± 31 pg/ml). Growth retardation of AD rats was demonstrated by lower weight (AD rats: 63.3 ± 4.8 g, control rats: 112.6 ± 4.7 g, and PF rats: 60.0 ± 3.8 g) and length (AD rats: 7.2 ± 0.2 cm, control rats: 11.1 ± 0.3 cm, and PF rats: 8.1 ± 0.3 cm) gains as well as lower osseous front advances (AD rats: 141 ± 13 μm/day, control rats: 293 ± 16 μm/day, and PF rats: 251 ± 10 μm/day). The processes of chondrocyte maturation and proliferation were impaired in AD rats, as shown by lower growth plate terminal chondrocyte height (21.7 ± 2.3 vs. 26.2 ± 1.9 and 23.9 ± 1.3 μm in control and PF rats) and proliferative activity index (AD rats: 30 ± 2%, control rats: 38 ± 2%, and PF rats: 42 ± 3%). The bone primary spongiosa structure of AD rats was markedly disorganized. In conclusion, adenine-induced CKD in young rats is associated with growth retardation and disturbed endochondral ossification. This animal protocol may be a useful new experimental model to study growth in CKD.

  13. External electric field promotes proton transfer in the radical cation of adenine-thymine

    NASA Astrophysics Data System (ADS)

    Zhang, Guiqing; Xie, Shijie

    2016-07-01

    According to pKa measurements, it has been predicted that proton transfer would not occur in the radical cation of adenine-thymine (A:T). However, recent theoretical calculations indicate that proton transfer takes place in the base pair in water below the room temperature. We have performed simulations of proton transfer in the cation of B-DNA stack composed of 10 A:T base pairs in water from 20 K to 300 K. Proton transfer occurs below the room temperature, meanwhile it could also be observed at the room temperature under the external electric field. Another case that interests us is that proton transfer bounces back after ˜300 fs from the appearance of proton transfer at low temperatures.

  14. Prebiotic synthesis of adenine and amino acids under Europa-like conditions

    NASA Technical Reports Server (NTRS)

    Levy, M.; Miller, S. L.; Brinton, K.; Bada, J. L.

    2000-01-01

    In order to simulate prebiotic synthetic processes on Europa and other ice-covered planets and satellites, we have investigated the prebiotic synthesis of organic compounds from dilute solutions of NH4CN frozen for 25 years at -20 and -78 degrees C. In addition, the aqueous products of spark discharge reactions from a reducing atmosphere were frozen for 5 years at -20 degrees C. We find that both adenine and guanine, as well as a simple set of amino acids dominated by glycine, are produced in substantial yields under these conditions. These results indicate that some of the key components necessary for the origin of life may have been available on Europa throughout its history and suggest that the circumstellar zone where life might arise may be wider than previously thought.

  15. Vacuum-ultraviolet circular dichroism reveals DNA duplex formation between short strands of adenine and thymine.

    PubMed

    Nielsen, Lisbeth Munksgaard; Hoffmann, Søren Vrønning; Brøndsted Nielsen, Steen

    2012-11-21

    Absorbance spectroscopy is used extensively to tell when two DNA single strands come together and form a double strand. Here we show that circular dichroism in the vacuum ultraviolet region provides an even stronger indication for duplex formation in the case of short strands of adenine and thymine (4 to 16 bases in each strand). Indeed, our results show that a strong positive CD band appears at 179 nm when double strands are formed. Melting experiments were done in aqueous solution with and without added Na(+) counter ions. With additional salt present a huge increase in the 179 nm CD band was observed when lowering the temperature. A 179 nm CD marker band for duplex formation can be used to measure the kinetics for the association of two single strands. Such experiments rely on large changes at one particular wavelength since it is too time-consuming to record a full-wavelength spectrum.

  16. Metal-adeninate vertices for the construction of an exceptionally porous metal-organic framework.

    PubMed

    An, Jihyun; Farha, Omar K; Hupp, Joseph T; Pohl, Ehmke; Yeh, Joanne I; Rosi, Nathaniel L

    2012-01-03

    Metal-organic frameworks comprising metal-carboxylate cluster vertices and long, branched organic linkers are the most porous materials known, and therefore have attracted tremendous attention for many applications, including gas storage, separations, catalysis and drug delivery. To increase metal-organic framework porosity, the size and complexity of linkers has increased. Here we present a promising alternative strategy for constructing mesoporous metal-organic frameworks that addresses the size of the vertex rather than the length of the organic linker. This approach uses large metal-biomolecule clusters, in particular zinc-adeninate building units, as vertices to construct bio-MOF-100, an exclusively mesoporous metal-organic framework. Bio-MOF-100 exhibits a high surface area (4,300 m(2) g(-1)), one of the lowest crystal densities (0.302 g cm(-3)) and the largest metal-organic framework pore volume reported to date (4.3 cm(3) g(-1)).

  17. The isolation and characterization of the Escherichia coli DNA adenine methylase (dam) gene.

    PubMed Central

    Brooks, J E; Blumenthal, R M; Gingeras, T R

    1983-01-01

    The E. coli dam (DNA adenine methylase) enzyme is known to methylate the sequence GATC. A general method for cloning sequence-specific DNA methylase genes was used to isolate the dam gene on a 1.14 kb fragment, inserted in the plasmid vector pBR322. Subsequent restriction mapping and subcloning experiments established a set of approximate boundaries of the gene. The nucleotide sequence of the dam gene was determined, and analysis of that sequence revealed a unique open reading frame which corresponded in length to that necessary to code for a protein the size of dam. Amino acid composition derived from this sequence corresponds closely to the amino acid composition of the purified dam protein. Enzymatic and DNA:DNA hybridization methods were used to investigate the possible presence of dam genes in a variety of prokaryotic organisms. PMID:6300769

  18. Synthesis and enzymatic incorporation of α-L-threofuranosyl adenine triphosphate (tATP).

    PubMed

    Zhang, Su; Chaput, John C

    2013-03-01

    Threose nucleic acid (TNA) is an artificial genetic polymer in which the natural ribose sugar found in RNA has been replaced with an unnatural threose sugar. TNA can be synthesized enzymatically using Therminator DNA polymerase to copy DNA templates into TNA. Here, we expand the substrate repertoire of Therminator DNA polymerase to include threofuranosyl adenine 3'-triphsophate (tATP). We chemically synthesized tATP by two different methods from the 2'-O-acetyl derivative. Enzyme-mediated polymerization reveals that tATP functions as an efficient substrate for Therminator DNA polymerase, indicating that tATP can replace the diaminopurine analogue (tDTP) in TNA transcription reactions. PMID:23352269

  19. Adaptive ligand binding by the purine riboswitch in the recognition of guanine and adenine analogs

    PubMed Central

    Gilbert, Sunny D.; Reyes, Francis E.; Edwards, Andrea L.; Batey, Robert T.

    2009-01-01

    SUMMARY Purine riboswitches discriminate between guanine and adenine by at least 10,000-fold based on the identity of a single pyrimidine (Y74) that forms a Watson-Crick base pair with the ligand. To understand how this high degree of specificity for closely related compounds is achieved through simple pairing, we investigated their interaction with purine analogs with varying functional groups at the 2- and 6-positions that have the potential to alter interactions with Y74. Using a combination of crystallographic and calorimetric approaches, we find that binding these purines is often facilitated by either small structural changes in the RNA or tautomeric changes in the ligand. This work also reveals that, along with base pairing, conformational restriction of Y74 significantly contributes to nucleobase selectivity. These results reveal that compounds that exploit the inherent local flexibility within riboswitch binding pockets can alter their ligand specificity. PMID:19523903

  20. Prebiotic Synthesis of Adenine and Amino Acids Under Europa-like Conditions

    NASA Technical Reports Server (NTRS)

    Levy, Matthew; Miller, Stanley L.; Brinton, Karen; Bada, Jeffrey L.

    2003-01-01

    In order to simulate prebiotic synthetic processes on Europa and other ice-covered planets and satellites. we have investigated the prebiotic synthesis of organic compounds from dilute solutions of NH4CN frozen for 25 year at -20 and -78 C. In addition the aqueous products of spark discharge reactions from a reducing atmosphere were frozen for 5 years at -20%. We find that both adenine and guanine, as well as a simple set of amino acids dominated by glycine, are produced in substantial yields under these conditions. These results indicate that some of the key components necessary for the origin of life may have been available on Europa throughout its history and suggest that the circumstellar zone where life might arise may be m der than previously thought.

  1. [Absolute bioavailability of the adenine derivative VMA-99-82 possessing antiviral activity].

    PubMed

    Smirnova, L A; Suchkov, E A; Riabukha, A F; Kuznetsov, K A; Ozerov, A A

    2013-01-01

    Investigation of the main pharmacokinetic parameters of adenine derivative VMA-99-82 in rats showed large values of the half-life (T1/2 = 11.03 h) and the mean retention time of drug molecules in the organism (MRT = 9.53 h). A high rate of the drug concentration decrease in the plasma determines a small value of the area under the pharmacokinetic curve (AUC = 74.96 mg h/ml). The total distribution volume (V(d) = 10.61 l/kg) is 15.8 times greater than the volume of extracellular fluid in the body of rat, which is indicative of a high ability of VMA-99-82 to be distributed and accumulated in the organs and tissues. The absolute bioavailability of VMA-99-82 is 66%. PMID:24605425

  2. Animal models of pediatric chronic kidney disease. Is adenine intake an appropriate model?

    PubMed

    Claramunt, Débora; Gil-Peña, Helena; Fuente, Rocío; Hernández-Frías, Olaya; Santos, Fernando

    2015-01-01

    Pediatric chronic kidney disease (CKD) has peculiar features. In particular, growth impairment is a major clinical manifestation of CKD that debuts in pediatric age because it presents in a large proportion of infants and children with CKD and has a profound impact on the self-esteem and social integration of the stunted patients. Several factors associated with CKD may lead to growth retardation by interfering with the normal physiology of growth plate, the organ where longitudinal growth rate takes place. The study of growth plate is hardly possible in humans and justifies the use of animal models. Young rats made uremic by 5/6 nephrectomy have been widely used as a model to investigate growth retardation in CKD. This article examines the characteristics of this model and analyzes the utilization of CKD induced by high adenine diet as an alternative research protocol.

  3. Intriguing radical-radical interactions among double-electron oxidized adenine-thymine base pairs

    NASA Astrophysics Data System (ADS)

    Wang, Mei; Zhao, Jing; Zhang, Laibin; Su, Xiyu; Su, Hanlei; Bu, Yuxiang

    2015-01-01

    We present a theoretical investigation of the structural and electronic properties of double-electron oxidized adenine-thymine base pair as well as its deprotonated Watson-Crick derivatives. Double-electron oxidation can destabilize the AT unit, leading to a barrier-hindered metastable A+T+ state with a dissociation channel featuring negative dissociation energy. This unusual energetic phenomenon originates from the competition of electrostatic repulsion and attractively hydrogen-bonding interaction co-existing between Arad + and Trad +. The associated double-proton-transfer process is also explored, suggesting a possible two-step mechanism. Magnetic coupling interactions of various diradical structures are controlled by both intra- and inter-molecular interactions.

  4. Sites of Adsorption of Adenine, Uracil, and Their Corresponding Derivatives on Sodium Montmorillonite

    NASA Astrophysics Data System (ADS)

    Perezgasga, L.; Serrato-Díaz, A.; Negrón-Mendoza, A.; Gal'N, L. De Pablo; Mosqueira, F. G.

    2005-04-01

    Clay minerals are considered important to chemical evolution processes due to their properties, ancient origin, and wide distribution. To extend the knowledge of their role in the prebiotic epoch, the adsorption sites of adenine, adenosine, AMP, ADP, ATP, Poly A, uracil, uridine, UMP, UDP, UTP and Poly U on sodium montmorillonite are investigated. X-ray diffraction, ultraviolet and infrared spectroscopy studies indicate that these molecules distribute into the interlamellar channel and the edge of the clay crystals. Monomers are adsorbed predominantly in the interlamellar channel, whereas polymers adsorb along the crystal edges. Such behavior is discussed mainly in terms of bulk pH, pKa of the adsorbate, and Van der Waals interactions.

  5. Similarities between UDP-Glucose and Adenine Nucleotide Release in Yeast

    PubMed Central

    Esther, Charles R.; Sesma, Juliana I.; Dohlman, Henrik G.; Ault, Addison D.; Clas, Marién L.; Lazarowski, Eduardo R.; Boucher, Richard C.

    2008-01-01

    Extracellular UDP-glucose is a natural purinergic receptor agonist, but its mechanisms of cellular release remain unclear. We studied these mechanisms in Saccharomyces cerevisiae, a simple model organism that releases ATP, another purinergic agonist. Similar to ATP, UDP-glucose was released by S. cerevisiae at a rate that was linear over time. However, unlike ATP release, UDP-glucose release was not dependent on glucose stimulation. This discrepancy was resolved by demonstrating the apparent glucose stimulation of ATP release reflected glucose-dependent changes in the intracellular pattern of adenine nucleotides, with AMP release dominating in the absence of glucose. Indeed, total adenine nucleotide release, like UDP-glucose release, did not vary with glucose concentration over the short term. The genetic basis of UDP-glucose release was explored through analysis of deletion mutants, aided by development of a novel bioassay for UDP-glucose based on signaling through heterologously expressed human P2Y14 receptors. Using this assay, an elevated rate of UDP-glucose release was demonstrated in mutants lacking the putative Golgi nucleotide sugar transporter YMD8. An increased rate of UDP-glucose release in ymd8Δ was reduced by deletion of the YEA4 UDP-N-acetylglucosamine or the HUT1 UDP-galactose transporters, and overexpression of YEA4 or HUT1 increased the rate of UDP-glucose release. These findings suggest an exocytotic release mechanism similar to that of ATP, a conclusion supported by decreased rates of ATP, AMP, and UDP-glucose release in response to the secretory inhibitor Brefeldin A. These studies demonstrate the involvement of the secretory pathway in nucleotide and nucleotide sugar efflux in yeast and offer a powerful model system for further investigation. PMID:18693752

  6. Acceleration of adventitious shoots by interaction between exogenous hormone and adenine sulphate in Althaea officinalis L.

    PubMed

    Naz, Ruphi; Anis, M

    2012-11-01

    In the current study attempts were made to investigate the effects of three different phases of callus induction followed by adventitious regeneration from leaf segments (central and lateral vein). Callus induction was observed in Murashige and Skoog's (MS) medium supplemented with 15.0 μM 2,4-dichloro phenoxy acetic acid (2,4-D). Adventitious shoot buds formation was achieved on MS medium supplemented with 7.5 μM 2,4-D and 20.0 μM AdS in liquid medium as it induced 19.2 ± 0.58 buds in central vein explants. Addition of different growth regulators (cytokinins-6-benzyladenine, kinetin and 2-isopentenyl adenine alone or in combination with auxins-indole-3-acetic acid, indole-3-butyric acid and α-naphthalene acetic acid, improved the shoot regeneration efficiency, in which 5.0 μM 6-benzyl adenine along with 0.25 μM α-naphthalene acetic acid was shown to be the most effective medium for maximum shoot regeneration (81.3 %) with 24.6 number of shoots and 4.4 ± 0.08 cm shoot length per explant. Leaf culture of central veins led to better shoot formation capacity in comparison to lateral vein. Rooting was readily achieved on the differentiated shoots on 1/2 MS medium augmented with 20.0 μM indole-3-butyric acid. The plants were successfully hardened off in sterile soilrite followed by their establishment in garden soil with 80 % survival rate.

  7. Regulation of Salmonella enterica pathogenicity island 1 by DNA adenine methylation.

    PubMed

    López-Garrido, Javier; Casadesús, Josep

    2010-03-01

    DNA adenine methylase (Dam(-)) mutants of Salmonella enterica are attenuated in the mouse model and present multiple virulence-related defects. Impaired interaction of Salmonella Dam(-) mutants with the intestinal epithelium has been tentatively correlated with reduced secretion of pathogenicity island 1 (SPI-1) effectors. In this study, we show that S. enterica Dam(-) mutants contain lowered levels of the SPI-1 transcriptional regulators HilA, HilC, HilD, and InvF. Epistasis analysis indicates that Dam-dependent regulation of SPI-1 requires HilD, while HilA, HilC, and InvF are dispensable. A transcriptional hilDlac fusion is expressed at similar levels in Dam(+) and Dam(-) hosts. However, lower levels of hilD mRNA are found in a Dam(-) background, thus providing unsuspected evidence that Dam methylation might exert post-transcriptional regulation of hilD expression. This hypothesis is supported by the following lines of evidence: (i) lowered levels of hilD mRNA are found in Salmonella Dam(-) mutants when hilD is transcribed from a heterologous promoter; (ii) increased hilD mRNA turnover is observed in Dam(-) mutants; (iii) lack of the Hfq RNA chaperone enhances hilD mRNA instability in Dam(-) mutants; and (iv) lack of the RNA degradosome components polynucleotide phosphorylase and ribonuclease E suppresses hilD mRNA instability in a Dam(-) background. Our report of Dam-dependent control of hilD mRNA stability suggests that DNA adenine methylation plays hitherto unknown roles in post-transcriptional control of gene expression.

  8. Herpes simplex type 1 defective interfering particles do not affect the antiviral activity of acyclovir, foscarnet and adenine arabinoside.

    PubMed

    Harmenberg, J G; Svensson, L T

    1988-03-01

    The concentration of defective interfering particles (DI-particles) of herpes simplex type 1 virus was analysed by electron microscopy and plaque titration. Fifteen consecutive passages of undiluted virus in green monkey kidney cells were followed. No relationship was found between the concentration of DI-particles and the activity of antiviral substances such as acyclovir, foscarnet and adenine arabinoside.

  9. Adenine nucleotide-dependent and redox-independent control of mitochondrial malate dehydrogenase activity in Arabidopsis thaliana.

    PubMed

    Yoshida, Keisuke; Hisabori, Toru

    2016-06-01

    Mitochondrial metabolism is important for sustaining cellular growth and maintenance; however, the regulatory mechanisms underlying individual processes in plant mitochondria remain largely uncharacterized. Previous redox-proteomics studies have suggested that mitochondrial malate dehydrogenase (mMDH), a key enzyme in the tricarboxylic acid (TCA) cycle and redox shuttling, is under thiol-based redox regulation as a target candidate of thioredoxin (Trx). In addition, the adenine nucleotide status may be another factor controlling mitochondrial metabolism, as respiratory ATP production in mitochondria is believed to be influenced by several environmental stimuli. Using biochemical and reverse-genetic approaches, we addressed the redox- and adenine nucleotide-dependent regulation of mMDH in Arabidopsis thaliana. Recombinant mMDH protein formed intramolecular disulfide bonds under oxidative conditions, but these bonds did not have a considerable effect on mMDH activity. Mitochondria-localized o-type Trx (Trx-o) did not facilitate re-reduction of oxidized mMDH. Determination of the in vivo redox state revealed that mMDH was stably present in the reduced form even in Trx-o-deficient plants. Accordingly, we concluded that mMDH is not in the class of redox-regulated enzymes. By contrast, mMDH activity was lowered by adenine nucleotides (AMP, ADP, and ATP). Each adenine nucleotide suppressed mMDH activity with different potencies and ATP exerted the largest inhibitory effect with a significantly lower K(I). Correspondingly, mMDH activity was inhibited by the increase in ATP/ADP ratio within the physiological range. These results suggest that mMDH activity is finely controlled in response to variations in mitochondrial adenine nucleotide balance. PMID:26946085

  10. Adenine nucleotide-dependent and redox-independent control of mitochondrial malate dehydrogenase activity in Arabidopsis thaliana.

    PubMed

    Yoshida, Keisuke; Hisabori, Toru

    2016-06-01

    Mitochondrial metabolism is important for sustaining cellular growth and maintenance; however, the regulatory mechanisms underlying individual processes in plant mitochondria remain largely uncharacterized. Previous redox-proteomics studies have suggested that mitochondrial malate dehydrogenase (mMDH), a key enzyme in the tricarboxylic acid (TCA) cycle and redox shuttling, is under thiol-based redox regulation as a target candidate of thioredoxin (Trx). In addition, the adenine nucleotide status may be another factor controlling mitochondrial metabolism, as respiratory ATP production in mitochondria is believed to be influenced by several environmental stimuli. Using biochemical and reverse-genetic approaches, we addressed the redox- and adenine nucleotide-dependent regulation of mMDH in Arabidopsis thaliana. Recombinant mMDH protein formed intramolecular disulfide bonds under oxidative conditions, but these bonds did not have a considerable effect on mMDH activity. Mitochondria-localized o-type Trx (Trx-o) did not facilitate re-reduction of oxidized mMDH. Determination of the in vivo redox state revealed that mMDH was stably present in the reduced form even in Trx-o-deficient plants. Accordingly, we concluded that mMDH is not in the class of redox-regulated enzymes. By contrast, mMDH activity was lowered by adenine nucleotides (AMP, ADP, and ATP). Each adenine nucleotide suppressed mMDH activity with different potencies and ATP exerted the largest inhibitory effect with a significantly lower K(I). Correspondingly, mMDH activity was inhibited by the increase in ATP/ADP ratio within the physiological range. These results suggest that mMDH activity is finely controlled in response to variations in mitochondrial adenine nucleotide balance.

  11. Characterization of an unbalanced de novo rearrangement, initially by dinucleotide repeat polymorphism typing and subsequent confirmation by FISH

    SciTech Connect

    Zhao, J.; Gordon, P.L.; Wilroy, R.S.

    1994-09-01

    Unbalanced de novo rearrangements not amenable to characterization by conventional cytogenetics can be elucidated using molecular techniques. By microsatellite polymorphism typing, we initially determined the origin and composition of an unbalanced de novo translocation (46,XX,15q+) in a child with multiple congenital anomalies. Microsatellite polymorphism for D5S208 (localized to 5p15) and polymerase chain reaction (PCR) analysis showed that the extra segment originated from the short arm of chromosome 5. Amplification of patient`s DNA with primers for dinucleotide repeats D5S350 and D5S118 showed that most of the short arm of chromosome 5 was present in three copies. The PCR findings were then reconfirmed by a battery of fluorescence in situ hybridizations (FISH). FISH analyses were further helpful in accurately defining the precise trisomic region as well as elucidating the dicentric nature of 46,XX,-15,+der dic (15)t(5;15)(q11.1;p12) de novo translocation. This work demonstrates the benefit of microsatellite polymorphism typing for the identification and characterization of de novo unbalanced rearrangements and apparent tandem duplications in which conventional cytogenetic techniques are usually uninformative. Such study design in cytogenetically equivocal cases could undoubtedly aid in patient management, family counseling and benefit gene mapping studies through accurate genotype-phenotype correlation.

  12. The cyclic di-nucleotide c-di-AMP is an allosteric regulator of metabolic enzyme function

    PubMed Central

    Precit, Mimi; Delince, Matthieu; Pensinger, Daniel; Huynh, TuAnh Ngoc; Jurado, Ashley R.; Goo, Young Ah; Sadilek, Martin; Iavarone, Anthony T.; Sauer, John-Demian; Tong, Liang; Woodward, Joshua J.

    2014-01-01

    SUMMARY Cyclic di-adenosine monophosphate (c-di-AMP) is a broadly conserved second messenger required for bacterial growth and infection. However, the molecular mechanisms of c-di-AMP signaling are still poorly understood. Using a chemical proteomics screen for c-di-AMP interacting proteins in the pathogen Listeria monocytogenes, we identified several broadly conserved protein receptors, including the central metabolic enzyme pyruvate carboxylase (LmPC). Biochemical and crystallographic studies of the LmPC-c-di-AMP interaction revealed a previously unrecognized allosteric regulatory site 25 Å from the active site. Mutations in this site disrupted c-di-AMP binding and affected enzyme catalysis of LmPC as well as PC from pathogenic Enterococcus faecalis. C-di-AMP depletion resulted in altered metabolic activity in L. monocytogenes. Correction of this metabolic imbalance rescued bacterial growth, reduced bacterial lysis, and resulted in enhanced bacterial burdens during infection. These findings greatly expand the c-di-AMP signaling repertoire and reveal a central metabolic regulatory role for a cyclic di-nucleotide. PMID:25215494

  13. Amperometric biosensors for glucose, lactate, and glycolate based on oxidases and redox-modified siloxane polymers

    NASA Astrophysics Data System (ADS)

    Hale, Paul D.; Inagaki, Toru; Lee, Hung Sui; Skotheim, Terje A.; Karan, Hiroko I.; Okamoto, Yoshi

    1989-06-01

    Amperometric biosensors based on flavin-containing oxidases undergo several steps which produce a measurable current that is related to the concentration of substrate. In the initial step, the substrate converts the oxidized flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) into the reduced form FADH sub 2 or FMNH sub 2. Because these cofactors are located well within the enzyme molecule, direct electron transfer to the surface of a conventional electrode does not occur to a measurable degree. A common method of facilitating this electron transfer is to introduce oxygen into the system because it is the natural acceptor for the oxidases; the oxygen is reduced by the FADH sub 2 or FMNH sub 2 to hydrogen peroxide, which can then be detected electrochemically. The major drawback to this approach is the fact that oxidation of hydrogen peroxide requires a large overpotential, thus making these sensors susceptible to interference from electroactive species. To lower the necessary applied potential, several non-physiological redox couples have been employed to shuttle electrons between the flavin moieties and the electrode. The present paper describes the development of amperometric biosensors based on flavin-containing enzymes and a family of polymeric mediators.

  14. Resonance Raman spectroscopy.

    PubMed

    Li, Jiang; Kitagawa, Teizo

    2014-01-01

    Flavin is a general name given to molecules having the heteroaromatic ring system of 7,8-dimethylisoalloxazine but practically means riboflavin (Rfl), flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN) in biological systems, whose structures are illustrated in Fig. 1, together with the atomic numbering scheme and ring numbering of the isoalloxazine moiety. As the isoalloxazine skeleton cannot be synthesized in human cells, it is obtained from diet as Rfl (vitamin B2). FAD and FMN can act as cofactors in flavoenzymes but Rfl does not. Most flavoenzymes catalyze redox reactions of substrates (Miura, Chem Rec 1:183-194, 2001). When O2 serves as the oxidant in the oxidation half cycle of an enzymic reaction, the enzyme is called "flavo-oxidase" but when others do, the enzyme is called "flavo-dehydrogenase." The difference between the two types of oxidative catalysis arises from delicate differences in the π-electron distributions in the isoalloxazine ring, which can be revealed by Raman spectroscopy (Miura, Chem Rec 1:183-194, 2001). Since a flavin is an extremely versatile molecule, the scientific field including chemistry, biochemistry, and enzymology is collectively called "flavonology." It was found recently, however, that the flavin also acts as a chromophore to initiate light-induced DNA repair and signal transductions (Sancar, Chem Rev 103:2203-2237, 2003).

  15. Determination of the g-matrix orientation in flavin radicals by high-field/high-frequency electron-nuclear double resonance.

    PubMed

    Kay, Christopher W M; Schleicher, Erik; Hitomi, Kenichi; Todo, Takeshi; Bittl, Robert; Weber, Stefan

    2005-11-01

    A high-microwave-frequency/high-magnetic-field pulsed electron-nuclear double resonance (ENDOR) study performed at 94 GHz on the flavin semiquinone cofactor of Xenopus laevis (6-4) photolyase in its neutral radical state is presented. Although the principal values of the flavin radical's g-matrix are not fully resolved in the 94-GHz EPR spectrum in a nonoriented sample, the orientation of the principal axes of g is obtained by exploiting the orientation selection of the proton ENDOR signals from the methyl protons at C-8alpha and the deuteron ENDOR signals from D-5 in an enzyme sample in deuterated buffer. This procedure for assigning the orientation of g relative to the molecular frame makes use of commercially available ENDOR instrumentation without the necessity to perform single-crystal studies.

  16. Extracellular Electron Transport-Mediated Fe(III) Reduction by a Community of Alkaliphilic Bacteria That Use Flavins as Electron Shuttles

    PubMed Central

    Fuller, Samuel J.; McMillan, Duncan G. G.; Renz, Marc B.; Schmidt, Martin

    2014-01-01

    The biochemical and molecular mechanisms used by alkaliphilic bacterial communities to reduce metals in the environment are currently unknown. We demonstrate that an alkaliphilic (pH > 9) consortium dominated by Tissierella, Clostridium, and Alkaliphilus spp. is capable of using iron (Fe3+) as a final electron acceptor under anaerobic conditions. Iron reduction is associated with the production of a freely diffusible species that, upon rudimentary purification and subsequent spectroscopic, high-performance liquid chromatography, and electrochemical analysis, has been identified as a flavin species displaying properties indistinguishable from those of riboflavin. Due to the link between iron reduction and the onset of flavin production, it is likely that riboflavin has an import role in extracellular metal reduction by this alkaliphilic community. PMID:24141133

  17. Structural and Functional Investigation of Flavin Binding Center of the NqrC Subunit of Sodium-Translocating NADH:Quinone Oxidoreductase from Vibrio harveyi

    PubMed Central

    Bertsova, Yulia; Polovinkin, Vitaly; Gushchin, Ivan; Ishchenko, Andrii; Kovalev, Kirill; Mishin, Alexey; Kachalova, Galina; Popov, Alexander; Bogachev, Alexander; Gordeliy, Valentin

    2015-01-01

    Na+-translocating NADH:quinone oxidoreductase (NQR) is a redox-driven sodium pump operating in the respiratory chain of various bacteria, including pathogenic species. The enzyme has a unique set of redox active prosthetic groups, which includes two covalently bound flavin mononucleotide (FMN) residues attached to threonine residues in subunits NqrB and NqrC. The reason of FMN covalent bonding in the s