Sample records for adenine dinucleotide cofactor

  1. The chemistry of nicotinamide adenine dinucleotide (NAD) analogues containing C-nucleosides related to nicotinamide riboside.

    PubMed

    Pankiewicz, Krzysztof W; Watanabe, Kyoichi A; Lesiak-Watanabe, Krystyna; Goldstein, Barry M; Jayaram, Hiremagalur N

    2002-04-01

    Oncolytic C-nucleosides, tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) and benzamide riboside (3-beta-D-ribofuranosylbenzamide) are converted in cell into active metabolites thiazole-4-carboxamide- and benzamide adenine dinucleotide, TAD and BAD, respectively. TAD and BAD as NAD analogues were found to bind at the nicotinamide adenine dinucleotide (cofactor NAD) site of inosine monophosphate dehydrogenase (IMPDH), an important target in cancer treatment. The synthesis and evaluation of anticancer activity of a number of C-nucleosides related to tiazofurin and nicotinamide riboside then followed and are reviewed herein. Interestingly, pyridine C-nucleosides (such as C-nicotinamide riboside) are not metabolized into the corresponding NAD analogues in cell. Their conversion by chemical methods is described. As dinucleotides these compounds show inhibition of IMPDH in low micromolar level. Also, the synthesis of BAD in metabolically stable bis(phosphonate) form is discussed indicating the usefulness of such preformed inhibitors in drug development. Among tiazofurin analogues, Franchetti and Grifantini found, that the replacement of the sulfur by oxygen (as in oxazafurin) but not the removal of nitrogen (tiophenfurin) of the thiazole ring resulted in inactive compounds. The anti cancer activity of their synthetic dinucleotide analogues indicate that inactive compounds are not only poorly metabolized in cell but also are weak inhibitors of IMPDH as dinucleotides.

  2. Reduction of mitomycin C is catalysed by human recombinant NRH:quinone oxidoreductase 2 using reduced nicotinamide adenine dinucleotide as an electron donating co-factor

    PubMed Central

    Jamieson, D; Tung, A T Y; Knox, R J; Boddy, A V

    2006-01-01

    NRH:Quinone Oxidoreductase 2 (NQO2) has been described as having no enzymatic activity with nicotinamide adenine dinucleotide (NADH) or NADPH as electron donating cosubstrates. Mitomycin C (MMC) is both a substrate for and a mechanistic inhibitor of the NQO2 homologue NQO1. NRH:quinone oxidoreductase 2 catalysed the reduction of MMC at pH 5.8 with NADH as a co-factor. This reaction results in species that inhibit the NQO2-mediated metabolism of CB1954. In addition, MMC caused an increase in DNA cross-links in a cell line transfected to overexpress NQO2 to an extent comparable to that observed with an isogenic NQO1-expressing cell line. These data indicate that NQO2 may contribute to the metabolism of MMC to cytotoxic species. PMID:17031400

  3. Enzyme-Mediated Conversion of Flavin Adenine Dinucleotide (FAD) to 8-Formyl FAD in Formate Oxidase Results in a Modified Cofactor with Enhanced Catalytic Properties.

    PubMed

    Robbins, John M; Souffrant, Michael G; Hamelberg, Donald; Gadda, Giovanni; Bommarius, Andreas S

    2017-07-25

    Flavins, including flavin adenine dinucleotide (FAD), are fundamental catalytic cofactors that are responsible for the redox functionality of a diverse set of proteins. Alternatively, modified flavin analogues are rarely found in nature as their incorporation typically results in inactivation of flavoproteins, thus leading to the disruption of important cellular pathways. Here, we report that the fungal flavoenzyme formate oxidase (FOX) catalyzes the slow conversion of noncovalently bound FAD to 8-formyl FAD and that this conversion results in a nearly 10-fold increase in formate oxidase activity. Although the presence of an enzyme-bound 8-formyl FMN has been reported previously as a result of site-directed mutagenesis studies of lactate oxidase, FOX is the first reported case of 8-formyl FAD in a wild-type enzyme. Therefore, the formation of the 8-formyl FAD cofactor in formate oxidase was investigated using steady-state kinetics, site-directed mutagenesis, ultraviolet-visible, circular dichroism, and fluorescence spectroscopy, liquid chromatography with mass spectrometry, and computational analysis. Surprisingly, the results from these studies indicate not only that 8-formyl FAD forms spontaneously and results in the active form of FOX but also that its autocatalytic formation is dependent on a nearby arginine residue, R87. Thus, this work describes a new enzyme cofactor and provides insight into the little-understood mechanism of enzyme-mediated 8α-flavin modifications.

  4. General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH

    DOE PAGES

    Brinkmann-Chen, Sabine; Flock, Tilman; Cahn, Jackson K. B.; ...

    2013-06-17

    To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymesmore » having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. As a result, high-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.« less

  5. Semisynthetic biosensors for mapping cellular concentrations of nicotinamide adenine dinucleotides.

    PubMed

    Sallin, Olivier; Reymond, Luc; Gondrand, Corentin; Raith, Fabio; Koch, Birgit; Johnsson, Kai

    2018-05-29

    We introduce a new class of semisynthetic fluorescent biosensors for the quantification of free nicotinamide adenine dinucleotide (NAD + ) and ratios of reduced to oxidized nicotinamide adenine dinucleotide phosphate (NADPH/NADP + ) in live cells. Sensing is based on controlling the spatial proximity of two synthetic fluorophores by binding of NAD(P) to the protein component of the sensor. The sensors possess a large dynamic range, can be excited at long wavelengths, are pH-insensitive, have tunable response range and can be localized in different organelles. Ratios of free NADPH/NADP + are found to be higher in mitochondria compared to those found in the nucleus and the cytosol. By recording free NADPH/NADP + ratios in response to changes in environmental conditions, we observe how cells can react to such changes by adapting metabolic fluxes. Finally, we demonstrate how a comparison of the effect of drugs on cellular NAD(P) levels can be used to probe mechanisms of action. © 2018, Sallin et al.

  6. Distinctive Spectral Features of Exciton and Excimer States in the Ultrafast Electronic Deactivation of the Adenine Dinucleotide

    NASA Astrophysics Data System (ADS)

    Stuhldreier, Mayra C.; Röttger, Katharina; Temps, Friedrich

    We report the observation by transient absorption spectroscopy of distinctive spectro-temporal signatures of delocalized exciton versus relaxed, weakly bound excimer states in the ultrafast electronic deactivation after UV photoexcitation of the adenine dinucleotide.

  7. Selectivity and activity of adenine dinucleotides at recombinant P2X2 and P2Y1 purinoceptors.

    PubMed Central

    Pintor, J.; King, B. F.; Miras-Portugal, M. T.; Burnstock, G.

    1996-01-01

    1. Adenine dinucleotides (Ap3A, x = 2-6) are naturally-occurring polyphosphated nucleotidic substances which are found in the CNS and are known to be released in a calcium-dependent manner from storage vesicles in brain synaptosomes. The selectivity and activity of adenine dinucleotides for neuronally-derived recombinant P2 purinoceptors were studied using P2X2 and P2Y1 subtypes expressed in Xenopus oocytes. 2. For the P2Y1 subtype derived from chick brain, Ap3A was equipotent and as active as ATP (EC50 values: 375 +/- 86 nM and 334 +/- 25 nM, respectively). Ap4A was a weak partial agonist and other dinucleotides were inactive as agonists. None of the inactive dinucleotides were antagonists nor modulated the activity of Ap3A and ATP. 3. For the P2X2 subtype derived from rat PC12 cells, Ap4A was as active as ATP but less potent (EC50 values: 15.2 +/- 1 microM and 3.7 +/- 0.7 microM, respectively). Other adenosine dinucleotides were inactive as either agonists or antagonists. 4. Ap5A (1-100 nM) potentiated ATP-responses at the P2X2 subtype, showing an EC50 of 2.95 +/- 0.7 nM for this modulatory effect. Ap5A (10 nM) shifted the concentration-response curves for ATP to the left by one-half log10 unit but did not alter the Hill co-efficient for ATP (nH = 2.1 +/- 0.1). Ap5A (10 nM) failed to potentiate Ap4A-responses but did enhance the efficacy of the P2 purinoceptor antagonist, suramin, by 12 fold at the P2X2 subtype. 5. In conclusion, the results show that ionotropic (P2X2) and metabotropic (P2Y1) ATP receptors which occur in the CNS are activated selectively by naturally-occurring adenine dinucleotides which are known to be released with nucleotides from storage vesicles. The observed potentiation of P2X2-responses by Ap5A, where co-released with ATP by brain synaptosomes, may have a functional bearing in purinergic signalling in the CNS. PMID:8922753

  8. An Ancient Fingerprint Indicates the Common Ancestry of Rossmann-Fold Enzymes Utilizing Different Ribose-Based Cofactors

    PubMed Central

    Laurino, Paola; Tóth-Petróczy, Ágnes; Meana-Pañeda, Rubén; Lin, Wei; Truhlar, Donald G.; Tawfik, Dan S.

    2016-01-01

    Nucleoside-based cofactors are presumed to have preceded proteins. The Rossmann fold is one of the most ancient and functionally diverse protein folds, and most Rossmann enzymes utilize nucleoside-based cofactors. We analyzed an omnipresent Rossmann ribose-binding interaction: a carboxylate side chain at the tip of the second β-strand (β2-Asp/Glu). We identified a canonical motif, defined by the β2-topology and unique geometry. The latter relates to the interaction being bidentate (both ribose hydroxyls interacting with the carboxylate oxygens), to the angle between the carboxylate and the ribose, and to the ribose’s ring configuration. We found that this canonical motif exhibits hallmarks of divergence rather than convergence. It is uniquely found in Rossmann enzymes that use different cofactors, primarily SAM (S-adenosyl methionine), NAD (nicotinamide adenine dinucleotide), and FAD (flavin adenine dinucleotide). Ribose-carboxylate bidentate interactions in other folds are not only rare but also have a different topology and geometry. We further show that the canonical geometry is not dictated by a physical constraint—geometries found in noncanonical interactions have similar calculated bond energies. Overall, these data indicate the divergence of several major Rossmann-fold enzyme classes, with different cofactors and catalytic chemistries, from a common pre-LUCA (last universal common ancestor) ancestor that possessed the β2-Asp/Glu motif. PMID:26938925

  9. Nicotinamide adenine dinucleotide biosynthesis promotes liver regeneration

    PubMed Central

    Mukherjee, Sarmistha; Chellappa, Karthikeyani; Moffitt, Andrea; Ndungu, Joan; Dellinger, Ryan W.; Davis, James G.; Agarwal, Beamon; Baur, Joseph A.

    2016-01-01

    The regenerative capacity of the liver is essential for recovery from surgical resection or injuries induced by trauma or toxins. During liver regeneration, the concentration of nicotinamide adenine dinucleotide (NAD) falls, at least in part due to metabolic competition for precursors. To test whether NAD availability restricts the rate of liver regeneration, we supplied nicotinamide riboside (NR), an NAD precursor, in the drinking water of mice subjected to partial hepatectomy. NR increased DNA synthesis, mitotic index, and mass restoration in the regenerating livers. Intriguingly, NR also ameliorated the steatosis that normally accompanies liver regeneration. To distinguish the role of hepatocyte NAD levels from any systemic effects of NR, we generated mice overexpressing Nicotinamide phosphoribosyltransferase (Nampt), a rate-limiting enzyme for NAD synthesis, specifically in the liver. Nampt overexpressing mice were mildly hyperglycemic at baseline and, similarly to the mice treated with NR, exhibited enhanced liver regeneration and reduced steatosis following partial hepatectomy. Conversely, mice lacking Nampt in hepatocytes exhibited impaired regenerative capacity that was completely rescued by administering NR. Conclusion NAD availability is limiting during liver regeneration and supplementation with precursors such as NR may be therapeutic in settings of acute liver injury. PMID:27809334

  10. Amperometric cholesterol biosensor based on in situ reconstituted cholesterol oxidase on an immobilized monolayer of flavin adenine dinucleotide cofactor.

    PubMed

    Vidal, Juan-C; Espuelas, Javier; Castillo, Juan-R

    2004-10-01

    A new amperometric biosensor for determining cholesterol based on deflavination of the enzyme cholesterol oxidase (ChOx) and subsequent reconstitution of the apo-protein with a complexed flavin adenine dinucleotide (FAD) monolayer is described. The charge transfer mediator pyrroquinoline quinone (PQQ) was covalently bound to a cystamine self-assembled monolayer (SAM) on an Au electrode. Boronic acid (BA) was then bound to PQQ using the carbodiimide procedure, and the BA ligand was complexed to the FAD molecules on which the apo-ChOx was subsequently reconstituted. The effective release of the FAD from the enzyme and the successful reconstitution were verified using molecular fluorescence and cyclic voltammetry. The optimal orientation of FAD toward the PQQ mediator and the distances between FAD and PQQ and between PQQ and electrode enhance the charge transfer, very high sensitivity (about 2,500 nAmM(-1)cm(-2)) being obtained for cholesterol determination. The biosensor is selective toward electroactive interferents (ascorbic acid and uric acid) and was tested in reference serum samples, demonstrating excellent accuracy (relative errors below 3% in all cases). The biosensor activity can be successfully regenerated in a simple process by successive reconstitution with batches of recently prepared apo-ChOx on the same immobilized Au/SAM-PQQ-BA-FAD monolayer (it was tested five times); the lifetime of the biosensor is about 45-60 days.

  11. Cofactor-Dependent Aldose Dehydrogenase of Rhodopseudomonas spheroides

    PubMed Central

    Niederpruem, Donald J.; Doudoroff, Michael

    1965-01-01

    Niederpruem, Donald J. (University of California, Berkeley), and Michael Doudoroff. Cofactor-dependent aldose dehydrogenase of Rhodopseudomonas spheroides. J. Bacteriol. 89:697–705. 1965.—Particulate enzyme preparations of cell extracts of Rhodopseudomonas spheroides possess constitutive dehydrogenase and oxidase activities for aldose sugars, reduced nicotinamide adenine dinucleotide (NADH2), and succinate. The dehydrogenation of aldoses requires an unidentified cofactor which is not required for the oxidation of succinate nor of NADH2. The cofactor is present in the particulate fraction of aerobic cells, but is unavailable to the enzyme system. It can be liberated by boiling or by treatment with salts at high concentration. The cofactor also appears in the soluble fraction of aerobic cells, but only after exponential growth has ceased. Extracts of cells grown anaerobically in the light possess the apoenzyme, but not the cofactor, for aldose oxidation. Cofactor activity was found in extracts of Bacterium anitratum (= Moraxella sp.) but not in Escherichia coli, Pseudomonas fluorescens, yeast, or mouse liver. In 0.075 m tris(hydroxymethyl)aminomethane-phosphoric acid buffer (pH 7.3), the oxidation of NADH2 was stimulated and succinoxidase was inhibited by high salt concentrations. PMID:14273648

  12. Nicotinamide adenine dinucleotide biosynthesis promotes liver regeneration.

    PubMed

    Mukherjee, Sarmistha; Chellappa, Karthikeyani; Moffitt, Andrea; Ndungu, Joan; Dellinger, Ryan W; Davis, James G; Agarwal, Beamon; Baur, Joseph A

    2017-02-01

    The regenerative capacity of the liver is essential for recovery from surgical resection or injuries induced by trauma or toxins. During liver regeneration, the concentration of nicotinamide adenine dinucleotide (NAD) falls, at least in part due to metabolic competition for precursors. To test whether NAD availability restricts the rate of liver regeneration, we supplied nicotinamide riboside (NR), an NAD precursor, in the drinking water of mice subjected to partial hepatectomy. NR increased DNA synthesis, mitotic index, and mass restoration in the regenerating livers. Intriguingly, NR also ameliorated the steatosis that normally accompanies liver regeneration. To distinguish the role of hepatocyte NAD levels from any systemic effects of NR, we generated mice overexpressing nicotinamide phosphoribosyltransferase, a rate-limiting enzyme for NAD synthesis, specifically in the liver. Nicotinamide phosphoribosyltransferase overexpressing mice were mildly hyperglycemic at baseline and, similar to mice treated with NR, exhibited enhanced liver regeneration and reduced steatosis following partial hepatectomy. Conversely, mice lacking nicotinamide phosphoribosyltransferase in hepatocytes exhibited impaired regenerative capacity that was completely rescued by administering NR. NAD availability is limiting during liver regeneration, and supplementation with precursors such as NR may be therapeutic in settings of acute liver injury. (Hepatology 2017;65:616-630). © 2016 by the American Association for the Study of Liver Diseases.

  13. Quantification of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in mammalian model cells by CE with LED-induced fluorescence detection.

    PubMed

    Hühner, Jens; Ingles-Prieto, Álvaro; Neusüß, Christian; Lämmerhofer, Michael; Janovjak, Harald

    2015-02-01

    Cultured mammalian cells essential are model systems in basic biology research, production platforms of proteins for medical use, and testbeds in synthetic biology. Flavin cofactors, in particular flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are critical for cellular redox reactions and sense light in naturally occurring photoreceptors and optogenetic tools. Here, we quantified flavin contents of commonly used mammalian cell lines. We first compared three procedures for extraction of free and noncovalently protein-bound flavins and verified extraction using fluorescence spectroscopy. For separation, two CE methods with different BGEs were established, and detection was performed by LED-induced fluorescence with limit of detections (LODs 0.5-3.8 nM). We found that riboflavin (RF), FMN, and FAD contents varied significantly between cell lines. RF (3.1-14 amol/cell) and FAD (2.2-17.0 amol/cell) were the predominant flavins, while FMN (0.46-3.4 amol/cell) was found at markedly lower levels. Observed flavin contents agree with those previously extracted from mammalian tissues, yet reduced forms of RF were detected that were not described previously. Quantification of flavins in mammalian cell lines will allow a better understanding of cellular redox reactions and optogenetic tools. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Development of a simple and efficient method for assaying cytidine monophosphate sialic acid synthetase activity using an enzymatic reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide converting system.

    PubMed

    Fujita, Akiko; Sato, Chihiro; Münster-Kühnel, Anja-K; Gerardy-Schahn, Rita; Kitajima, Ken

    2005-02-01

    A new reliable method to assay the activity of cytidine monophosphate sialic acid (CMP-Sia) synthetase (CSS) has been developed. The activation of sialic acids (Sia) to CMP-Sia is a prerequisite for the de novo synthesis of sialoglycoconjugates. In vertebrates, CSS has been cloned from human, mouse, and rainbow trout, and the crystal structure has been resolved for the mouse enzyme. The mouse and rainbow trout enzyme have been compared with respect to substrate specificity, demonstrating that the mouse enzyme exhibits a pronounced specificity for N-acetylneuraminic acid (Neu5Ac), while the rainbow trout CSS is equally active with either of three Sia species, Neu5Ac, N-glycolylneuraminic acid (Neu5Gc), and deaminoneuraminic acid (KDN). However, molecular details that explain the pronounced substrate specificities are unknown. Understanding the catalytic mechanisms of these enzymes is of major importance, since CSSs play crucial roles in cellular sialylation patterns and thus are potential drug targets in a number of pathophysiological situations. The availability of the cDNAs and the obtained structural data enable rational approaches; however, these efforts are limited by the lack of a reliable high-throughput assay system. Here we describe a new assay system that allows product quantification in a reduced nicotinamide adenine dinucleotide (NADH)-dependent color reaction. The activation reaction catalyzed by CSS, CTP+Sia-->CMP-Sia+pyrophosphate, was evaluated by a consumption of Sia, which corresponds to that of NADH on the following two successive reactions: (i) Sia-->pyruvate+ManNAc (or Man), catalyzed by a sialic acid lyase (SAL), and (ii) pyruvate+NADH-->lactate+oxidized nicotinamide adenine dinucleotide (NAD+), catalyzed by a lactate dehydrogenase (LDH). Consumption of NADH can be photometrically monitored on a microtiter plate reader for a number of test samples at the same time. Furthermore, based on the quantification of CSS used in the SAL/LDH assay

  15. Blue light induced reactive oxygen species from flavin mononucleotide and flavin adenine dinucleotide on lethality of HeLa cells.

    PubMed

    Yang, Ming-Yeh; Chang, Chih-Jui; Chen, Liang-Yü

    2017-08-01

    Photodynamic therapy (PDT) is a safe and non-invasive treatment for cancers and microbial infections. Various photosensitizers and light sources have been developed for clinical cancer therapies. Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are the cofactor of enzymes and are used as photosensitizers in this study. Targeting hypoxia and light-triggering reactive oxygen species (ROS) are experimental strategies for poisoning tumor cells in vitro. HeLa cells are committed to apoptosis when treated with FMN or FAD and exposed to visible blue light (the maximum emitted wavelength of blue light is 462nm). Under blue light irradiation at 3.744J/cm 2 (=0.52mW/cm 2 irradiated for 2h), the minimal lethal dose is 3.125μM and the median lethal doses (LD 50 ) for FMN and FAD are 6.5μM and 7.2μM, respectively. Individual exposure to visible blue light irradiation or riboflavin photosensitizers does not produce cytotoxicity and no side effects are observed in this study. The western blotting results also show that an intrinsic apoptosis pathway is activated by the ROS during photolysis of riboflavin analogues. Blue light triggers the cytotoxicity of riboflavins on HeLa cells in vitro. Based on these results, this is a feasible and efficient of PDT with an intrinsic photosensitizer for cancer research. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. 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. © 2016 The Author. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.

  17. Relationships between laser powers and photoacoustic signal intensities of flavin adenine dinucleotide and beta-carotene dissolved in solutions

    NASA Astrophysics Data System (ADS)

    Imakubo, Keiichi

    1994-10-01

    Ar ion laser-induced photoacoustic spectroscopy has been performed on 0.01 mu M flavin adenine dinucleotide in H2O and 0.01 mu M beta-carotene in n-hexane where the optical absorption spectroscopy is not applicable. On the basis of the linear relationships between laser powers and photoacoustic signal intensities up to 500 mW, it may be concluded that laser power ranging from 10 to 50 mW is required for the successful observation of photoacoustic signals without any photochemical or photobiological effects.

  18. The conserved baculovirus protein p33 (Ac92) is a flavin adenine dinucleotide-linked sulfhydryl oxidase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Long, C.M.; Rohrmann, G.F.; Merrill, G.F., E-mail: merrillg@onid.orst.ed

    2009-06-05

    Open reading frame 92 of the Autographa californica baculovirus (Ac92) is one of about 30 core genes present in all sequenced baculovirus genomes. Computer analyses predicted that the Ac92 encoded protein (called p33) and several of its baculovirus orthologs were related to a family of flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidases. Alignment of these proteins indicated that, although they were highly diverse, a number of amino acids in common with the Erv1p/Alrp family of sulfhydryl oxidases are present. Some of these conserved amino acids are predicted to stack against the isoalloxazine and adenine components of FAD, whereas others are involvedmore » in electron transfer. To investigate this relationship, Ac92 was expressed in bacteria as a His-tagged fusion protein, purified, and characterized both spectrophotometrically and for its enzymatic activity. The purified protein was found to have the color (yellow) and absorption spectrum consistent with it being a FAD-containing protein. Furthermore, it was demonstrated to have sulfhydryl oxidase activity using dithiothreitol and thioredoxin as substrates.« less

  19. The conserved baculovirus protein p33 (Ac92) is a flavin adenine dinucleotide-linked sulfhydryl oxidase.

    PubMed

    Long, C M; Rohrmann, G F; Merrill, G F

    2009-06-05

    Open reading frame 92 of the Autographa californica baculovirus (Ac92) is one of about 30 core genes present in all sequenced baculovirus genomes. Computer analyses predicted that the Ac92 encoded protein (called p33) and several of its baculovirus orthologs were related to a family of flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidases. Alignment of these proteins indicated that, although they were highly diverse, a number of amino acids in common with the Erv1p/Alrp family of sulfhydryl oxidases are present. Some of these conserved amino acids are predicted to stack against the isoalloxazine and adenine components of FAD, whereas others are involved in electron transfer. To investigate this relationship, Ac92 was expressed in bacteria as a His-tagged fusion protein, purified, and characterized both spectrophotometrically and for its enzymatic activity. The purified protein was found to have the color (yellow) and absorption spectrum consistent with it being a FAD-containing protein. Furthermore, it was demonstrated to have sulfhydryl oxidase activity using dithiothreitol and thioredoxin as substrates.

  20. Engineering cofactor flexibility enhanced 2,3-butanediol production in Escherichia coli.

    PubMed

    Liang, Keming; Shen, Claire R

    2017-12-01

    Enzymatic reduction of acetoin into 2,3-butanediol (2,3-BD) typically requires the reduced nicotinamide adenine dinucleotide (NADH) or its phosphate form (NADPH) as electron donor. Efficiency of 2,3-BD biosynthesis, therefore, is heavily influenced by the enzyme specificity and the cofactor availability which varies dynamically. This work describes the engineering of cofactor flexibility for 2,3-BD production by simultaneous overexpression of an NADH-dependent 2,3-BD dehydrogenase from Klebsiella pneumoniae (KpBudC) and an NADPH-specific 2,3-BD dehydrogenase from Clostridium beijerinckii (CbAdh). Co-expression of KpBudC and CbAdh not only enabled condition versatility for 2,3-BD synthesis via flexible utilization of cofactors, but also improved production stereo-specificity of 2,3-BD without accumulation of acetoin. With optimization of medium and fermentation condition, the co-expression strain produced 92 g/L of 2,3-BD in 56 h with 90% stereo-purity for (R,R)-isoform and 85% of maximum theoretical yield. Incorporating cofactor flexibility into the design principle should benefit production of bio-based chemical involving redox reactions.

  1. In vivo native fluorescence spectroscopy and nicotinamide adinine dinucleotide/flavin adenine dinucleotide reduction and oxidation states of oral submucous fibrosis for chemopreventive drug monitoring

    NASA Astrophysics Data System (ADS)

    Sivabalan, Shanmugam; Vedeswari, C. Ponranjini; Jayachandran, Sadaksharam; Koteeswaran, Dornadula; Pravda, Chidambaranathan; Aruna, Prakasa Rao; Ganesan, Singaravelu

    2010-01-01

    Native fluorescence spectroscopy has shown potential to characterize and diagnose oral malignancy. We aim at extending the native fluorescence spectroscopy technique to characterize normal and oral submucous fibrosis (OSF) patients under pre- and post-treated conditions, and verify whether this method could also be considered in the monitoring of therapeutic prognosis noninvasively. In this study, 28 normal subjects and 28 clinically proven cases of OSF in the age group of 20 to 40 years are diagnosed using native fluorescence spectroscopy. The OSF patients are given dexamethasone sodium phosphate and hyaluronidase twice a week for 6 weeks, and the therapeutic response is monitored using fluorescence spectroscopy. The fluorescence emission spectra of normal and OSF cases of both pre- and post-treated conditions are recorded in the wavelength region of 350 to 600 nm at an excitation wavelength of 330 nm. The statistical significance is verified using discriminant analysis. The oxidation-reduction ratio of the tissue is also calculated using the fluorescence emission intensities of flavin adenine dinucleotide and nicotinamide adinine dinucleotide at 530 and 440 nm, respectively, and they are compared with conventional physical clinical examinations. This study suggests that native fluorescence spectroscopy could also be extended to OSF diagnosis and therapeutic prognosis.

  2. Inhibition of NAD glycohydrolase and ADP-ribosyl transferases by carbocyclic analogues of oxidized nicotinamide adenine dinucleotide.

    PubMed

    Slama, J T; Simmons, A M

    1989-09-19

    Analogues of oxidized nicotinamide adenine dinucleotide (NAD+) in which a 2,3-dihydroxycyclopentane ring replaces the beta-D-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ have recently been synthesized [Slama, J. T., & Simmons, A. M. (1988) Biochemistry 27, 183]. Carbocyclic NAD+ analogues have been shown to inhibit NAD glycohydrolases and ADP-ribosyl transferases such as cholera toxin A subunit. In this study, the diastereomeric mixture of dinucleotides was separated, and the inhibitory capacity of each of the purified diastereomers was defined. The NAD+ analogue in which the D-dihydroxycyclopentane is substituted for the D-ribose is designated carba-NAD and was demonstrated to be a poor inhibitor of the Bungarus fasciatus venom NAD glycohydrolase. The diastereomeric dinucleotide pseudo-carbocyclic-NAD (psi-carba-NAD), containing L-dihydroxycyclopentane in place of the D-ribose of NAD+, was shown, however, to be a potent competitive inhibitor of the venom NAD glycohydrolase with an inhibitor dissociation constant (Ki) of 35 microM. This was surprising since psi-carba-NAD contains the carbocyclic analogue of the unnatural L-ribotide and was therefore expected to be a biologically inactive diastereomer. psi-Carba-NAD also competitively inhibited the insoluble brain NAD glycohydrolase from cow (Ki = 6.7 microM) and sheep (Ki = 31 microM) enzyme against which carba-NAD is ineffective. Sensitivity to psi-carba-NAD was found to parallel sensitivity to inhibition by isonicotinic acid hydrazide, another NADase inhibitor. psi-Carba-NAD is neither a substrate for nor an inhibitor of alcohol dehydrogenase, whereas carba-NAD is an efficient dehydrogenase substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

  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. Absorption and luminescence spectroscopy of mass-selected flavin adenine dinucleotide mono-anions

    NASA Astrophysics Data System (ADS)

    Giacomozzi, L.; Kjær, C.; Langeland Knudsen, J.; Andersen, L. H.; Brøndsted Nielsen, S.; Stockett, M. H.

    2018-06-01

    We report the absorption profile of isolated Flavin Adenine Dinucleotide (FAD) mono-anions recorded using photo-induced dissociation action spectroscopy. In this charge state, one of the phosphoric acid groups is deprotonated and the chromophore itself is in its neutral oxidized state. These measurements cover the first four optical transitions of FAD with excitation energies from 2.3 to 6.0 eV (210-550 nm). The S0 → S2 transition is strongly blue shifted relative to aqueous solution, supporting the view that this transition has a significant charge-transfer character. The remaining bands are close to their solution-phase positions. This confirms that the large discrepancy between quantum chemical calculations of vertical transition energies and solution-phase band maxima cannot be explained by solvent effects. We also report the luminescence spectrum of FAD mono-anions in vacuo. The gas-phase Stokes shift for S1 is 3000 cm-1, which is considerably larger than any previously reported for other molecular ions and consistent with a significant displacement of the ground and excited state potential energy surfaces. Consideration of the vibronic structure is thus essential for simulating the absorption and luminescence spectra of flavins.

  5. Eco-synthesis of graphene and its use in dihydronicotinamide adenine dinucleotide sensing.

    PubMed

    Amouzadeh Tabrizi, Mahmoud; Jalilzadeh Azar, Somayeh; Nadali Varkani, Javad

    2014-09-01

    In this paper, we report a green and eco-friendly approach to synthesize reduced graphene oxide (rGO) via a mild hydrothermal process using malt as a reduced agent. The proposed method is based on the reduction of graphene oxide (GO) in malt solution by making use of the reducing capability of phenolic compounds contained in malt solution. The obtained rGO was characterized by atomic force microscopy (AFM), ultraviolet-visible (UV-vis) absorption spectroscopy, X-ray diffraction spectroscopy (XRD), Raman spectroscopy, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Electrochemical impedance spectroscopy analysis revealed that the charge transfer resistance of rGO modified glassy carbon (GC) electrode was much lower than that of the GC electrode. The electrochemical behavior of dihydronicotinamide adenine dinucleotide (NADH) on rGO modified GC electrode was investigated by cyclic voltammetry and amperometry. Electrochemical experiments indicated that rGO/GC electrode exhibited excellent electrocatalytic activity toward the NADH, which can be attributed to excellent electrical conductivity and high specific surface area of the rGO composite. The resulting biosensor showed highly sensitive amperometric response to NADH with a low detection limit (0.33μM). Copyright © 2014 Elsevier Inc. All rights reserved.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 decarboxylatemore » 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.« less

  7. Roles of Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Oxidase in Angiogenesis: Isoform-Specific Effects

    PubMed Central

    Wang, Haibo; Hartnett, M. Elizabeth

    2017-01-01

    Angiogenesis is the formation of new blood vessels from preexisting ones and is implicated in physiologic vascular development, pathologic blood vessel growth, and vascular restoration. This is in contrast to vasculogenesis, which is de novo growth of vessels from vascular precursors, or from vascular repair that occurs when circulating endothelial progenitor cells home into an area and develop into blood vessels. The objective of this review is to discuss the isoform-specific role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) in physiologic and pathologic angiogenesis and vascular repair, but will not specifically address vasculogenesis. As the major source of reactive oxygen species (ROS) in vascular endothelial cells (ECs), NOX has gained increasing attention in angiogenesis. Activation of NOX leads to events necessary for physiologic and pathologic angiogenesis, including EC migration, proliferation and tube formation. However, activation of different NOX isoforms has different effects in angiogenesis. Activation of NOX2 promotes pathologic angiogenesis and vascular inflammation, but may be beneficial in revascularization in the hindlimb ischemic model. In contrast, activation of NOX4 appears to promote physiologic angiogenesis mainly by protecting the vasculature during ischemia, hypoxia and inflammation and by restoring vascularization, except in models of oxygen-induced retinopathy and diabetes where NOX4 activation leads to pathologic angiogenesis. PMID:28587189

  8. 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.

  9. Synthesis, conformational analysis, and biological activity of new analogues of thiazole-4-carboxamide adenine dinucleotide (TAD) as IMP dehydrogenase inhibitors.

    PubMed

    Franchetti, Palmarisa; Cappellacci, Loredana; Pasqualini, Michela; Petrelli, Riccardo; Jayaprakasan, Vetrichelvan; Jayaram, Hiremagalur N; Boyd, Donald B; Jain, Manojkumar D; Grifantini, Mario

    2005-03-15

    Thiazole-4-carboxamide adenine dinucleotide (TAD) analogues T-2'-MeAD (1) and T-3'-MeAD (2) containing, respectively, a methyl group at the ribose 2'-C-, and 3'-C-position of the adenosine moiety, were prepared as potential selective human inosine monophosphate dehydrogenase (IMPDH) type II inhibitors. The synthesis of heterodinucleotides was carried out by CDI-catalyzed coupling reaction of unprotected 2'-C-methyl- or 3'-C-methyl-adenosine 5'-monophosphate with 2',3'-O-isopropylidene-tiazofurin 5'-monophosphate, and then deisopropylidenation. Biological evaluation of dinucleotides 1 and 2 as inhibitors of recombinant human IMPDH type I and type II resulted in a good activity. Inhibition of both isoenzymes by T-2'-MeAD and T-3'-MeAD was noncompetitive with respect to NAD substrate. Binding of T-3'-MeAD was comparable to that of parent compound TAD, while T-2'-MeAD proved to be a weaker inhibitor. However, no significant difference was found in inhibition of the IMPDH isoenzymes. T-2'-MeAD and T-3'-MeAD were found to inhibit the growth of K562 cells (IC(50) 30.7 and 65.0muM, respectively).

  10. Minimizing photodecomposition of flavin adenine dinucleotide fluorescence by the use of pulsed LEDs.

    PubMed

    Rösner, J; Liotta, A; Angamo, E A; Spies, C; Heinemann, U; Kovács, R

    2016-11-01

    Dynamic alterations in flavin adenine dinucleotide (FAD) fluorescence permit insight into energy metabolism-dependent changes of intramitochondrial redox potential. Monitoring FAD fluorescence in living tissue is impeded by photobleaching, restricting the length of microfluorimetric recordings. In addition, photodecomposition of these essential electron carriers negatively interferes with energy metabolism and viability of the biological specimen. Taking advantage of pulsed LED illumination, here we determined the optimal excitation settings giving the largest fluorescence yield with the lowest photobleaching and interference with metabolism in hippocampal brain slices. The effects of FAD bleaching on energy metabolism and viability were studied by monitoring tissue pO 2 , field potentials and changes in extracellular potassium concentration ([K + ] o ). Photobleaching with continuous illumination consisted of an initial exponential decrease followed by a nearly linear decay. The exponential decay was significantly decelerated with pulsed illumination. Pulse length of 5 ms was sufficient to reach a fluorescence output comparable to continuous illumination, whereas further increasing duration increased photobleaching. Similarly, photobleaching increased with shortening of the interpulse interval. Photobleaching was partially reversible indicating the existence of a transient nonfluorescent flavin derivative. Pulsed illumination decreased FAD photodecomposition, improved slice viability and reproducibility of stimulus-induced FAD, field potential, [K + ] o and pO 2 changes as compared to continuous illumination. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

  11. Characterization of Two Mitochondrial Flavin Adenine Dinucleotide-Dependent Glycerol-3-Phosphate Dehydrogenases in Trypanosoma brucei

    PubMed Central

    Škodová, Ingrid; Verner, Zdeněk; Bringaud, Fréderic; Fabian, Peter

    2013-01-01

    Glycerol-3-phosphate dehydrogenases (G3PDHs) constitute a shuttle that serves for regeneration of NAD+ reduced during glycolysis. This NAD-dependent enzyme is employed in glycolysis and produces glycerol-3-phosphate from dihydroxyacetone phosphate, while its flavin adenine dinucleotide (FAD)-dependent homologue catalyzes a reverse reaction coupled to the respiratory chain. Trypanosoma brucei possesses two FAD-dependent G3PDHs. While one of them (mitochondrial G3PDH [mtG3PDH]) has been attributed to the mitochondrion and seems to be directly involved in G3PDH shuttle reactions, the function of the other enzyme (putative G3PDH [putG3PDH]) remains unknown. In this work, we used RNA interference and protein overexpression and tagging to shed light on the relative contributions of both FAD-G3PDHs to overall cellular metabolism. Our results indicate that mtG3PDH is essential for the bloodstream stage of T. brucei, while in the procyclic stage the enzyme is dispensable. Expressed putG3PDH-V5 was localized to the mitochondrion, and the data obtained by digitonin permeabilization, Western blot analysis, and immunofluorescence indicate that putG3PDH is located within the mitochondrion. PMID:24142106

  12. [Influence exogenous nicotinamide adenine dinucleotide (NAD+) on contractile and bioelectric activity of the rat heart].

    PubMed

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

    2014-04-01

    This study is aimed to the investigation of the nicotinamide adenine dinucleotide (NAD+) effects and mechanisms of action in a heart. NAD+ (mcM) induces multiphase alternation of contractile activity of isolated rat heart: short positive inotropic action is followed by a negative inotropic phase. NAD+ (1-100 mcM) induces decreasing of action potential duration (APD) in rat atrial myocardium (from 45 +/- 0.82 ms in control experiments to 39 +/- 1.05 (n = 8) and 32 +/- 2 (n = 8) during application of 10 and 100 mcM of NAD+, respectively). Significant APD increase (from 45 +/- 0.82 ms to 74 +/- 1.89 (n = 8) ms) was observed during washing out of NAD+ (100 mcM). ATP or adenosine was unable to increase APD both during application or washing out. NAD+ induced APD decrease was not suppressed by P1-antagonist theophylline. P1-purinoreceptor and metabolite independent direct action of NAD+ in rat heart is suggested. Activation of P2X or P2Y receptors, cyclic ADP-ribose accumulation in cardiomyocytes is proposed as a main mechanism of NAD(+)-induced effects in the heart.

  13. Visualization of Nicotine Adenine Dinucleotide Redox Homeostasis with Genetically Encoded Fluorescent Sensors.

    PubMed

    Zhao, Yuzheng; Zhang, Zhuo; Zou, Yejun; Yang, Yi

    2018-01-20

    Beyond their roles as redox currency in living organisms, pyridine dinucleotides (NAD + /NADH and NADP + /NADPH) are also precursors or cosubstrates of great significance in various physiologic and pathologic processes. Recent Advances: For many years, it was challenging to develop methodologies for monitoring pyridine dinucleotides in situ or in vivo. Recent advances in fluorescent protein-based sensors provide a rapid, sensitive, specific, and real-time readout of pyridine dinucleotide dynamics in single cells or in vivo, thereby opening a new era of pyridine dinucleotide bioimaging. In this article, we summarize the developments in genetically encoded fluorescent sensors for NAD + /NADH and NADP + /NADPH redox states, as well as their applications in life sciences and drug discovery. The strengths and weaknesses of individual sensors are also discussed. These sensors have the advantages of being specific and organelle targetable, enabling real-time monitoring and subcellular-level quantification of targeted molecules in living cells and in vivo. NAD + /NADH and NADP + /NADPH have distinct functions in metabolic and redox regulation, and thus, a comprehensive evaluation of metabolic and redox states must be multiplexed with a combination of various metabolite sensors in a single cell. Antioxid. Redox Signal. 28, 213-229.

  14. Stimulation of nicotinamide adenine dinucleotide biosynthetic pathways delays axonal degeneration after axotomy.

    PubMed

    Sasaki, Yo; Araki, Toshiyuki; Milbrandt, Jeffrey

    2006-08-16

    Axonal degeneration occurs in many neurodegenerative diseases and after traumatic injury and is a self-destructive program independent from programmed cell death. Previous studies demonstrated that overexpression of nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1) or exogenous application of nicotinamide adenine dinucleotide (NAD) can protect axons of cultured dorsal root ganglion (DRG) neurons from degeneration caused by mechanical or neurotoxic injury. In mammalian cells, NAD can be synthesized from multiple precursors, including tryptophan, nicotinic acid, nicotinamide, and nicotinamide riboside (NmR), via multiple enzymatic steps. To determine whether other components of these NAD biosynthetic pathways are capable of delaying axonal degeneration, we overexpressed each of the enzymes involved in each pathway and/or exogenously administered their respective substrates in DRG cultures and assessed their capacity to protect axons after axotomy. Among the enzymes tested, Nmnat1 had the strongest protective effects, whereas nicotinamide phosphoribosyl transferase and nicotinic acid phosphoribosyl transferase showed moderate protective activity in the presence of their substrates. Strong axonal protection was also provided by Nmnat3, which is predominantly located in mitochondria, and an Nmnat1 mutant localized to the cytoplasm, indicating that the subcellular location of NAD production is not crucial for protective activity. In addition, we showed that exogenous application of the NAD precursors that are the substrates of these enzymes, including nicotinic acid mononucleotide, nicotinamide mononucleotide, and NmR, can also delay axonal degeneration. These results indicate that stimulation of NAD biosynthetic pathways via a variety of interventions may be useful in preventing or delaying axonal degeneration.

  15. [Effect of flavin adenine dinucleotide on ultraviolet B induced damage in cultured human corneal epithelial cells].

    PubMed

    Sakamoto, Asuka; Nakamura, Masatsugu

    2012-01-01

    This study evaluated the effects of flavin adenine dinucleotide (FAD) on ultraviolet B (UV-B)-induced damage in cultured human corneal epithelial (HCE-T) cells. The cultured HCE-T cells were treated with 0.003125-0.05% FAD before exposure to 80 mJ/cm2 UV-B. Cell viability was measured 24 h after UV-B irradiation using the MTS assay. Reactive oxygen species (ROS) were detected 30 min after UV-B irradiation using 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester. Apoptosis was evaluated 4 h after UV-B irradiation in the caspase-3/7 activity assay. UV-B irradiation reduced cell viability and stimulated ROS production and caspase-3/7 activity in HCE-T cells. Pretreatment of UV-B irradiated HCE-T cells with FAD significantly attenuated cell viability reduction and inhibited the stimulation of both ROS production and caspase-3/7 activity due to UV-B exposure compared with those with vehicle (0% FAD). These results clarified that FAD inhibits ROS-mediated apoptosis by UV-B irradiation in HCE-T cells and suggest that FAD may be effective as a radical scavenger in UV-B-induced corneal damage.

  16. A lectin receptor kinase as a potential sensor for extracellular nicotinamide adenine dinucleotide in Arabidopsis thaliana

    PubMed Central

    Wang, Chenggang; Zhou, Mingqi; Zhang, Xudong; Yao, Jin; Zhang, Yanping; Mou, Zhonglin

    2017-01-01

    Nicotinamide adenine dinucleotide (NAD+) participates in intracellular and extracellular signaling events unrelated to metabolism. In animals, purinergic receptors are required for extracellular NAD+ (eNAD+) to evoke biological responses, indicating that eNAD+ may be sensed by cell-surface receptors. However, the identity of eNAD+-binding receptors still remains elusive. Here, we identify a lectin receptor kinase (LecRK), LecRK-I.8, as a potential eNAD+ receptor in Arabidopsis. The extracellular lectin domain of LecRK-I.8 binds NAD+ with a dissociation constant of 436.5 ± 104.8 nM, although much higher concentrations are needed to trigger in vivo responses. Mutations in LecRK-I.8 inhibit NAD+-induced immune responses, whereas overexpression of LecRK-I.8 enhances the Arabidopsis response to NAD+. Furthermore, LecRK-I.8 is required for basal resistance against bacterial pathogens, substantiating a role for eNAD+ in plant immunity. Our results demonstrate that lectin receptors can potentially function as eNAD+-binding receptors and provide direct evidence for eNAD+ being an endogenous signaling molecule in plants. DOI: http://dx.doi.org/10.7554/eLife.25474.001 PMID:28722654

  17. Simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and nicotinamide adenine dinucleotide in milk by a novel enzyme-coupled assay.

    PubMed

    Ummarino, Simone; Mozzon, Massimo; Zamporlini, Federica; Amici, Adolfo; Mazzola, Francesca; Orsomando, Giuseppe; Ruggieri, Silverio; Raffaelli, Nadia

    2017-04-15

    Nicotinamide riboside, the most recently discovered form of vitamin B3, and its phosphorylated form nicotinamide mononucleotide, have been shown to be potent supplements boosting intracellular nicotinamide adenine dinucleotide (NAD) levels, thus preventing or ameliorating metabolic and mitochondrial diseases in mouse models. Here we report for the first time on the simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and NAD in milk by means of a fluorometric, enzyme-coupled assay. Application of this assay to milk from different species revealed that the three vitamers were present in human and donkey milk, while being selectively distributed in the other milks. Human milk was the richest source of nicotinamide mononucleotide. Overall, the three vitamers accounted for a significant fraction of total vitamin B3 content. Pasteurization did not affect the bovine milk content of nicotinamide riboside, whereas UHT processing fully destroyed the vitamin. In human milk, NAD levels were significantly affected by the lactation time. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Role of Nicotinamide Adenine Dinucleotide and Related Precursors as Therapeutic Targets for Age-Related Degenerative Diseases: Rationale, Biochemistry, Pharmacokinetics, and Outcomes.

    PubMed

    Braidy, Nady; Berg, Jade; Clement, James; Khorshidi, Fatemeh; Poljak, Anne; Jayasena, Tharusha; Grant, Ross; Sachdev, Perminder

    2018-05-11

    Nicotinamide adenine dinucleotide (NAD + ) is an essential pyridine nucleotide that serves as an essential cofactor and substrate for a number of critical cellular processes involved in oxidative phosphorylation and ATP production, DNA repair, epigenetically modulated gene expression, intracellular calcium signaling, and immunological functions. NAD + depletion may occur in response to either excessive DNA damage due to free radical or ultraviolet attack, resulting in significant poly(ADP-ribose) polymerase (PARP) activation and a high turnover and subsequent depletion of NAD + , and/or chronic immune activation and inflammatory cytokine production resulting in accelerated CD38 activity and decline in NAD + levels. Recent studies have shown that enhancing NAD + levels can profoundly reduce oxidative cell damage in catabolic tissue, including the brain. Therefore, promotion of intracellular NAD + anabolism represents a promising therapeutic strategy for age-associated degenerative diseases in general, and is essential to the effective realization of multiple benefits of healthy sirtuin activity. The kynurenine pathway represents the de novo NAD + synthesis pathway in mammalian cells. NAD + can also be produced by the NAD + salvage pathway. Recent Advances: In this review, we describe and discuss recent insights regarding the efficacy and benefits of the NAD + precursors, nicotinamide (NAM), nicotinic acid (NA), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN), in attenuating NAD + decline in degenerative disease states and physiological aging. Results obtained in recent years have shown that NAD + precursors can play important protective roles in several diseases. However, in some cases, these precursors may vary in their ability to enhance NAD + synthesis via their location in the NAD + anabolic pathway. Increased synthesis of NAD + promotes protective cell responses, further demonstrating that NAD + is a regulatory molecule associated with several

  19. Flow-injection analysis with electrochemical detection of reduced nicotinamide adenine dinucleotide using 2,6-dichloroindophenol as a redox coupling agent.

    PubMed

    Tang, H T; Hajizadeh, K; Halsall, H B; Heineman, W R

    1991-01-01

    The determination of reduced nicotinamide adenine dinucleotide (NADH) by electrochemical oxidation requires a more positive potential than is predicted by the formal reduction potential for the NAD+/NADH couple. This problem is alleviated by use of 2,6-dichloroindophenol (DCIP) as a redox coupling agent for NADH. The electrochemical characteristics of DCIP at the glassy carbon electrode are examined by cyclic voltammetry and hydrodynamic voltammetry. NADH is determined by reaction with DCIP to form NAD+ and DCIPH2. DCIPH2 is then quantitated by flow-injection analysis with electrochemical detection by oxidation at a detector potential of +0.25 V at pH 7. NADH is determined over a linear range of 0.5 to 200 microM and with a detection limit of 0.38 microM. The lower detection potential for DCIPH2 compared to NADH helps to minimize interference from oxidizable components in serum samples.

  20. Comparison of oral nicotinamide adenine dinucleotide (NADH) versus conventional therapy for chronic fatigue syndrome.

    PubMed

    Santaella, María L; Font, Ivonne; Disdier, Orville M

    2004-06-01

    To compare effectiveness of oral therapy with reduced nicotinamide adenine dinucleotide (NADH) to conventional modalities of treatment in patients with chronic fatigue syndrome (CFS). CFS is a potentially disabling condition of unknown etiology. Although its clinical presentation is associated to a myriad of symptoms, fatigue is a universal and essential finding for its diagnosis. No therapeutic regimen has proven effective for this condition. A total of 31 patients fulfilling the Centers for Disease Control criteria for CFS, were randomly assigned to either NADH or nutritional supplements and psychological therapy for 24 months. A thorough medical history, physical examination and completion of a questionnaire on the severity of fatigue and other symptoms were performed each trimester of therapy. In addition, all of them underwent evaluation in terms of immunological parameters and viral antibody titers. Statistical analysis was applied to the demographic data, as well as to symptoms scores at baseline and at each trimester of therapy. The twelve patients who received NADH had a dramatic and statistically significant reduction of the mean symptom score in the first trimester (p < 0.001). However, symptom scores in the subsequent trimesters of therapy were similar in both treatment groups. Elevated IgG and Ig E antibody levels were found in a significant number of patients. Observed effectiveness of NADH over conventional treatment in the first trimester of the trial and the trend of improvement of that modality in the subsequent trimesters should be further assessed in a larger patient sample.

  1. Ca2+ release triggered by nicotinate adenine dinucleotide phosphate in intact sea urchin eggs.

    PubMed Central

    Perez-Terzic, C M; Chini, E N; Shen, S S; Dousa, T P; Clapham, D E

    1995-01-01

    Nicotinate adenine dinucleotide phosphate (NAADP) was recently identified [Lee and Aarhus (1995) J. Biol. Chem. 270, 2152-2157; Chini, Beers and Dousa (1995) J. Biol. Chem. 270, 3116-3223] as a potent Ca(2+)-releasing agent in sea urchin egg homogenates. NAADP triggered Ca2+ release by a mechanism that was distinct from inositol 1,4,5-trisphosphate (InsP3)- and cyclic ADP-ribose (cADPR)-induced Ca2+ release. When NAADP was microinjected into intact sea urchin eggs it induced a dose-dependent increase in cytoplasmic free Ca2+ which was independent of the extracellular [Ca2+]. The Ca2+ waves elicited by microinjections of NAADP originated at the site of injection and swept across the cytosol. As previously found in sea urchin egg homogenates, NAADP-induced Ca2+ release in intact eggs was not blocked by heparin or by prior desensitization to InsP3 or cADPR. Thio-NADP, a specific inhibitor of the NAADP-induced Ca2+ release in sea urchin homogenates [Chini, Beers and Dousa (1995) J. Biol. Chem. 270, 3116-3223] blocked NAADP (but not InsP3 or cADPR) injection-induced Ca2+ release in intact sea urchin eggs. Finally, fertilization of sea urchin eggs abrogated subsequent NAADP-induced Ca2+ release, suggesting that the NAADP-sensitive Ca2+ pool may participate in the fertilization response. This study demonstrates that NAADP acts as a selective Ca(2+)-releasing agonist in intact cells. Images Figure 2 PMID:8554544

  2. 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.

  3. Purification and characterization of the enzymes involved in nicotinamide adenine dinucleotide degradation by Penicillium brevicompactum NRC 829.

    PubMed

    Ali, Thanaa Hamed; El-Ghonemy, Dina Helmy

    2016-06-01

    The present study was conducted to investigate a new pathway for the degradation of nicotinamide adenine dinucleotide (NAD) by Penicillium brevicompactum NRC 829 extracts. Enzymes involved in the hydrolysis of NAD, i.e. alkaline phosphatase, aminohydrolase and glycohydrolase were determined. Alkaline phosphatase was found to catalyse the sequential hydrolysis of two phosphate moieties of NAD molecule to nicotinamide riboside plus adenosine. Adenosine was then deaminated by aminohydrolase to inosine and ammonia. While glycohydrolase catalyzed the hydrolysis of the nicotinamide-ribosidic bond of NAD+ to produce nicotinamide and ADP-ribose in equimolar amounts, enzyme purification through a 3-step purification procedure revealed the existence of two peaks of alkaline phosphatases, and one peak contained deaminase and glycohydrolase activities. NAD deaminase was purified to homogeneity as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with an apparent molecular mass of 91 kDa. Characterization and determination of some of NAD aminohydrolase kinetic properties were conducted due to its biological role in the regulation of cellular NAD level. The results also revealed that NAD did not exert its feedback control on nicotinamide amidase produced by P. brevicompactum.

  4. In situ chemichromic studies of interactions between a lutetium bis-octaalkyl-substituted phthalocyanine and selected biological cofactors

    PubMed Central

    Pal, C.; Cammidge, A. N.; Cook, M. J.; Sosa-Sanchez, J. L.; Sharma, A. K.; Ray, A. K.

    2012-01-01

    Spin-coated films, approximately 100 nm thick, of a newly synthesized bis[octakis(octyl)phthalocyaninato] lutetium(III) complex on ultrasonically cleaned glass substrates exhibit pronounced chemichromic behaviour with potential application in healthcare. In situ kinetic optical absorption spectroscopic measurements show that the phthalocyanine Q-band is red shifted by 60 nm upon oxidation arising from exposure to bromine vapour. Recovery to the original state is achieved by the treatment of the oxidized films with nicotinamide adenine dinucleotide and l-ascorbic acid (vitamin C) in an aqueous solution containing 1.5 M lithium perchlorate. The neutralization process is found to be governed by first-order kinetics. The linear increase of the reduction rate with increasing concentration of cofactors provides a basis for calibration of analyte concentrations ranging from 3.5 mM down to 0.03 mM. PMID:21676969

  5. The human amygdaloid complex: a cytologic and histochemical atlas using Nissl, myelin, acetylcholinesterase and nicotinamide adenine dinucleotide phosphate diaphorase staining.

    PubMed

    Sims, K S; Williams, R S

    1990-01-01

    We examined the distribution of acetylcholinesterase and nicotinamide adenine dinucleotide phosphate diaphorase enzyme activity in the human amygdala using histochemical techniques. Both methods revealed compartments of higher or lower enzyme activity, in cells or neuropil, which corresponded to the nuclear subdivisions of the amygdala as defined with classical Nissl and myelin methods. The boundaries between the histochemical compartments were usually so sharp that the identification of these nuclear subdivisions was enhanced. There was also variation of staining intensity within many of the nuclear subdivisions, such as the lateral and central nuclei, anterior amygdaloid area and the intercalated groups. This histochemical difference corresponded to more subtle differences in Nissl and myelin staining patterns, and suggests further structural subdivisions of potential functional significance. We present a revised scheme of anatomical parcellation of the human amygdala based upon serial analysis with all four techniques. Our expectation is that this will allow the delineation of a clearer homology between the cytoarchitectonic subdivisions of the human amygdala and those of experimental animals.

  6. Photosensitized oxidation of nicotinamide adenine dinucleotide by diethoxyphosphorus(V)tetraphenylporphyrin and its fluorinated derivative: Possibility of chain reaction

    NASA Astrophysics Data System (ADS)

    Hirakawa, Kazutaka; Murata, Atsushi

    2018-01-01

    Water-soluble porphyrins, diethoxyphosphorus(V)tetraphenylporphyrin (EtP(V)TPP) and its fluorinated analogue (FEtP(V)TPP), decreased the typical absorption around 340 nm of nicotinamide adenine dinucleotide (NADH) under visible light irradiation, indicating oxidative decomposition. A singlet oxygen quencher, sodium azide, and a triplet quencher, potassium iodide, slightly inhibited photosensitized NADH oxidation. However, these inhibitory effects were very small. Furthermore, the fluorescence lifetime of these P(V)porphyrins was decreased by NADH, suggesting the contribution of electron transfer to the singlet excited (S1) state of P(V)porphyrin. The redox potential measurement supports the electron transfer-mediated oxidation of NADH. The quantum yields of NADH photodecomposition by P(V)porphyrins could be estimated from the kinetic data and the effect of these quenchers on NADH oxidation. The obtained values suggest that the electron accepting by the S1 states of P(V)porphyrins triggers a chain reaction of NADH oxidation. This photosensitized reaction may play an important role in the photocytotoxicity of P(V)porphyrins. The axial ligand fluorination of P(V)porphyrins improved electron accepting ability. However, fluorination slightly suppressed static interaction with NADH, resulting in decreased oxidation quantum yield.

  7. Generation of Reduced Nicotinamide Adenine Dinucleotide for Nitrate Reduction in Green Leaves 1

    PubMed Central

    Klepper, Lowell; Flesher, Donna; Hageman, R. H.

    1971-01-01

    An in vivo assay of nitrate reductase activity was developed by vacuum infiltration of leaf discs or sections with a solution of 0.2 m KNO3 (with or without phosphate buffer, pH 7.5) and incubation of the infiltrated tissue and medium under essentially anaerobic conditions in the dark. Nitrite production, for computing enzyme activity, was determined on aliquots of the incubation media, removed at intervals. By adding, separately, various metabolites of the glycolytic, pentose phosphate, and citric acid pathways to the infiltrating media, it was possible to use the in vivo assay to determine the prime source of reduced nicotinamide adenine dinucleotide (NADH) required by the cytoplasmically located NADH-specific nitrate reductase. It was concluded that sugars that migrate from the chloroplast to the cytoplasm were the prime source of energy and that the oxidation of glyceraldehyde 3-phosphate was ultimately the in vivo source of NADH for nitrate reduction. This conclusion was supported by experiments that included: inhibition studies with iodoacetate; in vitro studies that established the presence and functionality of the requisite enzymes; and studies showing the effect of light (photosynthate) and exogenous carbohydrate on loss of endogenous nitrate from plant tissue. The level of nitrate reductase activity obtained with the in vitro assay is higher (2.5- to 20-fold) than with the in vivo assay for most plant species. The work done to date would indicate that the in vivo assays are proportional to the in vitro assays with respect to ranking genotypes for nitrate-reducing potential of a given species. The in vivo assay is especially useful in studying nitrate assimilation in species like giant ragweed from which only traces of active nitrate reductase can be extracted. PMID:16657841

  8. Cleavage of nicotinamide adenine dinucleotide by the ribosome-inactivating protein from Momordica charantia.

    PubMed

    Vinkovic, M; Dunn, G; Wood, G E; Husain, J; Wood, S P; Gill, R

    2015-09-01

    The interaction of momordin, a type 1 ribosome-inactivating protein from Momordica charantia, with NADP(+) and NADPH has been investigated by X-ray diffraction analysis of complexes generated by co-crystallization and crystal soaking. It is known that the proteins of this family readily cleave the adenine-ribose bond of adenosine and related nucleotides in the crystal, leaving the product, adenine, bound to the enzyme active site. Surprisingly, the nicotinamide-ribose bond of oxidized NADP(+) is cleaved, leaving nicotinamide bound in the active site in the same position but in a slightly different orientation to that of the five-membered ring of adenine. No binding or cleavage of NADPH was observed at pH 7.4 in these experiments. These observations are in accord with current views of the enzyme mechanism and may contribute to ongoing searches for effective inhibitors.

  9. UV-Vis Action Spectroscopy Reveals a Conformational Collapse in Hydrogen-Rich Dinucleotide Cation Radicals.

    PubMed

    Korn, Joseph A; Urban, Jan; Dang, Andy; Nguyen, Huong T H; Tureček, František

    2017-09-07

    We report the generation of deoxyriboadenosine dinucleotide cation radicals by gas-phase electron transfer to dinucleotide dications and their noncovalent complexes with crown ether ligands. Stable dinucleotide cation radicals of a novel hydrogen-rich type were generated and characterized by tandem mass spectrometry and UV-vis photodissociation (UVPD) action spectroscopy. Electron structure theory analysis indicated that upon electron attachment the dinucleotide dications underwent a conformational collapse followed by intramolecular proton migrations between the nucleobases to give species whose calculated UV-vis absorption spectra matched the UVPD action spectra. Hydrogen-rich cation radicals generated from chimeric riboadenosine 5'-diesters gave UVPD action spectra that pointed to novel zwitterionic structures consisting of aromatic π-electron anion radicals intercalated between stacked positively charged adenine rings. Analogies with DNA ionization are discussed.

  10. 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

  11. Sensitive colorimetric visualization of dihydronicotinamide adenine dinucleotide based on anti-aggregation of gold nanoparticles via boronic acid-diol binding.

    PubMed

    Liu, Shufeng; Du, Zongfeng; Li, Peng; Li, Feng

    2012-05-15

    A facile, highly sensitive colorimetric strategy for dihydronicotinamide adenine dinucleotide (NADH) detection is proposed based on anti-aggregation of gold nanoparticles (AuNPs) via boronic acid-diol binding chemistry. The aggregation agent, 4-mercaptophenylboronic acid (MPBA), has specific affinity for AuNPs through Au-S interaction, leading to the aggregation of AuNPs by self-dehydration condensation at a certain concentration, which is responsible for a visible color change of AuNPs from wine red to blue. With the addition of NADH, MPBA would prefer reacting with NADH to form stable borate ester via boronic acid-diol binding dependent on the pH and solvent, revealing an obvious color change from blue to red with increasing the concentration of NADH. The anti-aggregation effect of NADH on AuNPs was seen by the naked eye and monitored by UV-vis extinction spectra. The linear range of the colorimetric sensor for NADH is from 8.0 × 10(-9)M to 8.0 × 10(-6)M, with a low detection limit of 2.0 nM. The as-established colorimetric strategy opened a new avenue for NADH determination. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Photosensitized oxidation of nicotinamide adenine dinucleotide by diethoxyphosphorus(V)tetraphenylporphyrin and its fluorinated derivative: Possibility of chain reaction.

    PubMed

    Hirakawa, Kazutaka; Murata, Atsushi

    2018-01-05

    Water-soluble porphyrins, diethoxyphosphorus(V)tetraphenylporphyrin (EtP(V)TPP) and its fluorinated analogue (FEtP(V)TPP), decreased the typical absorption around 340nm of nicotinamide adenine dinucleotide (NADH) under visible light irradiation, indicating oxidative decomposition. A singlet oxygen quencher, sodium azide, and a triplet quencher, potassium iodide, slightly inhibited photosensitized NADH oxidation. However, these inhibitory effects were very small. Furthermore, the fluorescence lifetime of these P(V)porphyrins was decreased by NADH, suggesting the contribution of electron transfer to the singlet excited (S 1 ) state of P(V)porphyrin. The redox potential measurement supports the electron transfer-mediated oxidation of NADH. The quantum yields of NADH photodecomposition by P(V)porphyrins could be estimated from the kinetic data and the effect of these quenchers on NADH oxidation. The obtained values suggest that the electron accepting by the S 1 states of P(V)porphyrins triggers a chain reaction of NADH oxidation. This photosensitized reaction may play an important role in the photocytotoxicity of P(V)porphyrins. The axial ligand fluorination of P(V)porphyrins improved electron accepting ability. However, fluorination slightly suppressed static interaction with NADH, resulting in decreased oxidation quantum yield. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Coupling Drosophila melanogaster Cryptochrome Light Activation and Oxidation of the Kvβ Subunit Hyperkinetic NADPH Cofactor.

    PubMed

    Hong, Gongyi; Pachter, Ruth; Ritz, Thorsten

    2018-06-28

    Motivated by the observations on the involvement of light-induced processes in the Drosophila melanogaster cryptochrome (DmCry) in regulation of the neuronal firing rate, which is achieved by a redox-state change of its voltage-dependent K + channel Kvβ subunit hyperkinetic (Hk) reduced nicotinamide adenine dinucleotide phosphate (NADPH) cofactor, we propose in this work two hypothetical pathways that may potentially enable such coupling. In the first pathway, triggered by blue-light-induced formation of a radical pair [FAD •- TRP •+ ] in DmCry, the hole (TRP •+ ) may hop to Hk, for example, through a tryptophan chain and oxidize NADPH, possibly leading to inhibition of the N-terminus inactivation in the K + channel. In a second possible pathway, DmCry's FAD •- is reoxidized by molecular oxygen, producing H 2 O 2 , which then diffuses to Hk and oxidizes NADPH. In this work, by applying a combination of quantum and empirical-based methods for free-energy calculations, we find that the oxidation of NADPH by TRP •+ or H 2 O 2 and the reoxidation of FAD •- by O 2 are thermodynamically feasible. Our results may have an implication in identifying a magnetic sensing signal transduction pathway, specifically upon Drosophila's Hk NADPH cofactor oxidation, with a subsequent inhibition of the K + channel N-terminus inactivation gate, permitting K + flux.

  14. 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

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

    PubMed

    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; Lee, Ji-Young; Auwerx, Johan

    2016-04-01

    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 (Sirt1(hep-/-) ), 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. Our data warrant the future evaluation of NAD(+) boosting strategies to manage the development or progression of NAFLD. © 2015 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.

  16. [The release of flavin adenine dinucleotide upon local conformational transition in electron-transferring flavoprotein induced by trimethylamine dehydrogenase].

    PubMed

    Lomtev, A S; Bobrov, A G; Vekshin, N L

    2004-01-01

    The electron-transferring proteins, trimethylamine dehydrogenase (TMAD) and electron-transferring flavoprotein (ETF) from the bacterium Methylophilius methylotrophus, were studied in vitro by fluorescence spectroscopy. Flavin adenine dinucleotide (FAD) was found to be capable of a slow and spontaneous release from ETF, which is accompanied by an increase in flavin fluorescence. At a rather high ionic strength (0.1 M NaCl or 50 mM phosphate), the FAD release is sharply activated by TMAD preparations that induce a local conformational transition in ETF. The values of tryptophan fluorescence polarization and lifetime and the use of the Levshin-Perrin equation helped show that the size of protein particles remain unchanged upon the TMAD and ETF mixing; i.e., these proteins themselves do not form a stable complex with each other. The protein mixture did not release flavin from ETF in the presence of trimethylamine and formaldehyde. In this case, a stable complex between the proteins appeared to be formed under the action of formaldehyde. Upon a short-term incubation of ETF with ferricyanide, FAD was hydrolyzed to flavin mononucleotide (FMN) and AMP. This fact explains the previous detection of AMP in ETF preparations by some researches. A fluorescence method was proposed for distinguishing FAD from FMN in solution using ethylene glycol. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.

  17. Transcriptional regulation of nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase in murine hepatoma cells by 6-(methylsufinyl)hexyl isothiocyanate, an active principle of wasabi (Eutrema wasabi Maxim).

    PubMed

    Hou, D X; Fukuda, M; Fujii, M; Fuke, Y

    2000-12-20

    Wasabi is a very popular pungent spice in Japan. This study examined the ability of 6-(methylsufinyl)hexyl isothiocyanate (6-MITC), an active principle of wasabi, to induce the cellular expression of nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase (QR) in Hepa 1c1c7 cells. The cells were treated with various concentrations of 6-MITC, and were then assessed for cell growth, QR activity and QR mRNA expression. The induction of QR activity and QR mRNA expression was time- and dose-responsive over a narrow range of 0.1-5 microM, with declining induction at higher concentrations due to cell toxicity. Furthermore, transfection studies demonstrated that the induction of transcription of the QR gene by 6-MITC involved an antioxidant/electrophile-responsive element (ARE/EpRE) activation. Our results suggest a novel mechanism by which dietary wasabi 6-MITC may be implicated in cancer chemoprevention.

  18. Auxotrophic Actinobacillus pleurpneumoniae grows in multispecies biofilms without the need for nicotinamide-adenine dinucleotide (NAD) supplementation.

    PubMed

    Loera-Muro, Abraham; Jacques, Mario; Avelar-González, Francisco J; Labrie, Josée; Tremblay, Yannick D N; Oropeza-Navarro, Ricardo; Guerrero-Barrera, Alma L

    2016-06-27

    Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, which causes important worldwide economic losses in the swine industry. Several respiratory tract infections are associated with biofilm formation, and A. pleuropneumoniae has the ability to form biofilms in vitro. Biofilms are structured communities of bacterial cells enclosed in a self-produced polymer matrix that are attached to an abiotic or biotic surface. Virtually all bacteria can grow as a biofilm, and multi-species biofilms are the most common form of microbial growth in nature. The goal of this study was to determine the ability of A. pleuropneumoniae to form multi-species biofilms with other bacteria frequently founded in pig farms, in the absence of pyridine compounds (nicotinamide mononucleotide [NMN], nicotinamide riboside [NR] or nicotinamide adenine dinucleotide [NAD]) that are essential for the growth of A. pleuropneumoniae. For the biofilm assay, strain 719, a field isolate of A. pleuropneumoniae serovar 1, was mixed with swine isolates of Streptococcus suis, Bordetella bronchiseptica, Pasteurella multocida, Staphylococcus aureus or Escherichia coli, and deposited in 96-well microtiter plates. Based on the CFU results, A. pleuropneumoniae was able to grow with every species tested in the absence of pyridine compounds in the culture media. Interestingly, A. pleuropneumoniae was also able to form strong biofilms when mixed with S. suis, B. bronchiseptica or S. aureus. In the presence of E. coli, A. pleuropneumoniae only formed a weak biofilm. The live and dead populations, and the matrix composition of multi-species biofilms were also characterized using fluorescent markers and enzyme treatments. The results indicated that poly-N-acetyl-glucosamine remains the primary component responsible for the biofilm structure. In conclusion, A. pleuropneumoniae apparently is able to satisfy the requirement of pyridine compounds through of other swine pathogens by

  19. Correlating two-photon excited fluorescence imaging of breast cancer cellular redox state with seahorse flux analysis of normalized cellular oxygen consumption

    NASA Astrophysics Data System (ADS)

    Hou, Jue; Wright, Heather J.; Chan, Nicole; Tran, Richard; Razorenova, Olga V.; Potma, Eric O.; Tromberg, Bruce J.

    2016-06-01

    Two-photon excited fluorescence (TPEF) imaging of the cellular cofactors nicotinamide adenine dinucleotide and oxidized flavin adenine dinucleotide is widely used to measure cellular metabolism, both in normal and pathological cells and tissues. When dual-wavelength excitation is used, ratiometric TPEF imaging of the intrinsic cofactor fluorescence provides a metabolic index of cells-the "optical redox ratio" (ORR). With increased interest in understanding and controlling cellular metabolism in cancer, there is a need to evaluate the performance of ORR in malignant cells. We compare TPEF metabolic imaging with seahorse flux analysis of cellular oxygen consumption in two different breast cancer cell lines (MCF-7 and MDA-MB-231). We monitor metabolic index in living cells under both normal culture conditions and, for MCF-7, in response to cell respiration inhibitors and uncouplers. We observe a significant correlation between the TPEF-derived ORR and the flux analyzer measurements (R=0.7901, p<0.001). Our results confirm that the ORR is a valid dynamic index of cell metabolism under a range of oxygen consumption conditions relevant for cancer imaging.

  20. African Swine Fever Virus pB119L Protein Is a Flavin Adenine Dinucleotide-Linked Sulfhydryl Oxidase

    PubMed Central

    Rodríguez, Irene; Redrejo-Rodríguez, Modesto; Rodríguez, Javier M.; Alejo, Alí; Salas, José; Salas, María L.

    2006-01-01

    Protein pB119L of African swine fever virus belongs to the Erv1p/Alrp family of sulfhydryl oxidases and has been described as a late nonstructural protein required for correct virus assembly. To further our knowledge of the function of protein pB119L during the virus life cycle, we have investigated whether this protein possesses sulfhydryl oxidase activity, using a purified recombinant protein. We show that the purified protein contains bound flavin adenine dinucleotide and is capable of catalyzing the formation of disulfide bonds both in a protein substrate and in the small molecule dithiothreitol, the catalytic activity being comparable to that of the Erv1p protein. Furthermore, protein pB119L contains the cysteines of its active-site motif CXXC, predominantly in an oxidized state, and forms noncovalently bound dimers in infected cells. We also show in coimmunoprecipitation experiments that protein pB119L interacts with the viral protein pA151R, which contains a CXXC motif similar to that present in thioredoxins. Protein pA151R, in turn, was found to interact with the viral structural protein pE248R, which contains disulfide bridges and belongs to a class of myristoylated proteins related to vaccinia virus L1R, one of the substrates of the redox pathway encoded by this virus. These results suggest the existence in African swine fever virus of a system for the formation of disulfide bonds constituted at least by proteins pB119L and pA151R and identify protein pE248R as a possible final substrate of this pathway. PMID:16537584

  1. African swine fever virus pB119L protein is a flavin adenine dinucleotide-linked sulfhydryl oxidase.

    PubMed

    Rodríguez, Irene; Redrejo-Rodríguez, Modesto; Rodríguez, Javier M; Alejo, Alí; Salas, José; Salas, María L

    2006-04-01

    Protein pB119L of African swine fever virus belongs to the Erv1p/Alrp family of sulfhydryl oxidases and has been described as a late nonstructural protein required for correct virus assembly. To further our knowledge of the function of protein pB119L during the virus life cycle, we have investigated whether this protein possesses sulfhydryl oxidase activity, using a purified recombinant protein. We show that the purified protein contains bound flavin adenine dinucleotide and is capable of catalyzing the formation of disulfide bonds both in a protein substrate and in the small molecule dithiothreitol, the catalytic activity being comparable to that of the Erv1p protein. Furthermore, protein pB119L contains the cysteines of its active-site motif CXXC, predominantly in an oxidized state, and forms noncovalently bound dimers in infected cells. We also show in coimmunoprecipitation experiments that protein pB119L interacts with the viral protein pA151R, which contains a CXXC motif similar to that present in thioredoxins. Protein pA151R, in turn, was found to interact with the viral structural protein pE248R, which contains disulfide bridges and belongs to a class of myristoylated proteins related to vaccinia virus L1R, one of the substrates of the redox pathway encoded by this virus. These results suggest the existence in African swine fever virus of a system for the formation of disulfide bonds constituted at least by proteins pB119L and pA151R and identify protein pE248R as a possible final substrate of this pathway.

  2. β-Nicotinamide Adenine Dinucleotide (β-NAD) Inhibits ATP-Dependent IL-1β Release from Human Monocytic Cells.

    PubMed

    Hiller, Sebastian Daniel; Heldmann, Sarah; Richter, Katrin; Jurastow, Innokentij; Küllmar, Mira; Hecker, Andreas; Wilker, Sigrid; Fuchs-Moll, Gabriele; Manzini, Ivan; Schmalzing, Günther; Kummer, Wolfgang; Padberg, Winfried; McIntosh, J Michael; Damm, Jelena; Zakrzewicz, Anna; Grau, Veronika

    2018-04-10

    While interleukin-1β (IL-1β) is a potent pro-inflammatory cytokine essential for host defense, high systemic levels cause life-threatening inflammatory syndromes. ATP, a stimulus of IL-1β maturation, is released from damaged cells along with β-nicotinamide adenine dinucleotide (β-NAD). Here, we tested the hypothesis that β-NAD controls ATP-signaling and, hence, IL-1β release. Lipopolysaccharide-primed monocytic U937 cells and primary human mononuclear leukocytes were stimulated with 2'(3')- O -(4-benzoyl-benzoyl)ATP trieethylammonium salt (BzATP), a P2X7 receptor agonist, in the presence or absence of β-NAD. IL-1β was measured in cell culture supernatants. The roles of P2Y receptors, nicotinic acetylcholine receptors (nAChRs), and Ca 2+ -independent phospholipase A2 (iPLA2β, PLA2G6) were investigated using specific inhibitors and gene-silencing. Exogenous β-NAD signaled via P2Y receptors and dose-dependently (IC 50 = 15 µM) suppressed the BzATP-induced IL-1β release. Signaling involved iPLA2β, release of a soluble mediator, and nAChR subunit α9. Patch-clamp experiments revealed that β-NAD inhibited BzATP-induced ion currents. In conclusion, we describe a novel triple membrane-passing signaling cascade triggered by extracellular β-NAD that suppresses ATP-induced release of IL-1β by monocytic cells. This cascade links activation of P2Y receptors to non-canonical metabotropic functions of nAChRs that inhibit P2X7 receptor function. The biomedical relevance of this mechanism might be the control of trauma-associated systemic inflammation.

  3. Protective effect of nicotinamide adenine dinucleotide (NAD+) against spinal cord ischemia-reperfusion injury via reducing oxidative stress-induced neuronal apoptosis.

    PubMed

    Xie, Lei; Wang, Zhenfei; Li, Changwei; Yang, Kai; Liang, Yu

    2017-02-01

    As previous studies demonstrate that oxidative stress and apoptosis play crucial roles in ischemic pathogenesis and nicotinamide adenine dinucleotide (NAD + ) treatment attenuates oxidative stress-induced cell death among primary neurons and astrocytes as well as significantly reduce cerebral ischemic injury in rats. We used a spinal cord ischemia injury (SCII) model in rats to verify our hypothesis that NAD + could ameliorate oxidative stress-induced neuronal apoptosis. Adult male rats were subjected to transient spinal cord ischemia for 60min, and different doses of NAD + were administered intraperitoneally immediately after the start of reperfusion. Neurological function was determined by Basso, Beattie, Bresnahan (BBB) scores. The oxidative stress level was assessed by superoxide dismutase (SOD) activity and malondialdehyde (MDA) content. The degree of apoptosis was analyzed by deoxyuridinetriphosphate nick-end labeling (TUNEL) staining and protein levels of cleaved caspase-3 and AIF (apoptosis inducing factor). The results showed that NAD + at 50 or 100mg/kg significantly decreased the oxidative stress level and neuronal apoptosis in the spinal cord of ischemia-reperfusion rats compared with saline, as accompanied with the decreased oxidative stress, NAD + administration significantly restrained the neuronal apoptosis after ischemia injury while improved the neurological and motor function. These findings suggested that NAD + might protect against spinal cord ischemia-reperfusion via reducing oxidative stress-induced neuronal apoptosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. 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.

  5. Cofactor engineering to regulate NAD+/NADH ratio with its application to phytosterols biotransformation.

    PubMed

    Su, Liqiu; Shen, Yanbing; Zhang, Wenkai; Gao, Tian; Shang, Zhihua; Wang, Min

    2017-10-30

    Cofactor engineering is involved in the modification of enzymes related to nicotinamide adenine dinucleotides (NADH and NAD + ) metabolism, which results in a significantly altered spectrum of metabolic products. Cofactor engineering plays an important role in metabolic engineering but is rarely reported in the sterols biotransformation process owing to its use of multi-catabolic enzymes, which promote multiple consecutive reactions. Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are important steroid medicine intermediates that are obtained via the nucleus oxidation and the side chain degradation of phytosterols by Mycobacterium. Given that the biotransformation from phytosterols to AD (D) is supposed to be a NAD + -dependent process, this work utilized cofactor engineering in Mycobacterium neoaurum and investigated the effect on cofactor and phytosterols metabolism. Through the addition of the coenzyme precursor of nicotinic acid in the phytosterols fermentation system, the intracellular NAD + /NADH ratio and the AD (D) production of M. neoaurum TCCC 11978 (MNR M3) were higher than in the control. Moreover, the NADH: flavin oxidoreductase was identified and was supposed to exert a positive effect on cofactor regulation and phytosterols metabolism pathways via comparative proteomic profiling of MNR cultured with and without phytosterols. In addition, the NADH: flavin oxidoreductase and a water-forming NADH oxidase from Lactobacillus brevis, were successfully overexpressed and heterologously expressed in MNR M3 to improve the intracellular ratio of NAD + /NADH. After 96 h of cultivation, the expression of these two enzymes in MNR M3 resulted in the decrease in intracellular NADH level (by 51 and 67%, respectively) and the increase in NAD + /NADH ratio (by 113 and 192%, respectively). Phytosterols bioconversion revealed that the conversion ratio of engineered stains was ultimately improved by 58 and 147%, respectively. The highest AD (D

  6. A Bacterial Multidomain NAD-Independent d-Lactate Dehydrogenase Utilizes Flavin Adenine Dinucleotide and Fe-S Clusters as Cofactors and Quinone as an Electron Acceptor for d-Lactate Oxidization

    PubMed Central

    Jiang, Tianyi; Guo, Xiaoting; Yan, Jinxin; Zhang, Yingxin; Wang, Yujiao; Zhang, Manman; Sheng, Binbin; Ma, Cuiqing; Xu, Ping

    2017-01-01

    ABSTRACT Bacterial membrane-associated NAD-independent d-lactate dehydrogenase (Fe-S d-iLDH) oxidizes d-lactate into pyruvate. A sequence analysis of the enzyme reveals that it contains an Fe-S oxidoreductase domain in addition to a flavin adenine dinucleotide (FAD)-containing dehydrogenase domain, which differs from other typical d-iLDHs. Fe-S d-iLDH from Pseudomonas putida KT2440 was purified as a His-tagged protein and characterized in detail. This monomeric enzyme exhibited activities with l-lactate and several d-2-hydroxyacids. Quinone was shown to be the preferred electron acceptor of the enzyme. The two domains of the enzyme were then heterologously expressed and purified separately. The Fe-S cluster-binding motifs predicted by sequence alignment were preliminarily verified by site-directed mutagenesis of the Fe-S oxidoreductase domain. The FAD-containing dehydrogenase domain retained 2-hydroxyacid-oxidizing activity, although it decreased compared to the full Fe-S d-iLDH. Compared to the intact enzyme, the FAD-containing dehydrogenase domain showed increased catalytic efficiency with cytochrome c as the electron acceptor, but it completely lost the ability to use coenzyme Q10. Additionally, the FAD-containing dehydrogenase domain was no longer associated with the cell membrane, and it could not support the utilization of d-lactate as a carbon source. Based on the results obtained, we conclude that the Fe-S oxidoreductase domain functions as an electron transfer component to facilitate the utilization of quinone as an electron acceptor by Fe-S d-iLDH, and it helps the enzyme associate with the cell membrane. These functions make the Fe-S oxidoreductase domain crucial for the in vivo d-lactate utilization function of Fe-S d-iLDH. IMPORTANCE Lactate metabolism plays versatile roles in most domains of life. Lactate utilization processes depend on certain enzymes to oxidize lactate to pyruvate. In recent years, novel bacterial lactate-oxidizing enzymes have been

  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 Central

    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

    2015-01-01

    Pancreatic β cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca2+ action potentials due to the activation of voltage-dependent Ca2+ channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca2+ release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic β cells. NAADP-regulated Ca2+ release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca2+ from the endolysosomal system, resulting in localized Ca2+ signals. We show here that NAADP-mediated Ca2+ release from endolysosomal Ca2+ 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 Ca2+ release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca2+ signals, and insulin secretion. Our findings implicate NAADP-evoked Ca2+ release from acidic Ca2+ storage organelles in stimulus-secretion coupling in β cells. PMID:26152717

  8. Ultrafast photo-initiated molecular quantum dynamics in the DNA dinucleotide d(ApG) revealed by broadband transient absorption spectroscopy.

    PubMed

    Stuhldreier, Mayra C; Temps, Friedrich

    2013-01-01

    The ultrafast photo-initiated quantum dynamics of the adenine-guanine dinucleotide d(ApG) in aqueous solution (pH 7) has been studied by femtosecond time-resolved spectroscopy after excitation at lambda = 260 nm. The results reveal a hierarchy of processes on time scales from tau < 100 fs to tau > 100 ps. Characteristic spectro-temporal signatures are observed indicating the transformation of the molecules in the electronic relaxation from the photo-excited state to a long-lived exciplex. In particular, broadband UV/VIS excited-state absorption (ESA) measurements detected a distinctive absorption by the excited dinucleotide around lambda = 335 nm, approximately 0.5 eV to the blue compared to the maximum of the broad and unstructured ESA spectrum after excitation of an equimolar mixture of the mononucleotides dAMP and dGMP. A similar feature has been identified as signature of the excimer in the dynamics of the adenine dinucleotide d(ApA). The lifetime of the d(ApG) exciplex was found to be tau = 124 +/- 4 ps both from the ESA decay time and from the ground-state recovery time, far longer than the sub-picosecond lifetimes of excited dAMP or dGMP. Fluorescence-time profiles measured by the up-conversion technique indicate that the exciplex state is reached around approximately 6 ps after excitation. Very weak residual fluorescence at longer times red-shifted to the emission from the photo-excited state shows that the exciplex is almost optically dark, but still has enough oscillator strength to give rise to the dual fluorescence of the dinucleotide in the static fluorescence spectrum.

  9. Effects of Nicotinamide Adenine Dinucleotide (NAD(+)) and Diadenosine Tetraphosphate (Ap4A) on Electrical Activity of Working and Pacemaker Atrial Myocardium in Guinea Pigs.

    PubMed

    Pustovit, K B; Abramochkin, D V

    2016-04-01

    Effects of nucleotide polyphosphate compounds (nicotinamide adenine dinucleotide, NAD(+); diadenosine tetraphosphate, Ap4A) on the confi guration of action potentials were studied in isolated preparations of guinea pig sinoatrial node and right atrial appendage (auricle). In the working myocardium, NAD(+) and Ap4A in concentrations of 10(-5) and 10(-4) M had no effect on resting potential, but significantly reduced the duration of action potentials; the most pronounced decrease was found at 25% repolarization. In the primary pacemaker of the sinoatrial node, both concentrations of NAD(+) and Ap4A induced hyperpolarization and reduction in the rate of slow diastolic depolarization, but significant slowing of the sinus rhythm was produced by these substances only in the concentration of 10(-4) M. Moreover, AP shortening and marked acceleration of AP upstroke were observed in the pacemaker myocardium after application of polyphosphates. Comparative analysis of the effects of NAD(+) and Ap4A in the working and pacemaker myocardium drove us to a hypothesis on inhibitory effects of these substances on L-type calcium current accompanied by stimulation of one or several potassium currents, which induce enhancement of repolarization and hyperpolarization of membranes probably mediated by the activation of purine receptors.

  10. Rho-kinase inhibitor and nicotinamide adenine dinucleotide phosphate oxidase inhibitor prevent impairment of endothelium-dependent cerebral vasodilation by acute cigarette smoking in rats.

    PubMed

    Iida, Hiroki; Iida, Mami; Takenaka, Motoyasu; Fukuoka, Naokazu; Dohi, Shuji

    2008-06-01

    We previously reported that acute cigarette smoking can cause a dysfunction of endothelium-dependent vasodilation in cerebral vessels, and that blocking the angiotensin II (Ang II) type 1 (AT1) receptor with valsartan prevented this impairment. Our aim was to investigate the effects of a Rho-kinase inhibitor (fasudil) and a Nicotinamide Adenine Dinucleotide PHosphate (NADPH) oxidase inhibitor (apocynin) on smoking-induced endothelial dysfunction in cerebral arterioles. In Sprague-Dawley rats, we used a closed cranial window preparation to measure changes in pial vessel diameters following topical acetylcholine (ACh) before smoking. After one-minute smoking, we again examined the arteriolar responses to ACh. Finally, after intravenous fasudil or apocynin pre-treatment we re-examined the vasodilator responses to topical ACh (before and after cigarette smoking). Under control conditions, cerebral arterioles were dose-dependently dilated by topical ACh (10(-6) M and 10(-5) M). One hour after a one-minute smoking (1 mg-nicotine cigarette), 10(-5) M ACh constricted cerebral arterioles. However, one hour after a one-minute smoking, 10(-5) M ACh dilated cerebral pial arteries both in the fasudil pre-treatment and the apocynin pre-treatment groups, responses that were significantly different from those obtained without fasudil or apocynin pre-treatment. Thus, inhibition of Rho-kinase and NADPH oxidase activities may prevent the above smoking-induced impairment of endothelium-dependent vasodilation.

  11. Nicotinic Acid Adenine Dinucleotide Phosphate Plays a Critical Role in Naive and Effector Murine T Cells but Not Natural Regulatory T Cells*

    PubMed Central

    Ali, Ramadan A.; Camick, Christina; Wiles, Katherine; Walseth, Timothy F.; Slama, James T.; Bhattacharya, Sumit; Giovannucci, David R.; Wall, Katherine A.

    2016-01-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca2+ mobilizing second messenger discovered to date, has been implicated in Ca2+ signaling in some lymphomas and T cell clones. In contrast, the role of NAADP in Ca2+ signaling or the identity of the Ca2+ stores targeted by NAADP in conventional naive T cells is less clear. In the current study, we demonstrate the importance of NAADP in the generation of Ca2+ signals in murine naive T cells. Combining live-cell imaging methods and a pharmacological approach using the NAADP antagonist Ned-19, we addressed the involvement of NAADP in the generation of Ca2+ signals evoked by TCR stimulation and the role of this signal in downstream physiological end points such as proliferation, cytokine production, and other responses to stimulation. We demonstrated that acidic compartments in addition to the endoplasmic reticulum were the Ca2+ stores that were sensitive to NAADP in naive T cells. NAADP was shown to evoke functionally relevant Ca2+ signals in both naive CD4 and naive CD8 T cells. Furthermore, we examined the role of this signal in the activation, proliferation, and secretion of effector cytokines by Th1, Th2, Th17, and CD8 effector T cells. Overall, NAADP exhibited a similar profile in mediating Ca2+ release in effector T cells as in their counterpart naive T cells and seemed to be equally important for the function of these different subsets of effector T cells. This profile was not observed for natural T regulatory cells. PMID:26728458

  12. Uptake and Metabolism of Antibiotics Roseoflavin and 8-Demethyl-8-Aminoriboflavin in Riboflavin-Auxotrophic Listeria monocytogenes.

    PubMed

    Matern, Andreas; Pedrolli, Danielle; Großhennig, Stephanie; Johansson, Jörgen; Mack, Matthias

    2016-12-01

    The riboflavin analogs roseoflavin (RoF) and 8-demethyl-8-aminoriboflavin (AF) are produced by the bacteria Streptomyces davawensis and Streptomyces cinnabarinus Riboflavin analogs have the potential to be used as broad-spectrum antibiotics, and we therefore studied the metabolism of riboflavin (vitamin B 2 ), RoF, and AF in the human pathogen Listeria monocytogenes, a bacterium which is a riboflavin auxotroph. We show that the L. monocytogenes protein Lmo1945 is responsible for the uptake of riboflavin, RoF, and AF. Following import, these flavins are phosphorylated/adenylylated by the bifunctional flavokinase/flavin adenine dinucleotide (FAD) synthetase Lmo1329 and adenylylated by the unique FAD synthetase Lmo0728, the first monofunctional FAD synthetase to be described in bacteria. Lmo1329 generates the cofactors flavin mononucleotide (FMN) and FAD, whereas Lmo0728 produces FAD only. The combined activities of Lmo1329 and Lmo0728 are responsible for the intracellular formation of the toxic cofactor analogs roseoflavin mononucleotide (RoFMN), roseoflavin adenine dinucleotide (RoFAD), 8-demethyl-8-aminoriboflavin mononucleotide (AFMN), and 8-demethyl-8-aminoriboflavin adenine dinucleotide (AFAD). In vivo reporter gene assays and in vitro transcription/translation experiments show that the L. monocytogenes FMN riboswitch Rli96, which controls expression of the riboflavin transport gene lmo1945, is negatively affected by riboflavin/FMN and RoF/RoFMN but not by AF/AFMN. Treatment of L. monocytogenes with RoF or AF leads to drastically reduced FMN/FAD levels. We suggest that the reduced flavin cofactor levels in combination with concomitant synthesis of inactive cofactor analogs (RoFMN, RoFAD, AFMN, and AFAD) explain why RoF and AF contribute to antibiotic activity in L. monocytogenes IMPORTANCE: The riboflavin analogs roseoflavin (RoF) and 8-demethyl-8-aminoriboflavin (AF) are small molecules which are produced by Streptomyces davawensis and Streptomyces cinnabarinus

  13. Tissue-specific regulation of sirtuin and nicotinamide adenine dinucleotide biosynthetic pathways identified in C57Bl/6 mice in response to high-fat feeding.

    PubMed

    Drew, Janice E; Farquharson, Andrew J; Horgan, Graham W; Williams, Lynda M

    2016-11-01

    The sirtuin (SIRT)/nicotinamide adenine dinucleotide (NAD) system is implicated in development of type 2 diabetes (T2D) and diet-induced obesity, a major risk factor for T2D. Mechanistic links have not yet been defined. SIRT/NAD system gene expression and NAD/NADH levels were measured in liver, white adipose tissue (WAT) and skeletal muscle from mice fed either a low-fat diet or high-fat diet (HFD) for 3 days up to 16 weeks. An in-house custom-designed multiplex gene expression assay assessed all 7 mouse SIRTs (SIRT1-7) and 16 enzymes involved in conversion of tryptophan, niacin, nicotinamide riboside and metabolic precursors to NAD. Significantly altered transcription was correlated with body weight, fat mass, plasma lipids and hormones. Regulation of the SIRT/NAD system was associated with early (SIRT4, SIRT7, NAPRT1 and NMNAT2) and late phases (NMNAT3, NMRK2, ABCA1 and CD38) of glucose intolerance. TDO2 and NNMT were identified as markers of HFD consumption. Altered regulation of the SIRT/NAD system in response to HFD was prominent in liver compared with WAT or muscle. Multiple components of the SIRTs and NAD biosynthetic enzymes network respond to consumption of dietary fat. Novel molecular targets identified above could direct strategies for dietary/therapeutic interventions to limit metabolic dysfunction and development of T2D. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2015-01-01

    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. PMID:25813057

  15. Nicotinic Acid Adenine Dinucleotide Phosphate Plays a Critical Role in Naive and Effector Murine T Cells but Not Natural Regulatory T Cells.

    PubMed

    Ali, Ramadan A; Camick, Christina; Wiles, Katherine; Walseth, Timothy F; Slama, James T; Bhattacharya, Sumit; Giovannucci, David R; Wall, Katherine A

    2016-02-26

    Nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca(2+) mobilizing second messenger discovered to date, has been implicated in Ca(2+) signaling in some lymphomas and T cell clones. In contrast, the role of NAADP in Ca(2+) signaling or the identity of the Ca(2+) stores targeted by NAADP in conventional naive T cells is less clear. In the current study, we demonstrate the importance of NAADP in the generation of Ca(2+) signals in murine naive T cells. Combining live-cell imaging methods and a pharmacological approach using the NAADP antagonist Ned-19, we addressed the involvement of NAADP in the generation of Ca(2+) signals evoked by TCR stimulation and the role of this signal in downstream physiological end points such as proliferation, cytokine production, and other responses to stimulation. We demonstrated that acidic compartments in addition to the endoplasmic reticulum were the Ca(2+) stores that were sensitive to NAADP in naive T cells. NAADP was shown to evoke functionally relevant Ca(2+) signals in both naive CD4 and naive CD8 T cells. Furthermore, we examined the role of this signal in the activation, proliferation, and secretion of effector cytokines by Th1, Th2, Th17, and CD8 effector T cells. Overall, NAADP exhibited a similar profile in mediating Ca(2+) release in effector T cells as in their counterpart naive T cells and seemed to be equally important for the function of these different subsets of effector T cells. This profile was not observed for natural T regulatory cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Molecular simulation to investigate the cofactor specificity for pichia stipitis Xylose reductase.

    PubMed

    Xia, Xiao-Le; Cong, Shan; Weng, Xiao-Rong; Chen, Jin-Hua; Wang, Jing-Fang; Chou, Kuo-Chen

    2013-11-01

    Xylose is one of the most abundant carbohydrates in nature, and widely used to produce bioethanol via fermentation in industry. Xylulose can produce two key enzymes: xylose reductase and xylitol dehydrogenase. Owing to the disparate cofactor specificities of xylose reductase and xylitol dehydrogenase, intracellular redox imbalance is detected during the xylose fermentation, resulting in low ethanol yields. To overcome this barrier, a common strategy is applied to artificially modify the cofactor specificity of xylose reductase. In this study, we utilized molecular simulation approaches to construct a 3D (three-dimensional) structural model for the NADP-dependent Pichia stipitis xylose reductase (PsXR). Based on the 3D model, the favourable binding modes for both cofactors NAD and NADP were obtained using the flexible docking procedure and molecular dynamics simulation. Structural analysis of the favourable binding modes showed that the cofactor binding site of PsXR was composed of 3 major components: a hydrophilic pocket, a hydrophobic pocket as well as a linker channel between the aforementioned two pockets. The hydrophilic pocket could recognize the nicotinamide moiety of the cofactors by hydrogen bonding networks, while the hydrophobic pocket functioned to position the adenine moiety of the cofactors by hydrophobic and Π-Π stacking interactions. The linker channel contained some key residues for ligand-binding; their mutation could have impact to the specificity of PsXR. Finally, it was found that any of the two single mutations, K21A and K270N, might reverse the cofactor specificity of PsXR from major NADP- to NADdependent, which was further confirmed by the additional experiments. Our findings may provide useful insights into the cofactor specificity of PsXR, stimulating new strategies for better designing xylose reductase and improving ethanol production in industry.

  17. The distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) in the medulla oblongata, spinal cord, cranial and spinal nerves of frog, Microhyla ornata.

    PubMed

    Jadhao, Arun G; Biswas, Saikat P; Bhoyar, Rahul C; Pinelli, Claudia

    2017-04-01

    Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) enzymatic activity has been reported in few amphibian species. In this study, we report its unusual localization in the medulla oblongata, spinal cord, cranial nerves, spinal nerves, and ganglions of the frog, Microhyla ornata. In the rhombencephalon, at the level of facial and vagus nerves, the NADPH-d labeling was noted in the nucleus of the abducent and facial nerves, dorsal nucleus of the vestibulocochlear nerve, the nucleus of hypoglossus nerve, dorsal and lateral column nucleus, the nucleus of the solitary tract, the dorsal field of spinal grey, the lateral and medial motor fields of spinal grey and radix ventralis and dorsalis (2-10). Many ependymal cells around the lining of the fourth ventricle, both facial and vagus nerves and dorsal root ganglion, were intensely labeled with NADPH-d. Most strikingly the NADPH-d activity was seen in small and large sized motoneurons in both medial and lateral motor neuron columns on the right and left sides of the brain. This is the largest stained group observed from the caudal rhombencephalon up to the level of radix dorsalis 10 in the spinal cord. The neurons were either oval or elongated in shape with long processes and showed significant variation in the nuclear and cellular diameter. A massive NADPH-d activity in the medulla oblongata, spinal cord, and spinal nerves implied an important role of this enzyme in the neuronal signaling as well as in the modulation of motor functions in the peripheral nervous systems of the amphibians. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Effective immobilization of alcohol dehydrogenase on carbon nanoscaffolds for ethanol biofuel cell.

    PubMed

    Umasankar, Yogeswaran; Adhikari, Bal-Ram; Chen, Aicheng

    2017-12-01

    An efficient approach for immobilizing alcohol dehydrogenase (ADH) while enhancing its electron transfer ability has been developed using poly(2-(trimethylamino)ethyl methacrylate) (MADQUAT) cationic polymer and carbon nanoscaffolds. The carbon nanoscaffolds were comprised of single-walled carbon nanotubes (SWCNTs) wrapped with reduced graphene oxide (rGO). The ADH entrapped within the MADQUAT that was present on the carbon nanoscaffolds exhibited a high electron exchange capability with the electrode through its cofactor β-nicotinamide adenine dinucleotide hydrate and β-nicotinamide adenine dinucleotide reduced disodium salt hydrate (NAD + /NADH) redox reaction. The advantages of the carbon nanoscaffolds used as the support matrix and the MADQUAT employed for the entrapment of ADH versus physisorption were demonstrated via cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Our experimental results showed a higher electron transfer, electrocatalytic activity, and rate constant for MADQUAT entrapped ADH on the carbon nanoscaffolds. The immobilization of ADH using both MADQUAT and carbon nanoscaffolds exhibited strong potential for the development of an efficient bio-anode for ethanol powered biofuel cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Time-gated FLIM microscope for corneal metabolic imaging

    NASA Astrophysics Data System (ADS)

    Silva, Susana F.; Batista, Ana; Domingues, José Paulo; Quadrado, Maria João.; Morgado, António Miguel

    2016-03-01

    Detecting corneal cells metabolic alterations may prove a valuable tool in the early diagnosis of corneal diseases. Nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are autofluorescent metabolic co-factors that allow the assessment of metabolic changes through non-invasive optical methods. These co-factors exhibit double-exponential fluorescence decays, with well-separated short and lifetime components, which are related to their protein-bound and free-states. Corneal metabolism can be assessed by measuring the relative contributions of these two components. For that purpose, we have developed a wide-field time-gated fluorescence lifetime microscope based on structured illumination and one-photon excitation to record FAD lifetime images from corneas. NADH imaging was not considered as its UV excitation peak is regarded as not safe for in vivo measurements. The microscope relies on a pulsed blue diode laser (λ=443 nm) as excitation source, an ultra-high speed gated image intensifier coupled to a CCD camera to acquire fluorescence signals and a Digital Micromirror Device (DMD) to implement the Structured Illumination technique. The system has a lateral resolution better than 2.4 μm, a field of view of 160 per 120 μm and an optical sectioning of 6.91 +/- 0.45 μm when used with a 40x, 0.75 NA, Water Immersion Objective. With this setup we were able to measure FAD contributions from ex-vivo chicken corneas collected from a local slaughterhouse..

  20. The CoFactor database: organic cofactors in enzyme catalysis.

    PubMed

    Fischer, Julia D; Holliday, Gemma L; Thornton, Janet M

    2010-10-01

    Organic enzyme cofactors are involved in many enzyme reactions. Therefore, the analysis of cofactors is crucial to gain a better understanding of enzyme catalysis. To aid this, we have created the CoFactor database. CoFactor provides a web interface to access hand-curated data extracted from the literature on organic enzyme cofactors in biocatalysis, as well as automatically collected information. CoFactor includes information on the conformational and solvent accessibility variation of the enzyme-bound cofactors, as well as mechanistic and structural information about the hosting enzymes. The database is publicly available and can be accessed at http://www.ebi.ac.uk/thornton-srv/databases/CoFactor.

  1. Regulation of the Biosynthesis of Amino Acids of the Aspartate Family in Coliform Bacteria and Pseudomonads

    PubMed Central

    Cohen, G. N.; Stanier, R. Y.; Bras, Gisele Le

    1969-01-01

    The control of aspartokinase and homoserine dehydrogenase activities was compared in aerobic and fermentative pseudomonads (genera Pseudomonas and Aeromonas), and in coliform bacteria representative of the principal genera of the Enterobacteriaceae. Isofunctional aspartokinases subject to independent end-product control occur in the Enterobacteriaceae and in Aeromonas. In Pseudomonas, there appears to be a single aspartokinase, subject to concerted feedback inhibition by lysine and threonine. Within this genus, the sensitivity of aspartokinase to the single allosteric inhibitors varies considerably: the aspartokinase of the acidovorans group is little affected by the single inhibitors, whereas that of the fluorescent group is severely inhibited by either amino acid at high concentration. In all bacteria examined, homoserine dehydrogenase activity is inhibited by threonine; inhibition is more severe in aerobic pseudomonads than in the other groups. In most of the bacteria examined, either nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate can serve as a cofactor for this enzyme, though the relative activity with the two pyridine nucleotides varies widely. Aerobic pseudomonads of the acidovorans group contain a homoserine dehydrogenase that is absolutely specific for NAD. The taxonomic implications of these findings are discussed. PMID:4391829

  2. Absorption and emission spectroscopic characterization of BLUF protein Slr1694 from Synechocystis sp. PCC6803 with roseoflavin cofactor.

    PubMed

    Zirak, P; Penzkofer, A; Mathes, T; Hegemann, P

    2009-11-09

    The wild-type BLUF protein Slr1694 from Synechocystis sp. PCC6803 (BLUF=blue-light sensor using FAD) has flavin adenosine dinucleotide (FAD) as natural cofactor. This light sensor causes positive phototaxis of the marine cyanobacterium. In this study the FAD cofactor of the wild-type Slr1694 was replaced by roseoflavin (RoF) and the roseoflavin derivatives RoFMN and RoFAD during heterologous expression in a riboflavin auxotrophic E. coli strain. An absorption and emission spectroscopic characterization of the cofactor-exchanged-Slr1694 (RoSlr) was carried out both under dark conditions and under illuminated conditions. The behaviour of RoF embedded in RoSlr in aqueous solution at pH 8 is compared with the behaviour of RoF in aqueous solution. The fluorescence of RoF and RoSlr is quenched by photo-induced twisted intra-molecular charge transfer at room temperature with stronger effect for RoF. The fluorescence quenching is diminished at liquid nitrogen temperature. Light exposure of RoSlr causes irreversible conversion of the protein embedded roseoflavins to 8-methylamino-flavins, 8-dimethylamino-lumichrome and 8-methylamino-lumichrome.

  3. Increased rate of adenine incorporation into adenine nucleotide pool in erythrocytes of patients with chronic renal failure.

    PubMed

    Marlewski, M; Smolenski, R T; Szolkiewicz, M; Aleksandrowicz, Z; Rutkowski, B; Swierczynski, J

    2000-11-01

    Elevated purine nucleotide pool (mainly ATP) in erythrocytes of patients with chronic renal failure (CRF) is a known phenomenon, however the mechanism responsible for this abnormality is far from being clear. We hypothesize that the increased rate of adenine incorporation into adenine nucleotide pool is responsible for the elevated level of ATP in uremic erythrocytes. In chronically uremic patients we evaluated using HPLC technique: (a) plasma adenine concentration; (b) the rate of adenine incorporation into adenine nucleotide pool in uremic erythrocytes. Additionally, the effect of higher than physiological phosphate concentration (2.4 mM) and lower than physiological pH (7.1) on adenine incorporation into erythrocytes adenine nucleotide pool was investigated. Healthy volunteers with normal renal function served as control. The concentration of adenine in plasma of CRF patients was found to be significantly higher than in plasma of healthy subjects. In contrast, adenosine concentration was similar both in healthy humans and in CRF patients. In isolated erythrocytes of uremic patients (incubated in the medium pH 7.4, containing 1.2 mM inorganic phosphate) adenine was incorporated into adenine nucleotide pool at a rate approximately 2-fold higher than in erythrocytes from healthy subjects. The rate of adenosine incorporation into adenine nucleotide pool was similar in erythrocytes of both studied groups. Incubation of erythrocytes obtained from healthy subjects in the medium pH 7.4, containing 2.4 mM inorganic phosphate, caused the increase of adenine incorporation into adenine nucleotide pool by about 60%. Incubation of the cells in the pH 7.1 buffer containing 2. 4 mM inorganic phosphate increased the rate of adenine incorporation into adenylate approximately 2-fold as compared to erythrocytes incubated in the medium pH 7.4 containing 1.2 mM inorganic phosphate. Erythrocytes obtained from uremic patients and incubated in the pH 7.1 medium containing 2.4 m

  4. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Baig, M.; Brown, A.; Eswaramoorthy, S.

    Klebsiella pneumoniae, a gram-negative enteric bacterium, is found in nosocomial infections which are acquired during hospital stays for about 10% of hospital patients in the United States. The crystal structure of a putative oxidoreductase from K. pneumoniae has been determined. The structural information of this K. pneumoniae protein was used to understand its function. Crystals of the putative oxidoreductase enzyme were obtained by the sitting drop vapor diffusion method using Polyethylene glycol (PEG) 3350, Bis-Tris buffer, pH 5.5 as precipitant. These crystals were used to collect X-ray data at beam line X12C of the National Synchrotron Light Source (NSLS) atmore » Brookhaven National Laboratory (BNL). The crystal structure was determined using the SHELX program and refi ned with CNS 1.1. This protein, which is involved in the catalysis of an oxidation-reduction (redox) reaction, has an alpha/beta structure. It utilizes nicotinamide adenine dinucleotide phosphate (NADP) or nicotine adenine dinucleotide (NAD) to perform its function. This structure could be used to determine the active and co-factor binding sites of the protein, information that could help pharmaceutical companies in drug design and in determining the protein’s relationship to disease treatment such as that for pneumonia and other related pathologies.« less

  5. A Single Primary Blast-Induced Traumatic Brain Injury in a Rodent Model Causes Cell-Type Dependent Increase in Nicotinamide Adenine Dinucleotide Phosphate Oxidase Isoforms in Vulnerable Brain Regions.

    PubMed

    Rama Rao, Kakulavarapu V; Iring, Stephanie; Younger, Daniel; Kuriakose, Matthew; Skotak, Maciej; Alay, Eren; Gupta, Raj K; Chandra, Namas

    2018-06-12

    Blast-induced traumatic brain injury (bTBI) is a leading cause of morbidity in soldiers on the battlefield and in training sites with long-term neurological and psychological pathologies. Previous studies from our laboratory demonstrated activation of oxidative stress pathways after blast injury, but their distribution among different brain regions and their impact on the pathogenesis of bTBI have not been explored. The present study examined the protein expression of two isoforms: nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 and 2 (NOX1, NOX2), corresponding superoxide production, a downstream event of NOX activation, and the extent of lipid peroxidation adducts of 4-hydroxynonenal (4HNE) to a range of proteins. Brain injury was evaluated 4 h after the shock-wave exposure, and immunofluorescence signal quantification was performed in different brain regions. Expression of NOX isoforms displayed a differential increase in various brain regions: in hippocampus and thalamus, there was the highest increase of NOX1, whereas in the frontal cortex, there was the highest increase of NOX2 expression. Cell-specific analysis of changes in NOX expression with respect to corresponding controls revealed that blast resulted in a higher increase of NOX1 and NOX 2 levels in neurons compared with astrocytes and microglia. Blast exposure also resulted in increased superoxide levels in different brain regions, and such changes were reflected in 4HNE protein adduct formation. Collectively, this study demonstrates that primary blast TBI induces upregulation of NADPH oxidase isoforms in different regions of the brain parenchyma and that neurons appear to be at higher risk for oxidative damage compared with other neural cells.

  6. Molecular and Biochemical Characterization of a Cytokinin Oxidase from Maize1

    PubMed Central

    Bilyeu, Kristin D.; Cole, Jean L.; Laskey, James G.; Riekhof, Wayne R.; Esparza, Thomas J.; Kramer, Michelle D.; Morris, Roy O.

    2001-01-01

    It is generally accepted that cytokinin oxidases, which oxidatively remove cytokinin side chains to produce adenine and the corresponding isopentenyl aldehyde, play a major role in regulating cytokinin levels in planta. Partially purified fractions of cytokinin oxidase from various species have been studied for many years, but have yet to clearly reveal the properties of the enzyme or to define its biological significance. Details of the genomic organization of the recently isolated maize (Zea mays) cytokinin oxidase gene (ckx1) and some of its Arabidopsis homologs are now presented. Expression of an intronless ckx1 in Pichia pastoris allowed production of large amounts of recombinant cytokinin oxidase and facilitated detailed kinetic and cofactor analysis and comparison with the native enzyme. The enzyme is a flavoprotein containing covalently bound flavin adenine dinucleotide, but no detectable heavy metals. Expression of the oxidase in maize tissues is described. PMID:11154345

  7. Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Activates Global and Heterogeneous Local Ca2+ Signals from NAADP- and Ryanodine Receptor-gated Ca2+ Stores in Pulmonary Arterial Myocytes*

    PubMed Central

    Jiang, Yong-Liang; Lin, Amanda H. Y.; Xia, Yang; Lee, Suengwon; Paudel, Omkar; Sun, Hui; Yang, Xiao-Ru; Ran, Pixin; Sham, James S. K.

    2013-01-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+-mobilizing messenger that releases Ca2+ from endolysosomal organelles. Recent studies showed that NAADP-induced Ca2+ release is mediated by the two-pore channels (TPCs) TPC1 and TPC2. However, the expression of TPCs and the NAADP-induced local Ca2+ signals have not been examined in vascular smooth muscle. Here, we found that both TPC1 and TPC2 are expressed in rat pulmonary arterial smooth muscle cells (PASMCs), with TPC1 being the major subtype. Application of membrane-permeant NAADP acetoxymethyl ester to PASMCs elicited a biphasic increase in global [Ca2+]i, which was independent of extracellular Ca2+ and blocked by the NAADP antagonist Ned-19 or the vacuolar H+-ATPase inhibitor bafilomycin A1, indicating Ca2+ release from acidic endolysosomal Ca2+ stores. The Ca2+ response was unaffected by xestospongin C but was partially blocked by ryanodine or thapsigargin. NAADP triggered heterogeneous local Ca2+ signals, including a diffuse increase in cytosolic [Ca2+], Ca2+ sparks, Ca2+ bursts, and regenerative Ca2+ release. The diffuse Ca2+ increase and Ca2+ bursts were ryanodine-insensitive, presumably arising from different endolysosomal sources. Ca2+ sparks and regenerative Ca2+ release were inhibited by ryanodine, consistent with cross-activation of loosely coupled ryanodine receptors. Moreover, Ca2+ release stimulated by endothelin-1 was inhibited by Ned-19, ryanodine, or xestospongin C, suggesting that NAADP-mediated Ca2+ signals interact with both ryanodine and inositol 1,4,5-trisphosphate receptors during agonist stimulation. Our results show that NAADP mediates complex global and local Ca2+ signals. Depending on the physiological stimuli, these diverse Ca2+ signals may serve to regulate different cellular functions in PASMCs. PMID:23443655

  8. Biofuel cell anode: NAD +/glucose dehydrogenase-coimmobilized ketjenblack electrode

    NASA Astrophysics Data System (ADS)

    Miyake, T.; Oike, M.; Yoshino, S.; Yatagawa, Y.; Haneda, K.; Kaji, H.; Nishizawa, M.

    2009-09-01

    We have studied the coimmobilization of glucose dehydrogenase (GDH) and its cofactor, oxidized nicotinamide adenine dinucleotide (NAD +), on a ketjenblack (KB) electrode as a step toward a biofuel cell anode that works without mediators. A KB electrode was first treated with a sulfuric acid/nitric acid/water mixture to lower the overvoltage for NADH oxidation, and was next chemically modified with NAD + and GDH. The improved GDH/NAD +/KB electrode is found to oxidize glucose around 0 V vs. Ag/AgCl. A biofuel cell constructed with a bilirubin oxidase-immobilized KB cathode showed a maximum power density of 52 μW/cm 2 at 0.3 V.

  9. Binding of Substrate Locks the Electrochemistry of CRY-DASH into DNA Repair.

    PubMed

    Gindt, Yvonne M; Messyasz, Adriana; Jumbo, Pamela I

    2015-05-12

    VcCry1, a member of the CRY-DASH family, may serve two diverse roles in vivo, including blue-light signaling and repair of UV-damaged DNA. We have discovered that the electrochemistry of the flavin adenine dinucleotide cofactor of VcCry1 is locked to cycle only between the hydroquinone and neutral semiquinone states when UV-damaged DNA is present. Other potential substrates, including undamaged DNA and ATP, have no discernible effect on the electrochemistry, and the kinetics of the reduction is unaffected by damaged DNA. Binding of the damaged DNA substrate determines the role of the protein and prevents the presumed photochemistry required for blue-light signaling.

  10. 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.

  11. 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 Central

    Lorenz, Nicole; Olšovská, Jana; Šulc, Miroslav; Tudzynski, Paul

    2010-01-01

    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

  12. Adenine specific DNA chemical sequencing reaction.

    PubMed Central

    Iverson, B L; Dervan, P B

    1987-01-01

    Reaction of DNA with K2PdCl4 at pH 2.0 followed by a piperidine workup produces specific cleavage at adenine (A) residues. Product analysis revealed the K2PdCl4 reaction involves selective depurination at adenine, affording an excision reaction analogous to the other chemical DNA sequencing reactions. Adenine residues methylated at the exocyclic amine (N6) react with lower efficiency than unmethylated adenine in an identical sequence. This simple protocol specific for A may be a useful addition to current chemical sequencing reactions. Images PMID:3671067

  13. Enhancement of ethanol-oxygen biofuel cell output using a CNT based nano-composite as bioanode.

    PubMed

    Gouranlou, Farideh; Ghourchian, Hedayatollah

    2016-04-15

    The present research, describes preparation and application of a novel bioanode for ethanol-oxygen biofuel cells. We applied an enzyme based nanocomposite consisting of polymethylene green as electron transfer mediator, carboxylated-multiwall carbon nanotubes as electron transfer accelerator, alcohol dehydrogenase as biocatalyst and polydiallyldimethylammonium chloride as supporting agent. In the presence of β-nicotinamide adenine dinucleotide as cofactor, and ethanol as fuel, the feasibility of the bioanode for increasing the power was evaluated under the ambient conditions. In the optimum conditions the biofuel cell produced the power density of 1.713 mW cm(-2) and open circuit voltage of 0.281 V. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Two continuous coupled assays for ornithine-δ-aminotransferase.

    PubMed

    Juncosa, Jose I; Lee, Hyunbeom; Silverman, Richard B

    2013-09-15

    We have developed two new continuous coupled assays for ornithine-δ-aminotransferase (OAT) that are more sensitive than previous methods, measure activity in real time, and can be carried out in multiwell plates for convenience and high throughput. The first assay is based on the reduction of Δ(1)-pyrroline-5-carboxylate (P5C), generated from ornithine by OAT, using human pyrroline 5-carboxylate reductase 1 (PYCR1), which results in the concomitant oxidation of NADH (nicotinamide adenine dinucleotide, reduced form) to NAD⁺ (nicotinamide adenine dinucleotide, oxidized form). This procedure was found to be three times more sensitive than previous methods and is suitable for the study of small molecules as inhibitors or inactivators of OAT or as a method to determine OAT activity in unknown samples. The second method involves the detection of L-glutamate, produced during the regeneration of the cofactor pyridoxal 5'-phosphate (PLP) of OAT by an unamplified modification of the commercially available Amplex Red L-glutamate detection kit (Life Technologies). This assay is recommended for the determination of the substrate activity of small molecules against OAT; measuring the transformation of L-ornithine at high concentrations by this assay is complicated by the fact that it also acts as a substrate for the L-glutamate oxidase (GluOx) reporter enzyme. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Supramolecular polymer formation by cyclic dinucleotides and intercalators affects dinucleotide enzymatic processing

    PubMed Central

    Nakayama, Shizuka; Zhou, Jie; Zheng, Yue; Szmacinski, Henryk; Sintim, Herman O

    2016-01-01

    Background: Cyclic dinucleotides form supramolecular aggregates with intercalators, and this property could be utilized in nanotechnology and medicine. Methods & results: Atomic force microscopy and electrophoretic mobility shift assays were used to show that cyclic diguanylic acid (c-di-GMP) forms G-wires in the presence of intercalators. The average fluorescence lifetime of thiazole orange, when bound to c-di-GMP was greater than when bound to DNA G-quadruplexes or dsDNA. The stability of c-di-GMP supramolecular polymers is dependent on both the nature of the cation present and the intercalator. C-di-GMP or cyclic diadenylic acid/intercalator complexes are more resistant to cleavage by YybT, a phosphodiesterase, than the uncomplexed nucleotides. Conclusion: Cleavage of bacterial cyclic dinucleotides could be slowed down via complexation with small molecules and that this could be utilized for diverse applications in nanotechnology and medicine. PMID:28031943

  16. Cyclic Dinucleotides in Oral Bacteria and in Oral Biofilms.

    PubMed

    Gürsoy, Ulvi K; Gürsoy, Mervi; Könönen, Eija; Sintim, Herman O

    2017-01-01

    Oral cavity acts as a reservoir of bacterial pathogens for systemic infections and several oral microorganisms have been linked to systemic diseases. Quorum sensing and cyclic dinucleotides, two "decision-making" signaling systems, communicate to regulate physiological process in bacteria. Discovery of cyclic dinucleotides has a long history, but the progress in our understanding of how cyclic dinucleotides regulate bacterial lifestyle is relatively new. Oral microorganisms form some of the most intricate biofilms, yet c-di-GMP, and c-di-AMP signaling have been rarely studied in oral biofilms. Recent studies demonstrated that, with the aid of bacterial messenger molecules and their analogs, it is possible to activate host innate and adaptive immune responses and epithelial integrity with a dose that is relevant to inhibit bacterial virulence mechanisms, such as fimbriae and exopolysaccharide production, biofilm formation, and host cell invasion. The aim of this perspective article is to present available information on cyclic dinucleotides in oral bacteria and in oral biofilms. Moreover, technologies that can be used to detect cyclic dinucleotides in oral biofilms are described. Finally, directions for future research are highlighted.

  17. Accessing non-natural reactivity by irradiating nicotinamide-dependent enzymes with light

    NASA Astrophysics Data System (ADS)

    Emmanuel, Megan A.; Greenberg, Norman R.; Oblinsky, Daniel G.; Hyster, Todd K.

    2016-12-01

    Enzymes are ideal for use in asymmetric catalysis by the chemical industry, because their chemical compositions can be tailored to a specific substrate and selectivity pattern while providing efficiencies and selectivities that surpass those of classical synthetic methods. However, enzymes are limited to reactions that are found in nature and, as such, facilitate fewer types of transformation than do other forms of catalysis. Thus, a longstanding challenge in the field of biologically mediated catalysis has been to develop enzymes with new catalytic functions. Here we describe a method for achieving catalytic promiscuity that uses the photoexcited state of nicotinamide co-factors (molecules that assist enzyme-mediated catalysis). Under irradiation with visible light, the nicotinamide-dependent enzyme known as ketoreductase can be transformed from a carbonyl reductase into an initiator of radical species and a chiral source of hydrogen atoms. We demonstrate this new reactivity through a highly enantioselective radical dehalogenation of lactones—a challenging transformation for small-molecule catalysts. Mechanistic experiments support the theory that a radical species acts as an intermediate in this reaction, with NADH and NADPH (the reduced forms of nicotinamide adenine nucleotide and nicotinamide adenine dinucleotide phosphate, respectively) serving as both a photoreductant and the source of hydrogen atoms. To our knowledge, this method represents the first example of photo-induced enzyme promiscuity, and highlights the potential for accessing new reactivity from existing enzymes simply by using the excited states of common biological co-factors. This represents a departure from existing light-driven biocatalytic techniques, which are typically explored in the context of co-factor regeneration.

  18. Radioresistance of Adenine to Cosmic Rays.

    PubMed

    Vignoli Muniz, Gabriel S; Mejía, Christian F; Martinez, Rafael; Auge, Basile; Rothard, Hermann; Domaracka, Alicja; Boduch, Philippe

    2017-04-01

    The presence of nucleobases in carbonaceous meteorites on Earth is an indication of the existence of this class of molecules in outer space. However, space is permeated by ionizing radiation, which can have damaging effects on these molecules. Adenine is a purine nucleobase that amalgamates important biomolecules such as DNA, RNA, and ATP. Adenine has a unique importance in biochemistry and therefore life. The aim of this work was to study the effects of cosmic ray analogues on solid adenine and estimate its survival when exposed to corpuscular radiation. Adenine films were irradiated at GANIL (Caen, France) and GSI (Darmstadt, Germany) by 820 MeV Kr 33+ , 190 MeV Ca 10+ , 92 MeV Xe 23+ , and 12 MeV C 4+ ion beams at low temperature. The evolution of adenine molecules under heavy ion irradiation was studied by IR absorption spectroscopy as a function of projectile fluence. It was found that the adenine destruction cross section (σ d ) follows an electronic stopping power (S e ) power law under the form: CS e n ; C is a constant, and the exponential n is a dimensionless quantity. Using the equation above to fit our results, we determined σ d  = 4 × 10 -17 S e 1.17 , with S e in kiloelectronvolts per micrometer (keV μm -1 ). New IR absorption bands arise under irradiation of adenine and can be attributed to HCN, CN - , C 2 H 4 N 4 , CH 3 CN, and (CH 3 ) 3 CNC. These findings may help to understand the stability and chemistry related to complex organic molecules in space. The half-life of solid adenine exposed to the simulated interstellar medium cosmic ray flux was estimated as (10 ± 8) × 10 6 years. Key Words: Heavy ions-Infrared spectroscopy-Astrochemistry-Cosmic rays-Nucleobases-Adenine. Astrobiology 17, 298-308.

  19. Responses of Adenine Nucleotides in Germinating Soybean Embryonic Axes to Exogenously Applied Adenine and Adenosine

    PubMed Central

    Anderson, James D.

    1977-01-01

    The ATP content of soybean (Glycine max [L.] Merr. cv. Kent) axes incubated for 3 hours in 1 mm solutions of adenine and adenosine increased over 100% and 75%, respectively, over axes incubated in water. The increase in ATP was primarily due to the conversion of these purines to nucleotides via the nucleotide salvage pathway. The ATP formed was in a metabolically active pool because label from adenine was incorporated into acid-insoluble material. Adenine also increased the levels of GTP, UTP, and CTP, but not to the extent of the ATP level. PMID:16660165

  20. Radioresistance of Adenine to Cosmic Rays

    NASA Astrophysics Data System (ADS)

    Vignoli Muniz, Gabriel S.; Mejía, Christian F.; Martinez, Rafael; Auge, Basile; Rothard, Hermann; Domaracka, Alicja; Boduch, Philippe

    2017-04-01

    The presence of nucleobases in carbonaceous meteorites on Earth is an indication of the existence of this class of molecules in outer space. However, space is permeated by ionizing radiation, which can have damaging effects on these molecules. Adenine is a purine nucleobase that amalgamates important biomolecules such as DNA, RNA, and ATP. Adenine has a unique importance in biochemistry and therefore life. The aim of this work was to study the effects of cosmic ray analogues on solid adenine and estimate its survival when exposed to corpuscular radiation. Adenine films were irradiated at GANIL (Caen, France) and GSI (Darmstadt, Germany) by 820 MeV Kr33+, 190 MeV Ca10+, 92 MeV Xe23+, and 12 MeV C4+ ion beams at low temperature. The evolution of adenine molecules under heavy ion irradiation was studied by IR absorption spectroscopy as a function of projectile fluence. It was found that the adenine destruction cross section (σd) follows an electronic stopping power (Se) power law under the form: CSen; C is a constant, and the exponential n is a dimensionless quantity. Using the equation above to fit our results, we determined σd = 4 × 10-17 Se1.17, with Se in kiloelectronvolts per micrometer (keV μm-1). New IR absorption bands arise under irradiation of adenine and can be attributed to HCN, CN-, C2H4N4, CH3CN, and (CH3)3CNC. These findings may help to understand the stability and chemistry related to complex organic molecules in space. The half-life of solid adenine exposed to the simulated interstellar medium cosmic ray flux was estimated as (10 ± 8) × 106 years.

  1. Atomic substitution reveals the structural basis for substrate adenine recognition and removal by adenine DNA glycosylase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Seongmin; Verdine, Gregory L.; Harvard)

    2010-01-14

    Adenine DNA glycosylase catalyzes the glycolytic removal of adenine from the promutagenic A {center_dot} oxoG base pair in DNA. The general features of DNA recognition by an adenine DNA glycosylase, Bacillus stearothermophilus MutY, have previously been revealed via the X-ray structure of a catalytically inactive mutant protein bound to an A:oxoG-containing DNA duplex. Although the structure revealed the substrate adenine to be, as expected, extruded from the DNA helix and inserted into an extrahelical active site pocket on the enzyme, the substrate adenine engaged in no direct contacts with active site residues. This feature was paradoxical, because other glycosylases havemore » been observed to engage their substrates primarily through direct contacts. The lack of direct contacts in the case of MutY suggested that either MutY uses a distinctive logic for substrate recognition or that the X-ray structure had captured a noncatalytically competent state in lesion recognition. To gain further insight into this issue, we crystallized wild-type MutY bound to DNA containing a catalytically inactive analog of 2'-deoxyadenosine in which a single 2'-H atom was replaced by fluorine. The structure of this fluorinated lesion-recognition complex (FLRC) reveals the substrate adenine buried more deeply into the active site pocket than in the prior structure and now engaged in multiple direct hydrogen bonding and hydrophobic interactions. This structure appears to capture the catalytically competent state of adenine DNA glycosylases, and it suggests a catalytic mechanism for this class of enzymes, one in which general acid-catalyzed protonation of the nucleobase promotes glycosidic bond cleavage.« less

  2. The succinate dehydrogenase assembly factor, SdhE, is required for the flavinylation and activation of fumarate reductase in bacteria.

    PubMed

    McNeil, Matthew B; Hampton, Hannah G; Hards, Kiel J; Watson, Bridget N J; Cook, Gregory M; Fineran, Peter C

    2014-01-31

    The activity of the respiratory enzyme fumarate reductase (FRD) is dependent on the covalent attachment of the redox cofactor flavin adenine dinucleotide (FAD). We demonstrate that the FAD assembly factor SdhE, which flavinylates and activates the respiratory enzyme succinate dehydrogenase (SDH), is also required for the complete activation and flavinylation of FRD. SdhE interacted with, and flavinylated, the flavoprotein subunit FrdA, whilst mutations in a conserved RGxxE motif impaired the complete flavinylation and activation of FRD. These results are of widespread relevance because SDH and FRD play an important role in cellular energetics and are required for virulence in many important bacterial pathogens. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  3. Modulating NAD+ metabolism, from bench to bedside.

    PubMed

    Katsyuba, Elena; Auwerx, Johan

    2017-09-15

    Discovered in the beginning of the 20 th century, nicotinamide adenine dinucleotide (NAD + ) has evolved from a simple oxidoreductase cofactor to being an essential cosubstrate for a wide range of regulatory proteins that include the sirtuin family of NAD + -dependent protein deacylases, widely recognized regulators of metabolic function and longevity. Altered NAD + metabolism is associated with aging and many pathological conditions, such as metabolic diseases and disorders of the muscular and neuronal systems. Conversely, increased NAD + levels have shown to be beneficial in a broad spectrum of diseases. Here, we review the fundamental aspects of NAD + biochemistry and metabolism and discuss how boosting NAD + content can help ameliorate mitochondrial homeostasis and as such improve healthspan and lifespan. © 2017 The Authors.

  4. Cofactor specificity switch in Shikimate dehydrogenase by rational design and consensus engineering.

    PubMed

    García-Guevara, Fernando; Bravo, Iris; Martínez-Anaya, Claudia; Segovia, Lorenzo

    2017-08-01

    Consensus engineering has been used to design more stable variants using the most frequent amino acid at each site of a multiple sequence alignment; sometimes consensus engineering modifies function, but efforts have mainly been focused on studying stability. Here we constructed a consensus Rossmann domain for the Shikimate dehydrogenase enzyme; separately we decided to switch the cofactor specificity through rational design in the Escherichia coli Shikimate dehydrogenase enzyme and then analyzed the effect of consensus mutations on top of our design. We found that consensus mutations closest to the 2' adenine moiety increased the activity in our design. Consensus engineering has been shown to result in more stable proteins and our findings suggest it could also be used as a complementary tool for increasing or modifying enzyme activity during design. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  5. 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.

  6. The prokaryotic FAD synthetase family: a potential drug target.

    PubMed

    Serrano, Ana; Ferreira, Patricia; Martínez-Júlvez, Marta; Medina, Milagros

    2013-01-01

    Disruption of cellular production of the flavin cofactors, flavin adenine mononucleotide (FMN) and flavin adenine dinucleotide(FAD) will prevent the assembly of a large number of flavoproteins and flavoenzymes involved in key metabolic processes in all types of organisms. The enzymes responsible for FMN and FAD production in prokaryotes and eukaryotes exhibit various structural characteristics to catalyze the same chemistry, a fact that converts the prokaryotic FAD synthetase (FADS) in a potential drug target for the development of inhibitors endowed with anti-pathogenic activity. The first step before searching for selective inhibitors of FADS is to understand the structural and functional mechanisms for the riboflavin kinase and FMN adenylyltransferase activities of the prokaryotic enzyme, and particularly to identify their differential functional characteristics with regard to the enzymes performing similar functions in other organisms, particularly humans. In this paper, an overview of the current knowledge of the structure-function relationships in prokaryotic FADS has been presented, as well as of the state of the art in the use of these enzymes as drug targets.

  7. Engineering redox balance through cofactor systems.

    PubMed

    Chen, Xiulai; Li, Shubo; Liu, Liming

    2014-06-01

    Redox balance plays an important role in the production of enzymes, pharmaceuticals, and chemicals. To meet the demands of industrial production, it is desirable that microbes maintain a maximal carbon flux towards target metabolites with no fluctuations in redox. This requires functional cofactor systems that support dynamic homeostasis between different redox states or functional stability in a given redox state. Redox balance can be achieved by improving the self-balance of a cofactor system, regulating the substrate balance of a cofactor system, and engineering the synthetic balance of a cofactor system. This review summarizes how cofactor systems can be manipulated to improve redox balance in microbes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Probing conformational states of glutaryl-CoA dehydrogenase by fragment screening

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Begley, Darren W.; Davies, Douglas R.; Hartley, Robert C.

    Glutaric acidemia type 1 is an inherited metabolic disorder which can cause macrocephaly, muscular rigidity, spastic paralysis and other progressive movement disorders in humans. The defects in glutaryl-CoA dehydrogenase (GCDH) associated with this disease are thought to increase holoenzyme instability and reduce cofactor binding. Here, the first structural analysis of a GCDH enzyme in the absence of the cofactor flavin adenine dinucleotide (FAD) is reported. The apo structure of GCDH from Burkholderia pseudomallei reveals a loss of secondary structure and increased disorder in the FAD-binding pocket relative to the ternary complex of the highly homologous human GCDH. After conducting amore » fragment-based screen, four small molecules were identified which bind to GCDH from B. pseudomallei. Complex structures were determined for these fragments, which cause backbone and side-chain perturbations to key active-site residues. Structural insights from this investigation highlight differences from apo GCDH and the utility of small-molecular fragments as chemical probes for capturing alternative conformational states of preformed protein crystals.« less

  9. Cholesterol oxidase: ultrahigh-resolution crystal structure and multipolar atom model-based analysis.

    PubMed

    Zarychta, Bartosz; Lyubimov, Artem; Ahmed, Maqsood; Munshi, Parthapratim; Guillot, Benoît; Vrielink, Alice; Jelsch, Christian

    2015-04-01

    Examination of protein structure at the subatomic level is required to improve the understanding of enzymatic function. For this purpose, X-ray diffraction data have been collected at 100 K from cholesterol oxidase crystals using synchrotron radiation to an optical resolution of 0.94 Å. After refinement using the spherical atom model, nonmodelled bonding peaks were detected in the Fourier residual electron density on some of the individual bonds. Well defined bond density was observed in the peptide plane after averaging maps on the residues with the lowest thermal motion. The multipolar electron density of the protein-cofactor complex was modelled by transfer of the ELMAM2 charge-density database, and the topology of the intermolecular interactions between the protein and the flavin adenine dinucleotide (FAD) cofactor was subsequently investigated. Taking advantage of the high resolution of the structure, the stereochemistry of main-chain bond lengths and of C=O···H-N hydrogen bonds was analyzed with respect to the different secondary-structure elements.

  10. Small Cofactors May Assist Protein Emergence from RNA World: Clues from RNA-Protein Complexes

    PubMed Central

    Shen, Liang; Ji, Hong-Fang

    2011-01-01

    It is now widely accepted that at an early stage in the evolution of life an RNA world arose, in which RNAs both served as the genetic material and catalyzed diverse biochemical reactions. Then, proteins have gradually replaced RNAs because of their superior catalytic properties in catalysis over time. Therefore, it is important to investigate how primitive functional proteins emerged from RNA world, which can shed light on the evolutionary pathway of life from RNA world to the modern world. In this work, we proposed that the emergence of most primitive functional proteins are assisted by the early primitive nucleotide cofactors, while only a minority are induced directly by RNAs based on the analysis of RNA-protein complexes. Furthermore, the present findings have significant implication for exploring the composition of primitive RNA, i.e., adenine base as principal building blocks. PMID:21789260

  11. Multiple Animal Studies for Medical Chemical Defense Program in Soldier/ Patient Decontamination and Drug Development on Task Order 84-6: Pyruvate Dehydrogenase System for Determining the Effectiveness of Arsenic Antidotes

    DTIC Science & Technology

    1988-03-11

    adenine dinucleotide FAD = flavin-adenine dinucleotide iipS2 = lipoic acid lip(SH)2 = dihydrolipoic acid CoA = coenzyme A. SHepatic PDH complex activity...tissues has yet to be fully characterized, but it probably involves arsenic binding to the lipoic acid and dithiol moieties of the complex (Fluharty...covalently bound lipoic acid substrate of dihydrolipoyl transacetylase is greater per mole of L and CVAA than for sodium arsenite. This is possible

  12. Immobilization of flavin adenine dinucleotide (FAD) onto carbon cloth and its application as working electrode in an electroenzymatic bioreactor.

    PubMed

    Jayabalan, R; Sathishkumar, M; Jeong, E S; Mun, S P; Yun, S E

    2012-11-01

    A high porosity carbon cloth with immobilized FAD was employed as working electrode in electrochemical NADH-regeneration procedure. Carbon cloth was oxidized with hot acids to create surface carboxyl group and then coupled by adenine amino group of FAD with carbodiimide in the presence of N-hydroxysulfosuccinimide. The bioelectrocatalytic NADH-regeneration was coupled to the conversion of achiral substrate pyruvate into chiral product l-lactate by l-lactate dehydrogenase (l-LDH) within the same reactor. The conversion was completed at 96h in bioreactor with FAD-modified carbon cloth, resulting in about 6mM of l-lactate from 10mM of pyruvate. While with bare carbon cloth, the yield at 120h was around 5mM. Immobilized FAD on the surface of carbon cloth electrode facilitated it to carry electrons from electrode to electron transfer enzymes; thereby NADH-regeneration was accelerated to drive the enzymatic reaction efficiently. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Silver-induced reconstruction of an adeninate-based metal-organic framework for encapsulation of luminescent adenine-stabilized silver clusters.

    PubMed

    Jonckheere, Dries; Coutino-Gonzalez, Eduardo; Baekelant, Wouter; Bueken, Bart; Reinsch, Helge; Stassen, Ivo; Fenwick, Oliver; Richard, Fanny; Samorì, Paolo; Ameloot, Rob; Hofkens, Johan; Roeffaers, Maarten B J; De Vos, Dirk E

    2016-05-21

    Bright luminescent silver-adenine species were successfully stabilized in the pores of the MOF-69A (zinc biphenyldicarboxylate) metal-organic framework, starting from the intrinsically blue luminescent bio-MOF-1 (zinc adeninate 4,4'-biphenyldicarboxylate). Bio-MOF-1 is transformed to the MOF-69A framework by selectively leaching structural adenine linkers from the original framework using silver nitrate solutions in aqueous ethanol. Simultaneously, bright blue-green luminescent silver-adenine clusters are formed inside the pores of the recrystallized MOF-69A matrix in high local concentrations. The structural transition and concurrent changes in optical properties were characterized using a range of structural, physicochemical and spectroscopic techniques (steady-state and time-resolved luminescence, quantum yield determination, fluorescence microscopy). The presented results open new avenues for exploring the use of MOFs containing luminescent silver clusters for solid-state lighting and sensor applications.

  14. CpG Dinucleotide Frequencies Reveal the Role of Host Methylation Capabilities in Parvovirus Evolution

    PubMed Central

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

    2013-01-01

    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. PMID:24109231

  15. 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.

  16. Electrophoretically mediated microanalysis of a nicotinamide adenine dinucleotide-dependent enzyme and its facile multiplexing using an active pixel sensor UV detector.

    PubMed

    Urban, Pawel L; Goodall, David M; Bergström, Edmund T; Bruce, Neil C

    2007-08-31

    An electrophoretically mediated microanalysis (EMMA) method has been developed for yeast alcohol dehydrogenase and quantification of reactant and product cofactors, NAD and NADH. The enzyme substrate ethanol (1% (v/v)) was added to the buffer (50 mM borate, pH 8.8). Results are presented for parallel capillary electrophoresis with a novel miniature UV area detector, with an active pixel sensor imaging an array of two or six parallel capillaries connected via a manifold to a single output capillary in a commercial CE instrument, allowing conversions with five different yeast alcohol dehydrogenase concentrations to be quantified in a single experiment.

  17. Nitrite Formation from Hydroxylamine and Oximes by Pseudomonas aeruginosa

    PubMed Central

    Amarger, Noelle; Alexander, M.

    1968-01-01

    Nitrite was formed from hydroxylamine and several oximes by intact cells and extracts of Pseudomonas aeruginosa. The activity was induced by the presence of oximes in the culture medium. Nitroalkanes were not intermediates in the conversion of acetaldoxime, acetone oxime, or butanone oxime to nitrite, since nitromethane inhibited the formation of nitrite from the nitro compounds but not from the corresponding oximes. The oxime apparently functions as a constant source of hydroxylamine during growth of the bacterium. Hydroxylamine at low concentration was converted stoichiometrically to nitrite by extracts of the bacterium; high concentrations were inhibitory. Nicotinamide adenine dinucleotide phosphate, oxygen, and other unidentified cofactors were necessary for the reaction. Actively nitrifying extracts possessed no hydroxylamine-cytochrome c reductase activity. Hyponitrite, nitrous oxide, and nitric oxide were not metabolized. PMID:4384968

  18. Transport of adenine, hypoxanthine and uracil into Escherichia coli.

    PubMed Central

    Burton, K

    1977-01-01

    Uptake of adenine, hypoxanthine and uracil by an uncA strain of Escherichia coli is inhibited by uncouplers or when phosphate in the medium is replaced by less than 1 mM-arsenate, indicating a need for both a protonmotive force and phosphorylated metabolites. The rate of uptake of adenine or hypoxanthine was not markedly affected by a genetic deficiency of purine nucleoside phosphorylase. In two mutants with undetected adenine phosphoribosyltransferase, the rate of adenine uptake was about 30% of that in their parent strain, and evidence was obtained to confirm that adenine had then been utilized via purine nucleoside phosphorylase. In a strain deficient in both enzymes adenine uptake was about 1% of that shown by wild-type strains. Uptake of hypoxanthine was similarly limited in a strain lacking purine nucleoside phosphorylase, hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase. Deficiency of uracil phosphoribosyltransferase severely limits uracil uptake, but the defect can be circumvented by addition of inosine, which presumably provides ribose 1-phosphate for reversal of uridine phosphorylase. The results indicate that there are porter systems for adenine, hypoxanthine and uracil dependent on a protonmotive force and facilitated by intracellular metabolism of the free bases. PMID:413544

  19. Cofactor engineering for advancing chemical biotechnology.

    PubMed

    Wang, Yipeng; San, Ka-Yiu; Bennett, George N

    2013-12-01

    Cofactors provide redox carriers for biosynthetic reactions, catabolic reactions and act as important agents in transfer of energy for the cell. Recent advances in manipulating cofactors include culture conditions or additive alterations, genetic modification of host pathways for increased availability of desired cofactor, changes in enzyme cofactor specificity, and introduction of novel redox partners to form effective circuits for biochemical processes and biocatalysts. Genetic strategies to employ ferredoxin, NADH and NADPH most effectively in natural or novel pathways have improved yield and efficiency of large-scale processes for fuels and chemicals and have been demonstrated with a variety of microbial organisms. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Preparation, purification and analyses of thirteen alkali-stable dinucleotides from ribonucleic acid

    PubMed Central

    Trim, A. R.; Parker, Janet E.

    1970-01-01

    Of the 16 alkali-stable dinucleotides known to be obtained by hydrolysis of commercial yeast RNA with alkali, 13 were prepared in quantities of the order of 10mg or more. The samples, with only one exception, contain at least 90% of dinucleotide, and spectroscopic constants and nucleotide-sequence determinations, although not conclusive, indicate a high degree of purity of these products. The small dinucleotide fraction in 150g of RNA hydrolysed with alkali (1–2% of the total nucleotides) was separated from the mononucleotides by stepwise ion-exchange chromatography on DEAE-cellulose columns and resolved into seven fractions containing from one to four different dinucleotides by electrophoresis on paper at pH3.0. These fractions were resolved into their constituent dinucleotides by chromatography in ammonium sulphate. Contamination of the products by impurities from the paper was minimized by washing it before using it for chromatography or electrophoresis and, by using a thick grade of paper (Whatman no. 17), it was possible to handle and purify relatively large quantities of nucleotides. PMID:5435489

  1. Biomarkers and Biological Spectral Imaging

    DTIC Science & Technology

    2001-01-23

    Image t iZ ~ Rotator SSteping r De m oo ontroler Frmegabe (single ams) ... Software -= • . ... PCCm ue Data acquisition Rotator control PC Co puterImage...the depth of bum injury", Bums, 7, pp. 197-202, 1981. 7. R. A. De Blasi, M. Cope, C. Elwell, F. Safoue and M. Ferrari, "Noninvasive measurement of...nicotinamide adenine dinucleotide (NADH), oxidized flavin adenine dinucleotide (FAD) and porphyrins. A number of studies have shown that the measured

  2. Intrinsic fluorescence biomarkers in cells treated with chemopreventive drugs

    NASA Astrophysics Data System (ADS)

    Kirkpatrick, Nathaniel D.; Brands, William R.; Zou, Changping; Brewer, Molly A.; Utzinger, Urs

    2005-03-01

    Non-invasive monitoring of cellular metabolism offers promising insights into areas ranging from biomarkers for drug activity to cancer diagnosis. Fluorescence spectroscopy can be utilized in order to exploit endogenous fluorophores, typically metabolic co-factors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), and estimate the redox status of the sample. Fluorescence spectroscopy was applied to follow metabolic changes in epithelial ovarian cells as well as bladder epithelial cancer cells during treatment with a chemopreventive drug that initiates cellular quiescence. Fluorescence signals consistent with NADH, FAD, and tryptophan were measured to monitor cellular activity, redox status, and protein content. Cells were treated with varying concentrations of N-4-(hydroxyphenyl) retinamide (4-HPR) and measured in a stable environment with a sensitive fluorescence spectrometer. A subset of measurements was completed on a low concentration of cells to demonstrate feasibility for medical application such as in bladder or ovary washes. Results suggest that all of the cells responded with similar dose dependence but started at different estimated redox ratio baseline levels correlating with cell cycle, growth inhibition, and apoptosis assays. NADH and tryptophan related fluorescence changed significantly while FAD related fluorescence remained unaltered. Fluorescence data collected from approximately 1000 - 2000 cells, comparable to a bladder or ovary wash, was measurable and useful for future experiments. This study suggests that future intrinsic biomarker measurements may need to be most sensitive to changes in NADH and tryptophan related fluorescence while using FAD related fluorescence to help estimate the baseline redox ratio and predict response to chemopreventive agents.

  3. Optimization Strategies for Hardware-Based Cofactorization

    NASA Astrophysics Data System (ADS)

    Loebenberger, Daniel; Putzka, Jens

    We use the specific structure of the inputs to the cofactorization step in the general number field sieve (GNFS) in order to optimize the runtime for the cofactorization step on a hardware cluster. An optimal distribution of bitlength-specific ECM modules is proposed and compared to existing ones. With our optimizations we obtain a speedup between 17% and 33% of the cofactorization step of the GNFS when compared to the runtime of an unoptimized cluster.

  4. Dinucleotide Composition in Animal RNA Viruses Is Shaped More by Virus Family than by Host Species

    PubMed Central

    Di Giallonardo, Francesca; Schlub, Timothy E.; Shi, Mang

    2017-01-01

    ABSTRACT Viruses use the cellular machinery of their hosts for replication. It has therefore been proposed that the nucleotide and dinucleotide compositions of viruses should match those of their host species. If this is upheld, it may then be possible to use dinucleotide composition to predict the true host species of viruses sampled in metagenomic surveys. However, it is also clear that different taxonomic groups of viruses tend to have distinctive patterns of dinucleotide composition that may be independent of host species. To determine the relative strength of the effect of host versus virus family in shaping dinucleotide composition, we performed a comparative analysis of 20 RNA virus families from 15 host groupings, spanning two animal phyla and more than 900 virus species. In particular, we determined the odds ratios for the 16 possible dinucleotides and performed a discriminant analysis to evaluate the capability of virus dinucleotide composition to predict the correct virus family or host taxon from which it was isolated. Notably, while 81% of the data analyzed here were predicted to the correct virus family, only 62% of these data were predicted to their correct subphylum/class host and a mere 32% to their correct mammalian order. Similarly, dinucleotide composition has a weak predictive power for different hosts within individual virus families. We therefore conclude that dinucleotide composition is generally uniform within a virus family but less well reflects that of its host species. This has obvious implications for attempts to accurately predict host species from virus genome sequences alone. IMPORTANCE Determining the processes that shape virus genomes is central to understanding virus evolution and emergence. One question of particular importance is why nucleotide and dinucleotide frequencies differ so markedly between viruses. In particular, it is currently unclear whether host species or virus family has the biggest impact on dinucleotide

  5. Dinucleotide Composition in Animal RNA Viruses Is Shaped More by Virus Family than by Host Species.

    PubMed

    Di Giallonardo, Francesca; Schlub, Timothy E; Shi, Mang; Holmes, Edward C

    2017-04-15

    Viruses use the cellular machinery of their hosts for replication. It has therefore been proposed that the nucleotide and dinucleotide compositions of viruses should match those of their host species. If this is upheld, it may then be possible to use dinucleotide composition to predict the true host species of viruses sampled in metagenomic surveys. However, it is also clear that different taxonomic groups of viruses tend to have distinctive patterns of dinucleotide composition that may be independent of host species. To determine the relative strength of the effect of host versus virus family in shaping dinucleotide composition, we performed a comparative analysis of 20 RNA virus families from 15 host groupings, spanning two animal phyla and more than 900 virus species. In particular, we determined the odds ratios for the 16 possible dinucleotides and performed a discriminant analysis to evaluate the capability of virus dinucleotide composition to predict the correct virus family or host taxon from which it was isolated. Notably, while 81% of the data analyzed here were predicted to the correct virus family, only 62% of these data were predicted to their correct subphylum/class host and a mere 32% to their correct mammalian order. Similarly, dinucleotide composition has a weak predictive power for different hosts within individual virus families. We therefore conclude that dinucleotide composition is generally uniform within a virus family but less well reflects that of its host species. This has obvious implications for attempts to accurately predict host species from virus genome sequences alone. IMPORTANCE Determining the processes that shape virus genomes is central to understanding virus evolution and emergence. One question of particular importance is why nucleotide and dinucleotide frequencies differ so markedly between viruses. In particular, it is currently unclear whether host species or virus family has the biggest impact on dinucleotide frequencies and

  6. Catalytic carbene transfer allows the direct customization of cyclic purine dinucleotides.

    PubMed

    Fei, Na; Häussinger, Daniel; Blümli, Seraina; Laventie, Benoît-Joseph; Bizzini, Lorenzo D; Zimmermann, Kaspar; Jenal, Urs; Gillingham, Dennis

    2014-08-11

    We describe a simple method for the direct modification of nucleobases in cyclic purine dinucleotides, important signalling molecules in both prokaryotes and eukaryotes. The method tolerates all members of the cyclic dinucleotide family and could be used to modulate their function or introduce useful side-chains such as fluorophores and photo-crosslinking groups.

  7. 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

  8. Base Excision Repair of Tandem Modifications in a Methylated CpG Dinucleotide*

    PubMed Central

    Sassa, Akira; Çağlayan, Melike; Dyrkheeva, Nadezhda S.; Beard, William A.; Wilson, Samuel H.

    2014-01-01

    Cytosine methylation and demethylation in tracks of CpG dinucleotides is an epigenetic mechanism for control of gene expression. The initial step in the demethylation process can be deamination of 5-methylcytosine producing the TpG alteration and T:G mispair, and this step is followed by thymine DNA glycosylase (TDG) initiated base excision repair (BER). A further consideration is that guanine in the CpG dinucleotide may become oxidized to 7,8-dihydro-8-oxoguanine (8-oxoG), and this could affect the demethylation process involving TDG-initiated BER. However, little is known about the enzymology of BER of altered in-tandem CpG dinucleotides; e.g. Tp8-oxoG. Here, we investigated interactions between this altered dinucleotide and purified BER enzymes, the DNA glycosylases TDG and 8-oxoG DNA glycosylase 1 (OGG1), apurinic/apyrimidinic (AP) endonuclease 1, DNA polymerase β, and DNA ligases. The overall TDG-initiated BER of the Tp8-oxoG dinucleotide is significantly reduced. Specifically, TDG and DNA ligase activities are reduced by a 3′-flanking 8-oxoG. In contrast, the OGG1-initiated BER pathway is blocked due to the 5′-flanking T:G mispair; this reduces OGG1, AP endonuclease 1, and DNA polymerase β activities. Furthermore, in TDG-initiated BER, TDG remains bound to its product AP site blocking OGG1 access to the adjacent 8-oxoG. These results reveal BER enzyme specificities enabling suppression of OGG1-initiated BER and coordination of TDG-initiated BER at this tandem alteration in the CpG dinucleotide. PMID:24695738

  9. Genetics Home Reference: molybdenum cofactor deficiency

    MedlinePlus

    ... called molybdenum cofactor. Molybdenum cofactor, which contains the element molybdenum, is essential to the function of several ... Citation on PubMed or Free article on PubMed Central Reiss J, Gross-Hardt S, Christensen E, Schmidt P, ...

  10. Dinucleotide controlled null models for comparative RNA gene prediction.

    PubMed

    Gesell, Tanja; Washietl, Stefan

    2008-05-27

    Comparative prediction of RNA structures can be used to identify functional noncoding RNAs in genomic screens. It was shown recently by Babak et al. [BMC Bioinformatics. 8:33] that RNA gene prediction programs can be biased by the genomic dinucleotide content, in particular those programs using a thermodynamic folding model including stacking energies. As a consequence, there is need for dinucleotide-preserving control strategies to assess the significance of such predictions. While there have been randomization algorithms for single sequences for many years, the problem has remained challenging for multiple alignments and there is currently no algorithm available. We present a program called SISSIz that simulates multiple alignments of a given average dinucleotide content. Meeting additional requirements of an accurate null model, the randomized alignments are on average of the same sequence diversity and preserve local conservation and gap patterns. We make use of a phylogenetic substitution model that includes overlapping dependencies and site-specific rates. Using fast heuristics and a distance based approach, a tree is estimated under this model which is used to guide the simulations. The new algorithm is tested on vertebrate genomic alignments and the effect on RNA structure predictions is studied. In addition, we directly combined the new null model with the RNAalifold consensus folding algorithm giving a new variant of a thermodynamic structure based RNA gene finding program that is not biased by the dinucleotide content. SISSIz implements an efficient algorithm to randomize multiple alignments preserving dinucleotide content. It can be used to get more accurate estimates of false positive rates of existing programs, to produce negative controls for the training of machine learning based programs, or as standalone RNA gene finding program. Other applications in comparative genomics that require randomization of multiple alignments can be considered. SISSIz

  11. Structure of Escherichia coli AdhP (ethanol-inducible dehydrogenase) with bound NAD.

    PubMed

    Thomas, Leonard M; Harper, Angelica R; Miner, Whitney A; Ajufo, Helen O; Branscum, Katie M; Kao, Lydia; Sims, Paul A

    2013-07-01

    The crystal structure of AdhP, a recombinantly expressed alcohol dehydrogenase from Escherichia coli K-12 (substrain MG1655), was determined to 2.01 Å resolution. The structure, which was solved using molecular replacement, also included the structural and catalytic zinc ions and the cofactor nicotinamide adenine dinucleotide (NAD). The crystals belonged to space group P21, with unit-cell parameters a = 68.18, b = 118.92, c = 97.87 Å, β = 106.41°. The final R factor and Rfree were 0.138 and 0.184, respectively. The structure of the active site of AdhP suggested a number of residues that may participate in a proton relay, and the overall structure of AdhP, including the coordination to structural and active-site zinc ions, is similar to those of other tetrameric alcohol dehydrogenase enzymes.

  12. The requirement for bivalent cations in formation of nicotinamide–adenine dinucleotide by nicotinamide mononucleotide adenylyltransferase of pig-liver nuclei

    PubMed Central

    Jackson, J. F.; Atkinson, M. R.

    1966-01-01

    1. The requirement for bivalent cations in catalysis of NAD formation from ATP and NMN in the presence of NMN adenylyltransferase of pig-liver nuclei was studied. Rates of NAD formation in the presence of the activating cations Cd2+, Mn2+, Mg2+, Zn2+, Co2+ and Ni2+ were approximately a linear function of heats of hydration of the corresponding ions. Ba2+, Sr2+, Ca2+, Cu2+ and Be2+ did not activate the enzyme; Be2+ inhibited the reaction in the presence of Mg2+ and, to a greater extent, in the presence of Ni2+. 2. Michaelis constants for NAD formation, measured in a coupled assay with NMN adenylyltransferase and alcohol dehydrogenase at pH8·0 and 25°, in the presence of 3mm concentrations of the unvaried reactants, were 88±7μm-ATP, 42±4μm-NMN and 85±4μm-Mg2+. The results at this pH and at pH7·5 were consistent with mechanisms in which Mg2+–ATP complex is a reactant and free ATP a competitive inhibitor. 3. Formation of nicotinamide–hypoxanthine dinucleotide from NMN and ITP in the presence of the transferase was also more rapid with Ni2+ and Co2+ than with Mg2+. PMID:4291356

  13. Structure of conjugated polyketone reductase from Candida parapsilosis IFO 0708 reveals conformational changes for substrate recognition upon NADPH binding.

    PubMed

    Qin, Hui-Min; Yamamura, Akihiro; Miyakawa, Takuya; Kataoka, Michihiko; Nagai, Takahiro; Kitamura, Nahoko; Urano, Nobuyuki; Maruoka, Shintaro; Ohtsuka, Jun; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2014-01-01

    Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to D-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2'-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone.

  14. Methods for transfer a saliva based alcohol content test to a dermal patch

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Silks, III, Louis A.

    Detection and quantitation of ethanol which is highly sensitive, specific, and efficient has been a commercial target for sometime. Clearly analytical methods are useful such as gas and liquid chromatography, mass spectrometry, and NMR spectroscopy. However, those methods are best used in the laboratory and a less useful for detection and quantitation of ethanol in the field. Enzymes have been employed for the detection and quantitation of EtOH. Enzymes are proteins that perform a particular task in a bio-catalytic way. Most of the chemistry that these enzymes do are frequently exquisitely specific in that only one alcohol reacts and onlymore » one product is produced. One enzyme molecule can catalyze the reaction of numerous substrate molecules which in itself is an amplification of the recognition signal. Alcohol dehydrogenase (ADH) and alcohol oxidase (AO) are two possible enzymatic targets for EtOH sensor development.1 The ADH oxidizes the alcohol using a co-factor nicotinamide adenine dinucleotide. This co-factor needs to be within close proximity of the ADH. AO also oxidizes the ethanol using molecular oxygen giving rise to the production of the aldehyde and hydrogen peroxide.« less

  15. Depletion of CpG Dinucleotides in Papillomaviruses and Polyomaviruses: A Role for Divergent Evolutionary Pressures.

    PubMed

    Upadhyay, Mohita; Vivekanandan, Perumal

    2015-01-01

    Papillomaviruses and polyomaviruses are small ds-DNA viruses infecting a wide-range of vertebrate hosts. Evidence supporting co-evolution of the virus with the host does not fully explain the evolutionary path of papillomaviruses and polyomaviruses. Studies analyzing CpG dinucleotide frequencies in virus genomes have provided interesting insights on virus evolution. CpG dinucleotide depletion has not been extensively studied among papillomaviruses and polyomaviruses. We sought to analyze the relative abundance of dinucleotides and the relative roles of evolutionary pressures in papillomaviruses and polyomaviruses. We studied 127 full-length sequences from papillomaviruses and 56 full-length sequences from polyomaviruses. We analyzed the relative abundance of dinucleotides, effective codon number (ENC), differences in synonymous codon usage. We examined the association, if any, between the extent of CpG dinucleotide depletion and the evolutionary lineage of the infected host. We also investigated the contribution of mutational pressure and translational selection to the evolution of papillomaviruses and polyomaviruses. All papillomaviruses and polyomaviruses are CpG depleted. Interestingly, the evolutionary lineage of the infected host determines the extent of CpG depletion among papillomaviruses and polyomaviruses. CpG dinucleotide depletion was more pronounced among papillomaviruses and polyomaviruses infecting human and other mammals as compared to those infecting birds. Our findings demonstrate that CpG depletion among papillomaviruses is linked to mutational pressure; while CpG depletion among polyomaviruses is linked to translational selection. We also present evidence that suggests methylation of CpG dinucleotides may explain, at least in part, the depletion of CpG dinucleotides among papillomaviruses but not polyomaviruses. The extent of CpG depletion among papillomaviruses and polyomaviruses is linked to the evolutionary lineage of the infected host. Our

  16. Nicotinamide adenine dinucleotide is transported into mammalian mitochondria.

    PubMed

    Davila, Antonio; Liu, Ling; Chellappa, Karthikeyani; Redpath, Philip; Nakamaru-Ogiso, Eiko; Paolella, Lauren M; Zhang, Zhigang; Migaud, Marie E; Rabinowitz, Joshua D; Baur, Joseph A

    2018-06-12

    Mitochondrial NAD levels influence fuel selection, circadian rhythms, and cell survival under stress. It has alternately been argued that NAD in mammalian mitochondria arises from import of cytosolic nicotinamide (NAM), nicotinamide mononucleotide (NMN), or NAD itself. We provide evidence that murine and human mitochondria take up intact NAD. Isolated mitochondria preparations cannot make NAD from NAM, and while NAD is synthesized from NMN, it does not localize to the mitochondrial matrix or effectively support oxidative phosphorylation. Treating cells with nicotinamide riboside that is isotopically labeled on the nicotinamide and ribose moieties results in the appearance of doubly labeled NAD within mitochondria. Analogous experiments with doubly labeled nicotinic acid riboside (labeling cytosolic NAD without labeling NMN) demonstrate that NAD(H) is the imported species. Our results challenge the long-held view that the mitochondrial inner membrane is impermeable to pyridine nucleotides and suggest the existence of an unrecognized mammalian NAD (or NADH) transporter. © 2018, Davila et al.

  17. Synthesis of adenine-modified reduced graphene oxide nanosheets.

    PubMed

    Cao, Huaqiang; Wu, Xiaoming; Yin, Gui; Warner, Jamie H

    2012-03-05

    We report here a facile strategy to synthesize the nanocomposite of adenine-modified reduced graphene oxide (AMG) via reaction between adenine and GOCl which is generated from SOCl(2) reacted with graphite oxide (GO). The as-synthesized AMG was characterized by transmission electron microscopy (TEM), atomic force microscopy (AFM), UV-vis absorption spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, Raman spectroscopy, thermogravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV), and galvanostatic discharge analysis. The AMG owns about one adenine group per 53 carbon atoms on a graphene sheet, which improves electronic conductivity compared with reduced graphene oxide (RGO). The AMG displays enhanced supercapacitor performance compared with RGO accompanying good stability and good cycling behavior in the supercapacitor.

  18. Studying Catabolism of Protein ADP-Ribosylation.

    PubMed

    Palazzo, Luca; James, Dominic I; Waddell, Ian D; Ahel, Ivan

    2017-01-01

    Protein ADP-ribosylation is a conserved posttranslational modification that regulates many major cellular functions, such as DNA repair, transcription, translation, signal transduction, stress response, cell division, aging, and cell death. Protein ADP-ribosyl transferases catalyze the transfer of an ADP-ribose (ADPr) group from the β-nicotinamide adenine dinucleotide (β-NAD + ) cofactor onto a specific target protein with the subsequent release of nicotinamide. ADP-ribosylation leads to changes in protein structure, function, stability, and localization, thus defining the appropriate cellular response. Signaling processes that are mediated by modifications need to be finely tuned and eventually silenced and one of the ways to achieve this is through the action of enzymes that remove (reverse) protein ADP-ribosylation in a timely fashion such as PARG, TARG1, MACROD1, and MACROD2. Here, we describe several basic methods used to study the enzymatic activity of de-ADP-ribosylating enzymes.

  19. More to NAD+ than meets the eye: A regulator of metabolic pools and gene expression in Arabidopsis.

    PubMed

    Gakière, Bertrand; Fernie, Alisdair R; Pétriacq, Pierre

    2018-01-05

    Since its discovery more than a century ago, nicotinamide adenine dinucleotide (NAD + ) is recognised as a fascinating cornerstone of cellular metabolism. This ubiquitous energy cofactor plays vital roles in metabolic pathways and regulatory processes, a fact emphasised by the essentiality of a balanced NAD + metabolism for normal plant growth and development. Research on the role of NAD in plants has been predominantly carried out in the model plant Arabidopsis thaliana (Arabidopsis) with emphasis on the redox properties and cellular signalling functions of the metabolite. This review examines the current state of knowledge concerning how NAD can regulate both metabolic pools and gene expression in Arabidopsis. Particular focus is placed on recent studies highlighting the complexity of metabolic regulations involving NAD, more particularly in the mitochondrial compartment, and of signalling roles with respect to interactions with environmental fluctuations most specifically those involving plant immunity. Copyright © 2018 Elsevier Inc. All rights reserved.

  20. Non-enzymatic glycation reduces heparin cofactor II anti-thrombin activity.

    PubMed

    Ceriello, A; Marchi, E; Barbanti, M; Milani, M R; Giugliano, D; Quatraro, A; Lefebvre, P

    1990-04-01

    The effects of non-enzymatic glycation on heparin cofactor II activity, at glucose concentrations which might be expected in physiological or diabetic conditions have been evaluated in this study. Radiolabelled glucose incorporation was associated with a loss of heparin cofactor anti-thrombin activity. The heparin cofactor heparin and dermatan sulfate-dependent inhibition of thrombin was significantly reduced, showing a remarkable decrease of the maximum second order rate constant. This study shows that heparin cofactor can be glycated at glucose concentrations found in the blood, and that this phenomenon produces a loss of heparin cofactor-antithrombin activity. These data suggest, furthermore, a possible link between heparin cofactor glycation and the pathogenesis of thrombosis in diabetes mellitus.

  1. Glutamate Dehydrogenase from Apodachlya (Oomycetes) 1

    PubMed Central

    Price, Jeffrey S.; Gleason, Frank H.

    1972-01-01

    A glutamate dehydrogenase specific for nicotinamide-adenine-dinucleotide has been purified 50-fold from Apodachlya brachynema (Leptomitales). Certain physical, chemical, and kinetic properties of this enzyme have been studied, particularly specificity for coenzymes and substrates. With glucose as the sole carbon source, the synthesis of glutamate dehydrogenase was repressed, whereas glutamate, proline, alanine, or ornithine plus aspartate as sole carbon sources induced synthesis of the enzyme. These data indicate that the function of this enzyme is primarily degradative, although there is no evidence for a nicotinamide-adenine-dinucleotide-phosphate-specific biosynthetic glutamate dehydrogenase in Apodachlya. PMID:16657902

  2. Adenine and 2-aminopurine: paradigms of modern theoretical photochemistry.

    PubMed

    Serrano-Andrés, Luis; Merchán, Manuela; Borin, Antonio C

    2006-06-06

    Distinct photophysical behavior of nucleobase adenine and its constitutional isomer, 2-aminopurine, has been studied by using quantum chemical methods, in particular an accurate ab initio multiconfigurational second-order perturbation theory. After light irradiation, the efficient, ultrafast energy dissipation observed for nonfluorescent 9H-adenine is explained here by the nonradiative internal conversion process taking place along a barrierless reaction path from the initially populated 1(pipi* La) excited state toward a low-lying conical intersection (CI) connected with the ground state. In contrast, the strong fluorescence recorded for 2-aminopurine at 4.0 eV with large decay lifetime is interpreted by the presence of a minimum in the 1(pipi* La) hypersurface lying below the lowest CI and the subsequent potential energy barrier required to reach the funnel to the ground state. Secondary deactivation channels were found in the two systems related to additional CIs involving the 1(pipi* Lb) and 1(npi*) states. Although in 9H-adenine a population switch between both states is proposed, in 7H-adenine this may be perturbed by a relatively larger barrier to access the 1(npi*) state, and, therefore, the 1(pipi* Lb) state becomes responsible for the weak fluorescence measured in aqueous adenine at approximately 4.5 eV. In contrast to previous models that explained fluorescence quenching in adenine, unlike in 2-aminopurine, on the basis of the vibronic coupling of the nearby 1(pipi*) and 1(npi*) states, the present results indicate that the 1(npi*) state does not contribute to the leading photophysical event and establish the prevalence of a model based on the CI concept in modern photochemistry.

  3. One-pot synthesis of fluorescent polysaccharides: adenine grafted agarose and carrageenan.

    PubMed

    Oza, Mihir D; Prasad, Kamalesh; Siddhanta, A K

    2012-08-01

    New fluorescent polysaccharides were synthesized by grafting the nucleobase adenine on to the backbones of agarose and κ-carrageenan, which were characterized by FT-IR, (13)C NMR, TGA, XRD, UV, and fluorescence properties. The synthesis involved a rapid water based potassium persulfate (KPS) initiated method under microwave irradiation. The emission spectra of adenine grafted agarose and κ-carrageenan were recorded in aqueous (5×10(-5) M) solution, exhibiting λ(em,max) 347 nm by excitation at 261 nm, affording ca. 30% and 40% enhanced emission intensities, respectively compared to that of pure adenine solution in the same concentration. Similar emission intensity was recorded in the pure adenine solution at its molar equivalent concentrations present in the 5×10(-5) M solution of the agarose and carrageenan grafted products, that is, 3.28×10(-5) M and 4.5×10(-5) M respectively. These fluorescent adenine grafted products may have potential utility in various sensor applications. Copyright © 2012. Published by Elsevier Ltd.

  4. 3-base periodicity in coding DNA is affected by intercodon dinucleotides

    PubMed Central

    Sánchez, Joaquín

    2011-01-01

    All coding DNAs exhibit 3-base periodicity (TBP), which may be defined as the tendency of nucleotides and higher order n-tuples, e.g. trinucleotides (triplets), to be preferentially spaced by 3, 6, 9 etc, bases, and we have proposed an association between TBP and clustering of same-phase triplets. We here investigated if TBP was affected by intercodon dinucleotide tendencies and whether clustering of same-phase triplets was involved. Under constant protein sequence intercodon dinucleotide frequencies depend on the distribution of synonymous codons. So, possible effects were revealed by randomly exchanging synonymous codons without altering protein sequences to subsequently document changes in TBP via frequency distribution of distances (FDD) of DNA triplets. A tripartite positive correlation was found between intercodon dinucleotide frequencies, clustering of same-phase triplets and TBP. So, intercodon C|A (where “|” indicates the boundary between codons) was more frequent in native human DNA than in the codon-shuffled sequences; higher C|A frequency occurred along with more frequent clustering of C|AN triplets (where N jointly represents A, C, G and T) and with intense CAN TBP. The opposite was found for C|G, which was less frequent in native than in shuffled sequences; lower C|G frequency occurred together with reduced clustering of C|GN triplets and with less intense CGN TBP. We hence propose that intercodon dinucleotides affect TBP via same-phase triplet clustering. A possible biological relevance of our findings is briefly discussed. PMID:21814388

  5. A survey of synthetic nicotinamide cofactors in enzymatic processes.

    PubMed

    Paul, Caroline E; Hollmann, Frank

    2016-06-01

    Synthetic nicotinamide cofactors are analogues of the natural cofactors used by oxidoreductases as redox intermediates. Their ability to be fine-tuned makes these biomimetics an attractive alternative to the natural cofactors in terms of stability, reactivity, and cost. The following mini-review focuses on the current state of the art of those biomimetics in enzymatic processes.

  6. Adenine and 2-aminopurine: Paradigms of modern theoretical photochemistry

    PubMed Central

    Serrano-Andrés, Luis; Merchán, Manuela; Borin, Antonio C.

    2006-01-01

    Distinct photophysical behavior of nucleobase adenine and its constitutional isomer, 2-aminopurine, has been studied by using quantum chemical methods, in particular an accurate ab initio multiconfigurational second-order perturbation theory. After light irradiation, the efficient, ultrafast energy dissipation observed for nonfluorescent 9H-adenine is explained here by the nonradiative internal conversion process taking place along a barrierless reaction path from the initially populated 1(ππ* La) excited state toward a low-lying conical intersection (CI) connected with the ground state. In contrast, the strong fluorescence recorded for 2-aminopurine at 4.0 eV with large decay lifetime is interpreted by the presence of a minimum in the 1(ππ* La) hypersurface lying below the lowest CI and the subsequent potential energy barrier required to reach the funnel to the ground state. Secondary deactivation channels were found in the two systems related to additional CIs involving the 1(ππ* Lb) and 1(nπ*) states. Although in 9H-adenine a population switch between both states is proposed, in 7H-adenine this may be perturbed by a relatively larger barrier to access the 1(nπ*) state, and, therefore, the 1(ππ* Lb) state becomes responsible for the weak fluorescence measured in aqueous adenine at ≈4.5 eV. In contrast to previous models that explained fluorescence quenching in adenine, unlike in 2-aminopurine, on the basis of the vibronic coupling of the nearby 1(ππ*) and 1(nπ*) states, the present results indicate that the 1(nπ*) state does not contribute to the leading photophysical event and establish the prevalence of a model based on the CI concept in modern photochemistry. PMID:16731617

  7. Elicitors and co-factors in food-induced anaphylaxis in adults

    PubMed Central

    2013-01-01

    Food-induced anaphylaxis (FIA) in adults is often insufficiently diagnosed. One reason is related to the presence of co-factors like exercise, alcohol, additives and non-steroidal anti-inflammatory drugs. The objective of this analysis was to retrospectively investigate the role of co-factors in patients with FIA. 93 adult patients with suspected FIA underwent double-blind, placebo-controlled food challenges with suspected allergens and co-factors. The elicitors of anaphylaxis were identified in 44/93 patients. 27 patients reacted to food allergens upon challenge, 15 patients reacted only when a co-factor was co-exposed with the allergen. The most common identified allergens were celery (n = 7), soy, wheat (n = 4 each) and lupine (n = 3). Among the co-factors food additives (n = 8) and physical exercise (n = 6) were most frequent. In 10 patients more than one co-factor and/or more than one food allergen was necessary to elicit a positive reaction. The implementation of co-factors into the challenge protocol increases the identification rate of elicitors in adult food anaphylactic patients. PMID:24262093

  8. The catalase activity of diiron adenine deaminase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kamat S. S.; Swaminathan S.; Holmes-Hampton, G. P.

    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 spectrometrymore » 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.« less

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kamat, S.S.; Swaminathan, S.; Bagaria, A.

    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. Themore » 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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    S Kamat; A Bagaria; D Kumaran

    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{supmore » -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

  11. Absorption and fluorescence spectroscopic characterisation of the circadian blue-light photoreceptor cryptochrome from Drosophila melanogaster (dCry)

    NASA Astrophysics Data System (ADS)

    Shirdel, J.; Zirak, P.; Penzkofer, A.; Breitkreuz, H.; Wolf, E.

    2008-09-01

    The absorption and fluorescence behaviour of the circadian blue-light photoreceptor cryptochrome from Drosophila melanogaster (dCry) in a pH 8 aqueous buffer solution is studied. The flavin adenine dinucleotide (FAD) cofactor of dCry is identified to be present in its oxidized form (FAD ox), and the 5,10-methenyltetrahydrofolate (MTHF) cofactor is found to be hydrolyzed and oxidized to 10-formyldihydrofolate (10-FDHF). The absorption and the fluorescence behaviour of dCry is investigated in the dark-adapted (receptor) state, the light-adapted (signalling) state, and under long-time violet light exposure. Photo-excitation of FAD ox in dCry causes a reductive electron transfer to the formation of anionic FAD semiquinone (FAD rad - ), and photo-excitation of the generated FAD rad - causes an oxidative electron transfer to the back formation of FAD ox. In light adapted dCry a photo-induced equilibrium between FAD ox and FAD rad - exists. The photo-cycle dynamics of signalling state formation and recovery is discussed. Quantum yields of photo-induced signalling state formation of about 0.2 and of photo-induced back-conversion of about 0.2 are determined. A recovery of FAD rad - to FAD ox in the dark with a time constant of 1.6 min at room temperature is found.

  12. Efficient Enzymatic Preparation of (13) N-Labelled Amino Acids: Towards Multipurpose Synthetic Systems.

    PubMed

    da Silva, Eunice S; Gómez-Vallejo, Vanessa; Baz, Zuriñe; Llop, Jordi; López-Gallego, Fernando

    2016-09-12

    Nitrogen-13 can be efficiently produced in biomedical cyclotrons in different chemical forms, and its stable isotopes are present in the majority of biologically active molecules. Hence, it may constitute a convenient alternative to Fluorine-18 and Carbon-11 for the preparation of positron-emitter-labelled radiotracers; however, its short half-life demands for the development of simple, fast, and efficient synthetic processes. Herein, we report the one-pot, enzymatic and non-carrier-added synthesis of the (13) N-labelled amino acids l-[(13) N]alanine, [(13) N]glycine, and l-[(13) N]serine by using l-alanine dehydrogenase from Bacillus subtilis, an enzyme that catalyses the reductive amination of α-keto acids by using nicotinamide adenine dinucleotide (NADH) as the redox cofactor and ammonia as the amine source. The integration of both l-alanine dehydrogenase and formate dehydrogenase from Candida boidinii in the same reaction vessel to facilitate the in situ regeneration of NADH during the radiochemical synthesis of the amino acids allowed a 50-fold decrease in the concentration of the cofactor without compromising reaction yields. After optimization of the experimental conditions, radiochemical yields were sufficient to carry out in vivo imaging studies in small rodents. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Adenine alleviates iron overload by cAMP/PKA mediated hepatic hepcidin in mice.

    PubMed

    Zhang, Yingqi; Wang, Xudong; Wu, Qian; Wang, Hao; Zhao, Lu; Wang, Xinhui; Mu, Mingdao; Xie, Enjun; He, Xuyan; Shao, Dandan; Shang, Yanna; Lai, Yongrong; Ginzburg, Yelena; Min, Junxia; Wang, Fudi

    2018-03-30

    Hemochromatosis is prevalent and often associated with high rates of morbidity and mortality worldwide. The safe alternative iron-reducing approaches are urgently needed in order to better control iron overload. Our unbiased vitamin screen for modulators of hepcidin, a master iron regulatory hormone, identifies adenine (vitamin B4) as a potent hepcidin agonist. Adenine significantly induced hepcidin mRNA level and promoter activity activation in human cell lines, possibly through BMP/SMAD pathway. Further studies in mice validated the effect of adenine on hepcidin upregulation. Consistently, adenine dietary supplement in mice led to an increase of hepatic hepcidin expression compared with normal diet-fed mice via BMP/SMAD pathway. Notably, adenine-rich diet significantly ameliorated iron overload accompanied by the enhanced hepcidin expression in both high iron-fed mice and in Hfe -/- mice, a murine model of hereditary hemochromatosis. To further validate this finding, we selected pharmacological inhibitors against BMP (LDN193189). We found LDN193189 strongly blocked the hepcidin induction by adenine. Moreover, we uncovered an essential role of cAMP/PKA-dependent axis in triggering adenine-induced hepcidin expression in primary hepatocytes by using 8 br cAMP, a cAMP analog, and H89, a potent inhibitor for PKA signaling. These findings suggest a potential therapeutic role of adenine for hereditary hemochromatosis. © 2018 Wiley Periodicals, Inc.

  14. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  15. Nitrogenase Cofactor: Inspiration for Model Chemistry.

    PubMed

    Djurdjevic, Ivana; Einsle, Oliver; Decamps, Laure

    2017-07-04

    The cofactor of nitrogenase is the largest and most intricate metal cluster known in nature. Its reactivity, mode of action and even the precise binding site of substrate remain a matter of debate. For decades, synthetic chemists have taken inspiration from the exceptional structural, electronic and catalytic features of the cofactor and have tried to either mimic the unique topology of the entire site, or to extract its functional principles and build them into novel catalysts that achieve the same-or very similar-astounding transformations. We review some of the available model chemistry as it represents the various approaches that have been taken from studying the cofactor, to eventually summarize the current state of knowledge on catalysis by nitrogenase and highlight the mutually beneficial role of model chemistry and enzymology in bioinorganic chemistry. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Oxidation of ethane by an Acremonium species.

    PubMed Central

    Davies, J S; Wellman, A M; Zajic, J E

    1976-01-01

    Ethane oxidation was studied in ethane-grown resting cells (mycelia) of an Acremonium sp. and in cell-free preparations of such mycelia. From resting cell experiments evidence was found for a pathway of ethane oxidation via ethanol, acetaldehyde, and acetic acid. In vitro studies indicated that ethane-oxidizing activity in such mycelia occurred predominantly in the microsomal fraction of crude homogenates. Microsomal preparations were inactive in the absence of added coenzyme. Marked stimulation of activity was obtained in such preparations with reduced nicotinamide adenine dinucleotide phosphate and to a much lesser degree with nicotinamide adenine dinucleotide phosphate. Ethane oxidation was inhibited by sodium azide and carbon monoxide. PMID:9900

  17. Therapeutic effect of enhancing endothelial nitric oxide synthase (eNOS) expression and preventing eNOS uncoupling

    PubMed Central

    Förstermann, Ulrich; Li, Huige

    2011-01-01

    Nitric oxide (NO) produced by the endothelium is an important protective molecule in the vasculature. It is generated by the enzyme endothelial NO synthase (eNOS). Similar to all NOS isoforms, functional eNOS transfers electrons from nicotinamide adenine dinucleotide phosphate (NADPH), via the flavins flavin adenine dinucleotide and flavin mononucleotide in the carboxy-terminal reductase domain, to the heme in the amino-terminal oxygenase domain. Here, the substrate L-arginine is oxidized to L-citrulline and NO. Cardiovascular risk factors such as diabetes mellitus, hypertension, hypercholesterolaemia or cigarette smoking reduce bioactive NO. These risk factors lead to an enhanced production of reactive oxygen species (ROS) in the vessel wall. NADPH oxidases represent major sources of this ROS and have been found upregulated in the presence of cardiovascular risk factors. NADPH-oxidase-derived superoxide avidly reacts with eNOS-derived NO to form peroxynitrite (ONOO-). The essential NOS cofactor (6R-)5,6,7,8-tetrahydrobiopterin (BH4) is highly sensitive to oxidation by this ONOO-. In BH4 deficiency, oxygen reduction uncouples from NO synthesis, thereby converting NOS to a superoxide-producing enzyme. Among conventional drugs, compounds interfering with the renin-angiotensin-aldosterone system and statins can reduce vascular oxidative stress and increase bioactive NO. In recent years, we have identified a number of small molecules that have the potential to prevent eNOS uncoupling and, at the same time, enhance eNOS expression. These include the protein kinase C inhibitor midostaurin, the pentacyclic triterpenoids ursolic acid and betulinic acid, the eNOS enhancing compounds AVE9488 and AVE3085, and the polyphenolic phytoalexin trans-resveratrol. Such compounds enhance NO production from eNOS also under pathophysiological conditions and may thus have therapeutic potential. PMID:21198553

  18. Quantitative Analysis of NAD Synthesis-Breakdown Fluxes.

    PubMed

    Liu, Ling; Su, Xiaoyang; Quinn, William J; Hui, Sheng; Krukenberg, Kristin; Frederick, David W; Redpath, Philip; Zhan, Le; Chellappa, Karthikeyani; White, Eileen; Migaud, Marie; Mitchison, Timothy J; Baur, Joseph A; Rabinowitz, Joshua D

    2018-05-01

    The redox cofactor nicotinamide adenine dinucleotide (NAD) plays a central role in metabolism and is a substrate for signaling enzymes including poly-ADP-ribose-polymerases (PARPs) and sirtuins. NAD concentration falls during aging, which has triggered intense interest in strategies to boost NAD levels. A limitation in understanding NAD metabolism has been reliance on concentration measurements. Here, we present isotope-tracer methods for NAD flux quantitation. In cell lines, NAD was made from nicotinamide and consumed largely by PARPs and sirtuins. In vivo, NAD was made from tryptophan selectively in the liver, which then excreted nicotinamide. NAD fluxes varied widely across tissues, with high flux in the small intestine and spleen and low flux in the skeletal muscle. Intravenous administration of nicotinamide riboside or mononucleotide delivered intact molecules to multiple tissues, but the same agents given orally were metabolized to nicotinamide in the liver. Thus, flux analysis can reveal tissue-specific NAD metabolism. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. Plasticity, dynamics, and inhibition of emerging tetracycline-resistance enzymes

    PubMed Central

    Park, Jooyoung; Gasparrini, Andrew J.; Reck, Margaret R.; Symister, Chanez T.; Elliott, Jennifer L.; Vogel, Joseph P.; Wencewicz, Timothy A.; Dantas, Gautam; Tolia, Niraj H.

    2017-01-01

    While tetracyclines are an important class of antibiotics in agriculture and the clinic, their efficacy is threatened by increasing resistance. Resistance to tetracyclines can occur through efflux, ribosomal protection, or enzymatic inactivation. Surprisingly, tetracycline enzymatic inactivation has remained largely unexplored despite providing the distinct advantage of antibiotic clearance. The tetracycline destructases are a recently-discovered family of tetracycline-inactivating flavoenzymes from pathogens and soil metagenomes with a high potential for broad dissemination. Here, we show tetracycline destructases accommodate tetracycline-class antibiotics in diverse and novel orientations for catalysis, and antibiotic binding drives unprecedented structural dynamics facilitating tetracycline inactivation. We identify a key inhibitor binding mode that locks the flavin adenine dinucleotide cofactor in an inactive state, functionally rescuing tetracycline activity. Our results reveal the potential of a novel tetracycline/tetracycline destructase inhibitor combination therapy strategy to overcome resistance by enzymatic inactivation and restore the use of an important class of antibiotics. PMID:28481346

  20. Plasticity, dynamics, and inhibition of emerging tetracycline resistance enzymes.

    PubMed

    Park, Jooyoung; Gasparrini, Andrew J; Reck, Margaret R; Symister, Chanez T; Elliott, Jennifer L; Vogel, Joseph P; Wencewicz, Timothy A; Dantas, Gautam; Tolia, Niraj H

    2017-07-01

    Although tetracyclines are an important class of antibiotics for use in agriculture and the clinic, their efficacy is threatened by increasing resistance. Resistance to tetracyclines can occur through efflux, ribosomal protection, or enzymatic inactivation. Surprisingly, tetracycline enzymatic inactivation has remained largely unexplored, despite providing the distinct advantage of antibiotic clearance. The tetracycline destructases are a recently discovered family of tetracycline-inactivating flavoenzymes from pathogens and soil metagenomes that have a high potential for broad dissemination. Here, we show that tetracycline destructases accommodate tetracycline-class antibiotics in diverse and novel orientations for catalysis, and antibiotic binding drives unprecedented structural dynamics facilitating tetracycline inactivation. We identify a key inhibitor binding mode that locks the flavin adenine dinucleotide cofactor in an inactive state, functionally rescuing tetracycline activity. Our results reveal the potential of a new tetracycline and tetracycline destructase inhibitor combination therapy strategy to overcome resistance by enzymatic inactivation and restore the use of an important class of antibiotics.

  1. Quantification of ethanol in plasma by electrochemical detection with an unmodified screen printed carbon electrode

    NASA Astrophysics Data System (ADS)

    Tian, Gang; Zhang, Xiao-Qing; Zhu, Ming-Song; Zhang, Zhong; Shi, Zheng-Hu; Ding, Min

    2016-03-01

    Simple, rapid and accurate detection of ethanol concentration in blood is very crucial in the diagnosis and management of potential acute ethanol intoxication patients. A novel electrochemical detection method was developed for the quantification of ethanol in human plasma with disposable unmodified screen-printed carbon electrode (SPCE) without sample preparation procedure. Ethanol was detected indirectly by the reaction product of ethanol dehydrogenase (ADH) and cofactor nicotinamide adenine dinucleotide (NAD+). Method validation indicated good quantitation precisions with intra-day and inter-day relative standard deviations of ≤9.4% and 8.0%, respectively. Ethanol concentration in plasma is linear ranging from 0.10 to 3.20 mg/mL, and the detection limit is 40.0 μg/mL (S/N > 3). The method shows satisfactory correlation with the reference method of headspace gas chromatography in twenty human plasma samples (correlation coefficient 0.9311). The proposed method could be applied to diagnose acute ethanol toxicity or ethanol-related death.

  2. NAD+ and SIRT3 control microtubule dynamics and reduce susceptibility to antimicrotubule agents

    PubMed Central

    Harkcom, William T.; Ghosh, Ananda K.; Sung, Matthew S.; Matov, Alexandre; Brown, Kevin D.; Giannakakou, Paraskevi; Jaffrey, Samie R.

    2014-01-01

    Nicotinamide adenine dinucleotide (NAD+) is an endogenous enzyme cofactor and cosubstrate that has effects on diverse cellular and physiologic processes, including reactive oxygen species generation, mitochondrial function, apoptosis, and axonal degeneration. A major goal is to identify the NAD+-regulated cellular pathways that may mediate these effects. Here we show that the dynamic assembly and disassembly of microtubules is markedly altered by NAD+. Furthermore, we show that the disassembly of microtubule polymers elicited by microtubule depolymerizing agents is blocked by increasing intracellular NAD+ levels. We find that these effects of NAD+ are mediated by the activation of the mitochondrial sirtuin sirtuin-3 (SIRT3). Overexpression of SIRT3 prevents microtubule disassembly and apoptosis elicited by antimicrotubule agents and knockdown of SIRT3 prevents the protective effects of NAD+ on microtubule polymers. Taken together, these data demonstrate that NAD+ and SIRT3 regulate microtubule polymerization and the efficacy of antimicrotubule agents. PMID:24889606

  3. Crystal structure of 4-hydroxybutyryl-CoA dehydratase: radical catalysis involving a [4Fe-4S] cluster and flavin.

    PubMed

    Martins, Berta M; Dobbek, Holger; Cinkaya, Irfan; Buckel, Wolfgang; Messerschmidt, Albrecht

    2004-11-02

    Dehydratases catalyze the breakage of a carbon-oxygen bond leading to unsaturated products via the elimination of water. The 1.6-A resolution crystal structure of 4-hydroxybutyryl-CoA dehydratase from the gamma-aminobutyrate-fermenting Clostridium aminobutyricum represents a new class of dehydratases with an unprecedented active site architecture. A [4Fe-4S](2+) cluster, coordinated by three cysteine and one histidine residues, is located 7 A from the Re-side of a flavin adenine dinucleotide (FAD) moiety. The structure provides insight into the function of these ubiquitous prosthetic groups in the chemically nonfacile, radical-mediated dehydration of 4-hydroxybutyryl-CoA. The substrate can be bound between the [4Fe-4S](2+) cluster and the FAD with both cofactors contributing to its radical activation and catalytic conversion. Our results raise interesting questions regarding the mechanism of acyl-CoA dehydrogenases, which are involved in fatty acid oxidation, and address the divergent evolution of the ancestral common gene.

  4. Quantification of ethanol in plasma by electrochemical detection with an unmodified screen printed carbon electrode

    PubMed Central

    Tian, Gang; Zhang, Xiao-Qing; Zhu, Ming-Song; Zhang, Zhong; Shi, Zheng-Hu; Ding, Min

    2016-01-01

    Simple, rapid and accurate detection of ethanol concentration in blood is very crucial in the diagnosis and management of potential acute ethanol intoxication patients. A novel electrochemical detection method was developed for the quantification of ethanol in human plasma with disposable unmodified screen-printed carbon electrode (SPCE) without sample preparation procedure. Ethanol was detected indirectly by the reaction product of ethanol dehydrogenase (ADH) and cofactor nicotinamide adenine dinucleotide (NAD+). Method validation indicated good quantitation precisions with intra-day and inter-day relative standard deviations of ≤9.4% and 8.0%, respectively. Ethanol concentration in plasma is linear ranging from 0.10 to 3.20 mg/mL, and the detection limit is 40.0 μg/mL (S/N > 3). The method shows satisfactory correlation with the reference method of headspace gas chromatography in twenty human plasma samples (correlation coefficient 0.9311). The proposed method could be applied to diagnose acute ethanol toxicity or ethanol-related death. PMID:27006081

  5. The RFK catalytic cycle of the pathogen Streptococcus pneumoniae shows species-specific features in prokaryotic FMN synthesis.

    PubMed

    Sebastián, María; Velázquez-Campoy, Adrián; Medina, Milagros

    2018-12-01

    Emergence of multidrug-resistant bacteria forces us to explore new therapeutic strategies, and proteins involved in key metabolic pathways are promising anti-bacterial targets. Bifunctional flavin-adenine dinucleotide (FAD) synthetases (FADS) are prokaryotic enzymes that synthesise the flavin mononucleotide (FMN) and FAD cofactors. The FADS from the human pathogen Streptococcus pneumoniae (SpnFADS)-causative agent of pneumonia in humans - shows relevant catalytic dissimilarities compared to other FADSs. Here, by integrating thermodynamic and kinetic data, we present a global description of the riboflavin kinase activity of SpnFADS, as well as of the inhibition mechanisms regulating this activity. Our data shed light on biophysical determinants that modulate species-specific conformational changes leading to catalytically competent conformations, as well as binding rates and affinities of substrates versus products. This knowledge paves the way for the development of tools - that taking advantage of the regulatory dissimilarities during FMN biosynthesis in different species - might be used in the discovery of specific anti-pneumococcal drugs.

  6. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying the peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  7. Constraints on texture zero and cofactor zero models for neutrino mass

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Whisnant, K.; Liao, Jiajun; Marfatia, D.

    2014-06-24

    Imposing a texture or cofactor zero on the neutrino mass matrix reduces the number of independent parameters from nine to seven. Since five parameters have been measured, only two independent parameters would remain in such models. We find the allowed regions for single texture zero and single cofactor zero models. We also find strong similarities between single texture zero models with one mass hierarchy and single cofactor zero models with the opposite mass hierarchy. We show that this correspondence can be generalized to texture-zero and cofactor-zero models with the same homogeneous costraints on the elements and cofactors.

  8. Dissociative Excitation of Adenine by Electron Impact

    NASA Astrophysics Data System (ADS)

    McConkey, J. William; Trocchi, Joshuah; Dech, Jeffery; Kedzierski, Wladek

    2017-04-01

    Dissociative excitation of adenine (C6H5NH2) into excited atomic fragments has been studied in the electron impact energy range from threshold to 300 eV. A crossed beam system coupled to a vacuum ultraviolet (VUV) monochromator is used to study emissions in the wavelength range from 110 to 200 nm. The beam of adenine vapor from a stainless steel oven is crossed at right angles by the electron beam and the resultant UV radiation is detected in a mutually orthogonal direction. The strongest feature in the spectrum is H Lyman- α. Financial support from NSERC and CFI, Canada, is gratefully acknowledged.

  9. Polynucleotide: adenosine glycosidase activity of saporin-L1: effect on DNA, RNA and poly(A).

    PubMed Central

    Barbieri, L; Valbonesi, P; Gorini, P; Pession, A; Stirpe, F

    1996-01-01

    The ribosome-inactivating proteins (RIPs) are a family of plant enzymes for which a unique activity has been determined: rRNA N-glycosidase, which removes adenine at a specific universally conserved position (A4324 in the case of rat ribosomes). Here we report that saporin-L1, a RIP from the leaves of Saponaria officinalis, recognizes other substrates, including RNAs from different sources, DNA and poly(A). Saporin-L1 depurinated DNA extensively and released adenine from all adenine-containing polynucleotides tested. Adenine was the only base released from DNA or artificial polynucleotides. The characteristics of the reactions catalysed by saporin-L1 have been determined: optimal pH and temperature, ionic requirements, and the kinetic parameters Km and kcat. The reaction proceeded without cofactors, at low ionic strength, in the absence of Mg2+ and K+. Saporin-L1 had no activity towards various adenine-containing non-polynucleotide compounds (cytokinins, cofactors, nucleotides). This plant protein may now be classified as a polynucleotide: adenosine glycosidase. PMID:8912688

  10. Butyrate influences intracellular levels of adenine and adenine derivatives in the fungus Penicillium restrictum.

    PubMed

    Zutz, Christoph; Chiang, Yi Ming; Faehnrich, Bettina; Bacher, Markus; Hellinger, Roland; Kluger, Bernhard; Wagner, Martin; Strauss, Joseph; Rychli, Kathrin

    2017-04-01

    Butyrate, a small fatty acid, has an important role in the colon of ruminants and mammalians including the inhibition of inflammation and the regulation of cell proliferation. There is also growing evidence that butyrate is influencing the histone structure in mammalian cells by inhibition of histone deacetylation. Butyrate shows furthermore an antimicrobial activity against fungi, yeast and bacteria, which is linked to its toxicity at a high concentration. In fungi there are indications that butyrate induces the production of secondary metabolites potentially via inhibition of histone deacetylases. However, information about the influence of butyrate on growth, primary metabolite production and metabolism, besides lipid catabolism, in fungi is scarce. We have identified the filamentous fungus Penicillium (P.) restrictum as a susceptible target for butyrate treatment in an antimicrobial activity screen. The antimicrobial activity was detected only in the mycelium of the butyrate treated culture. We investigated the effect of butyrate ranging from low (0.001mM) to high (30mM), potentially toxic, concentrations on biomass and antimicrobial activity. Butyrate at high concentrations (3 and 30mM) significantly reduced the fungal biomass. In contrast P. restrictum treated with 0.03mM of butyrate showed the highest antimicrobial activity. We isolated three antimicrobial active compounds, active against Staphylococcus aureus, from P. restrictum cellular extracts treated with butyrate: adenine, its derivate hypoxanthine and the nucleoside derivate adenosine. Production of all three compounds was increased at low butyrate concentrations. Furthermore we found that butyrate influences the intracellular level of the adenine nucleoside derivate cAMP, an important signalling molecule in fungi and various organisms. In conclusion butyrate treatment increases the intracellular levels of adenine and its respective derivatives. Copyright © 2017 Elsevier GmbH. All rights reserved.

  11. Adenine formation from adenosine by mycoplasmas: adenosine phosphorylase activity.

    PubMed Central

    Hatanaka, M; Del Giudice, R; Long, C

    1975-01-01

    Mammalian cells have enzymes to convert adenosine to inosine by deamination and inosine to hypoxanthine by phosphorolysis, but they do not possess the enzymes necessary to form the free base, adenine, from adenosine. Mycoplasmas grown in broth or in cell cultures can produce adenine from adenosine. This activity was detected in a variety of mycoplasmatales, and the enzyme was shown to be adenosine phosphorylase. Adenosine formation from adenine and ribose 1-phosphate, the reverse reaction of adenine formation from adenosine, was also observed with the mycoplasma enzyme. Adenosine phosphorylase is apparently common to the mycoplasmatales but it is not universal, and the organisms can be divided into three groups on the basis of their use of adenosine as substrate. Thirteen of 16 Mycoplasma, Acholeplasma, and Siroplasma species tested exhibit adenosine phosphorylase activity. M. lipophilium differed from the other mycoplasmas and shared with mammalian cells the ability to convert adenosine to inosine by deamination. M. pneumoniae and the unclassified M. sp. 70-159 showed no reaction with adenosine. Adenosine phosphorylase activity offers an additional method for the detection of mycoplasma contamination of cells. The patterns of nucleoside metabolism will provide additional characteristics for identification of mycoplasmas and also may provide new insight into the classification of mycoplasmas. PMID:236559

  12. A disposable tear glucose biosensor--part 3: assessment of enzymatic specificity.

    PubMed

    Lan, Kenneth; McAferty, Kenyon; Shah, Pankti; Lieberman, Erica; Patel, Dharmendra R; Cook, Curtiss B; La Belle, Jeffrey T

    2011-09-01

    A concept for a tear glucose sensor based on amperometric measurement of enzymatic oxidation of glucose was previously presented, using glucose dehydrogenase flavin adenine dinucleotide (GDH-FAD) as the enzyme. Glucose dehydrogenase flavin adenine dinucleotide is further characterized in this article and evaluated for suitability in glucose-sensing applications in purified tear-like saline, with specific attention to the effect of interfering substances only. These interferents are specifically saccharides that could interact with the enzymatic activity seen in the sensor's performance. Bench top amperometric glucose assays were performed using an assay solution of GDH-FAD and ferricyanide redox mediator with samples of glucose, mannose, lactose, maltose, galactose, fructose, sucrose, and xylose at varying concentrations to evaluate specificity, linear dynamic range, signal size, and signal-to-noise ratio. A comparison study was done by substituting an equivalent activity unit concentration of glucose oxidase (GOx) for GDH-FAD. Glucose dehydrogenase flavin adenine dinucleotide was found to be more sensitive than GOx, producing larger oxidation currents than GOx on an identical glucose concentration gradient, and GDH-FAD exhibited larger slope response (-5.65 × 10(-7) versus -3.11 × 10(-7) A/mM), signal-to-noise ratio (18.04 versus 2.62), and linear dynamic range (0-30 versus 0-10 mM), and lower background signal (-7.12 versus -261.63 nA) than GOx under the same assay conditions. GDH-FAD responds equally to glucose and xylose but is otherwise specific for glucose. Glucose dehydrogenase flavin adenine dinucleotide compares favorably with GOx in many sensor-relevant attributes and may enable measurement of glucose concentrations both higher and lower than those measurable by GOx. GDH-FAD is a viable enzyme to use in the proposed amperometric tear glucose sensor system and perhaps also in detecting extreme hypoglycemia or hyperglycemia in blood. © 2011 Diabetes

  13. A Disposable Tear Glucose Biosensor—Part 3: Assessment of Enzymatic Specificity

    PubMed Central

    Lan, Kenneth; McAferty, Kenyon; Shah, Pankti; Lieberman, Erica; Patel, Dharmendra R; Cook, Curtiss B; La Belle, Jeffrey T

    2011-01-01

    Background A concept for a tear glucose sensor based on amperometric measurement of enzymatic oxidation of glucose was previously presented, using glucose dehydrogenase flavin adenine dinucleotide (GDH-FAD) as the enzyme. Glucose dehydrogenase flavin adenine dinucleotide is further characterized in this article and evaluated for suitability in glucose-sensing applications in purified tear-like saline, with specific attention to the effect of interfering substances only. These interferents are specifically saccharides that could interact with the enzymatic activity seen in the sensor's performance. Methods Bench top amperometric glucose assays were performed using an assay solution of GDH-FAD and ferricyanide redox mediator with samples of glucose, mannose, lactose, maltose, galactose, fructose, sucrose, and xylose at varying concentrations to evaluate specificity, linear dynamic range, signal size, and signal-to-noise ratio. A comparison study was done by substituting an equivalent activity unit concentration of glucose oxidase (GOx) for GDH-FAD. Results Glucose dehydrogenase flavin adenine dinucleotide was found to be more sensitive than GOx, producing larger oxidation currents than GOx on an identical glucose concentration gradient, and GDH-FAD exhibited larger slope response (-5.65 × 10-7 versus -3.11 × 10-7 A/mM), signal-to-noise ratio (18.04 versus 2.62), and linear dynamic range (0–30 versus 0–10 mM), and lower background signal (-7.12 versus -261.63 nA) than GOx under the same assay conditions. GDH-FAD responds equally to glucose and xylose but is otherwise specific for glucose. Conclusion Glucose dehydrogenase flavin adenine dinucleotide compares favorably with GOx in many sensor-relevant attributes and may enable measurement of glucose concentrations both higher and lower than those measurable by GOx. GDH-FAD is a viable enzyme to use in the proposed amperometric tear glucose sensor system and perhaps also in detecting extreme hypoglycemia or

  14. Profiling Redox and Energy Coenzymes in Whole Blood, Tissue and Cells Using NMR Spectroscopy.

    PubMed

    Gowda, G A Nagana

    2018-05-14

    Coenzymes of cellular redox reactions and cellular energy, as well as antioxidants mediate biochemical reactions fundamental to the functioning of all living cells. Conventional analysis methods lack the opportunity to evaluate these important redox and energy coenzymes and antioxidants in a single step. Major coenzymes include redox coenzymes: NAD⁺ (oxidized nicotinamide adenine dinucleotide), NADH (reduced nicotinamide adenine dinucleotide), NADP⁺ (oxidized nicotinamide adenine dinucleotide phosphate) and NADPH (reduced nicotinamide adenine dinucleotide phosphate); energy coenzymes: ATP (adenosine triphosphate), ADP (adenosine diphosphate) and AMP (adenosine monophosphate); and antioxidants: GSSG (oxidized glutathione) and GSH (reduced glutathione). We show here that a simple ¹H NMR experiment can measure these coenzymes and antioxidants in tissue and whole blood apart from a vast pool of other metabolites. In addition, focused on the goal of identification of coenzymes in subcellular fractions, we demonstrate analysis of coenzymes in the cytoplasm using breast cancer cells. Owing to their unstable nature, or low concentrations, most of the coenzymes either evade detection or lose their integrity when established sample preparation and analysis methods are used. To overcome this challenge, here we describe the development of new methods to detect these molecules without affecting the integrity of other metabolites. We used an array of 1D and 2D NMR methods, chemical shift databases, pH measurements and spiking with authentic compounds to establish the identity of peaks for the coenzymes and antioxidants in NMR spectra. Interestingly, while none of the coenzymes and antioxidants were detected in plasma, they were abundant in whole blood. Considering that the coenzymes and antioxidants represent a sensitive measure of human health and risk for numerous diseases, the presented NMR methods to measure them in one step potentially open new opportunities in the

  15. Multiple isotope effects with alternative dinucleotide substrates as a probe of the malic enzyme reaction

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Weiss, P.M.; Urbauer, J.L.; Cleland, W.W.

    1991-06-11

    Deuterium isotope effects and {sup 13}C isotope effects with deuterium- and protium-labeled malate have been obtained for both NAD- and NADP-malic enzymes by using a variety of alternative dinucleotide substrates. With nicotinamide-containing dinucleotides as the oxidizing substrate, the {sup 13}C effect decreases when deuterated malate is the substrate compared to the value obtained with protium-labeled malate. These data are consistent with a stepwise chemical mechanism in which hydride transfer precedes decarboxylation of the oxalacetate intermediate as previously proposed. When dinucleotide substrates such as thio-NAD, 3-nicotinamide rings are used, the {sup 13}C effect increases when deuterated malate is the substrate comparedmore » to the value obtained with protium-labeled malate. These data, at face value, are consistent with a change in mechanism from stepwise to concerted for the oxidative decarboxylation portion of the mechanism. However, the increase in the deuterium isotope effect from 1.5 to 3 with a concomitant decrease in the {sup 13}C isotope effect from 1.034 to 1.003 as the dinucleotide substrate is changed suggests that the reaction may still be stepwise with the non-nicotinamide dinucleotides. A more likely explanation is that a {beta}-secondary {sup 13}C isotope effect accompanies hydride transfer as a result of hyperconjugation of the {beta}-carboxyl of malate as the transition state for the hydride transfer step is approached.« less

  16. Was adenine the first purine?

    NASA Technical Reports Server (NTRS)

    Schwartz, Alan W.; Bakker, C. G.

    1989-01-01

    Oligomerization of HCN (1 molar) in the presence of added formaldehyde (0.5 molar) produced an order of magnitude more 8-hydroxymethyladenine than adenine or any other biologically significant purine. This result suggests that on the prebiotic earth, nucleoside analogs may have been synthesized directly in more complex mixtures of HCN with other aldehydes.

  17. Singlet Oxygen Generation by UVA Light Exposure of Endogenous Photosensitizers

    PubMed Central

    Baier, Jürgen; Maisch, Tim; Maier, Max; Engel, Eva; Landthaler, Michael; Bäumler, Wolfgang

    2006-01-01

    UVA light (320–400 nm) has been shown to produce deleterious biological effects in tissue due to the generation of singlet oxygen by substances like flavins or urocanic acid. Riboflavin, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), β-nicotinamide adenine dinucleotide (NAD), and β-nicotinamide adenine dinucleotide phosphate (NADP), urocanic acid, or cholesterol in solution were excited at 355 nm. Singlet oxygen was directly detected by time-resolved measurement of its luminescence at 1270 nm. NAD, NADP, and cholesterol showed no luminescence signal possibly due to the very low absorption coefficient at 355 nm. Singlet oxygen luminescence of urocanic acid was clearly detected but the signal was too weak to quantify a quantum yield. The quantum yield of singlet oxygen was precisely determined for riboflavin (ΦΔ = 0.54 ± 0.07), FMN (ΦΔ = 0.51 ± 0.07), and FAD (ΦΔ = 0.07 ± 0.02). In aerated solution, riboflavin and FMN generate more singlet oxygen than exogenous photosensitizers such as Photofrin, which are applied in photodynamic therapy to kill cancer cells. With decreasing oxygen concentration, the quantum yield of singlet oxygen generation decreased, which must be considered when assessing the role of singlet oxygen at low oxygen concentrations (inside tissue). PMID:16751234

  18. Biochemical and Structural Characterisation of DNA Ligases from Bacteria and Archaea.

    PubMed

    Pergolizzi, Giulia; Wagner, Gerd K; Bowater, Richard Peter

    2016-08-31

    DNA ligases are enzymes that seal breaks in the backbones of DNA, leading to them being essential for the survival of all organisms. DNA ligases have been studied from many different types of cells and organisms and shown to have diverse sizes and sequences, with well conserved specific sequences that are required for enzymatic activity. A significant number of DNA ligases have been isolated or prepared in recombinant forms and, here, we review their biochemical and structural characterisation. All DNA ligases contain an essential lysine that transfers an adenylate group from a co-factor to the 5'-phosphate of the DNA end that will ultimately be joined to the 3'-hydroxyl of the neighbouring DNA strand. The essential DNA ligases in bacteria use nicotinamide adenine dinucleotide ( β -NAD + ) as their co-factor whereas those that are essential in other cells use adenosine-5'-triphosphate (ATP) as their co-factor. This observation suggests that the essential bacterial enzyme could be targeted by novel antibiotics and the complex molecular structure of β -NAD + affords multiple opportunities for chemical modification. Several recent studies have synthesised novel derivatives and their biological activity against a range of DNA ligases has been evaluated as inhibitors for drug discovery and/or non-natural substrates for biochemical applications. Here, we review the recent advances that herald new opportunities to alter the biochemical activities of these important enzymes. The recent development of modified derivatives of nucleotides highlights that the continued combination of structural, biochemical and biophysical techniques will be useful in targeting these essential cellular enzymes. ©2016 The Author(s).

  19. The glmS Ribozyme Cofactor is a General Acid-Base Catalyst

    PubMed Central

    Viladoms, Julia; Fedor, Martha J.

    2012-01-01

    The glmS ribozyme is the first natural self-cleaving ribozyme known to require a cofactor. The D-glucosamine-6-phosphate (GlcN6P) cofactor has been proposed to serve as a general acid, but its role in the catalytic mechanism has not been established conclusively. We surveyed GlcN6P-like molecules for their ability to support self-cleavage of the glmS ribozyme and found a strong correlation between the pH dependence of the cleavage reaction and the intrinsic acidity of the cofactors. For cofactors with low binding affinities the contribution to rate enhancement was proportional to their intrinsic acidity. This linear free-energy relationship between cofactor efficiency and acid dissociation constants is consistent with a mechanism in which the cofactors participate directly in the reaction as general acid-base catalysts. A high value for the Brønsted coefficient (β ~ 0.7) indicates that a significant amount of proton transfer has already occurred in the transition state. The glmS ribozyme is the first self-cleaving RNA to use an exogenous acid-base catalyst. PMID:23113700

  20. The glmS ribozyme cofactor is a general acid-base catalyst.

    PubMed

    Viladoms, Júlia; Fedor, Martha J

    2012-11-21

    The glmS ribozyme is the first natural self-cleaving ribozyme known to require a cofactor. The d-glucosamine-6-phosphate (GlcN6P) cofactor has been proposed to serve as a general acid, but its role in the catalytic mechanism has not been established conclusively. We surveyed GlcN6P-like molecules for their ability to support self-cleavage of the glmS ribozyme and found a strong correlation between the pH dependence of the cleavage reaction and the intrinsic acidity of the cofactors. For cofactors with low binding affinities, the contribution to rate enhancement was proportional to their intrinsic acidity. This linear free-energy relationship between cofactor efficiency and acid dissociation constants is consistent with a mechanism in which the cofactors participate directly in the reaction as general acid-base catalysts. A high value for the Brønsted coefficient (β ~ 0.7) indicates that a significant amount of proton transfer has already occurred in the transition state. The glmS ribozyme is the first self-cleaving RNA to use an exogenous acid-base catalyst.

  1. Sequencing of adenine in DNA by scanning tunneling microscopy

    NASA Astrophysics Data System (ADS)

    Tanaka, Hiroyuki; Taniguchi, Masateru

    2017-08-01

    The development of DNA sequencing technology utilizing the detection of a tunnel current is important for next-generation sequencer technologies based on single-molecule analysis technology. Using a scanning tunneling microscope, we previously reported that dI/dV measurements and dI/dV mapping revealed that the guanine base (purine base) of DNA adsorbed onto the Cu(111) surface has a characteristic peak at V s = -1.6 V. If, in addition to guanine, the other purine base of DNA, namely, adenine, can be distinguished, then by reading all the purine bases of each single strand of a DNA double helix, the entire base sequence of the original double helix can be determined due to the complementarity of the DNA base pair. Therefore, the ability to read adenine is important from the viewpoint of sequencing. Here, we report on the identification of adenine by STM topographic and spectroscopic measurements using a synthetic DNA oligomer and viral DNA.

  2. Molybdenum cofactor in chlorate-resistant and nitrate reductase-deficient insertion mutants of Escherichia coli.

    PubMed Central

    Miller, J B; Amy, N K

    1983-01-01

    We examined molybdenum cofactor activity in chlorate-resistant (chl) and nitrate reductase-deficient (nar) insertion mutants and wild-type strains of Escherichia coli K-12. The bacterial molybdenum cofactor was assayed by its ability to restore activity to the cofactor-deficient nitrate reductase found in the nit-1 strain of Neurospora crassa. In the wild-type E. coli strains, molybdenum cofactor was synthesized constitutively and found in both cytoplasmic and membrane fractions. Cofactor was found in two forms: the demolybdo form required additional molybdate in the assay mix for detection, whereas the molybdenum-containing form was active without additional molybdate. The chlA and chlE mutants had no detectable cofactor. The chlB and the narG, narI, narK, and narL (previously designated chlC) strains had cofactor levels similar to those of the wild-type strains, except the chlB strains had two to threefold more membrane-bound cofactor. Cofactor levels in the chlD and chlG strains were sensitive to molybdate. When grown in 1 microM molybdate, the chlD strains had only 15 to 20% of the wild-type levels of the demolybdo and molybdenum-containing forms of the cofactor. In contrast, the chlG strains had near wild-type levels of demolybdo cofactor when grown in 1 microM molybdate, but none of the molybdenum-containing form of the cofactor. Near wild-type levels of both forms of the cofactor were restored to the chlD and chlG strains by growth in 1 mM molybdate. PMID:6307982

  3. Nicotinamide nucleotide transhydrogenase from Rhodobacter capsulatus; the H+/H- ratio and the activation state of the enzyme during reduction of acetyl pyridine adenine dinucleotide.

    PubMed

    Palmer, T; Jackson, J B

    1992-02-21

    Chromatophores from Rhodobacter capsulatus were incubated in the dark with NADPH and acetylpyridineadenine dinucleotide (AcPdAD+) in the presence of different concentrations of myxothiazol. The transhydrogenase activity was monitored until an appropriate mass action ratio, [AcPdAD+][NADPH]/[AcPdADH][NADP+], was reached. The sample was then illuminated and the initial rate of either AcPdAD+ reduction by NADPH or AcPdADH oxidation by NADP+ was recorded. The ratio of H+ translocated per H- equivalent transferred by transhydrogenase was calculated from the value of the membrane potential (delta pH = 0) at which illumination caused no net reaction in either direction. The mean value for the H+/H- ratio was 0.55. At greater values of [AcPdAD+][NADPH]/[AcPdADH][NADP+] than were employed in the above experiments and over a wider range of concentrations of myxothiazol, it was found that incremental increases in the membrane potential always gave rise to a decrease, never an increase in the rate of AcPdAD+ reduction. In contrast to the H(+)-ATP synthase, there is no evidence of any activation/deactivation of H(+)-transhydrogenase by the protonmotive force.

  4. Characterization of plant carotenoid cyclases as members of the flavoprotein family functioning with no net redox change.

    PubMed

    Mialoundama, Alexis Samba; Heintz, Dimitri; Jadid, Nurul; Nkeng, Paul; Rahier, Alain; Deli, Jozsef; Camara, Bilal; Bouvier, Florence

    2010-07-01

    The later steps of carotenoid biosynthesis involve the formation of cyclic carotenoids. The reaction is catalyzed by lycopene beta-cyclase (LCY-B), which converts lycopene into beta-carotene, and by capsanthin-capsorubin synthase (CCS), which is mainly dedicated to the synthesis of kappa-cyclic carotenoids (capsanthin and capsorubin) but also has LCY-B activity. Although the peptide sequences of plant LCY-Bs and CCS contain a putative dinucleotide-binding motif, it is believed that these two carotenoid cyclases proceed via protic activation and stabilization of resulting carbocation intermediates. Using pepper (Capsicum annuum) CCS as a prototypic carotenoid cyclase, we show that the monomeric protein contains one noncovalently bound flavin adenine dinucleotide (FAD) that is essential for enzyme activity only in the presence of NADPH, which functions as the FAD reductant. The reaction proceeds without transfer of hydrogen from the dinucleotide cofactors to beta-carotene or capsanthin. Using site-directed mutagenesis, amino acids potentially involved in the protic activation were identified. Substitutions of alanine, lysine, and arginine for glutamate-295 in the conserved 293-FLEET-297 motif of pepper CCS or LCY-B abolish the formation of beta-carotene and kappa-cyclic carotenoids. We also found that mutations of the equivalent glutamate-196 located in the 194-LIEDT-198 domain of structurally divergent bacterial LCY-B abolish the formation of beta-carotene. The data herein reveal plant carotenoid cyclases to be novel enzymes that combine characteristics of non-metal-assisted terpene cyclases with those attributes typically found in flavoenzymes that catalyze reactions, with no net redox, such as type 2 isopentenyl diphosphate isomerase. Thus, FAD in its reduced form could be implicated in the stabilization of the carbocation intermediate.

  5. Purification and Characterization of Active-Site Components of the Putative p-Cresol Methylhydroxylase Membrane Complex from Geobacter metallireducens▿

    PubMed Central

    Johannes, Jörg; Bluschke, Alexander; Jehmlich, Nico; von Bergen, Martin; Boll, Matthias

    2008-01-01

    p-Cresol methylhydroxylases (PCMH) from aerobic and facultatively anaerobic bacteria are soluble, periplasmic flavocytochromes that catalyze the first step in biological p-cresol degradation, the hydroxylation of the substrate with water. Recent results suggested that p-cresol degradation in the strictly anaerobic Geobacter metallireducens involves a tightly membrane-bound PCMH complex. In this work, the soluble components of this complex were purified and characterized. The data obtained suggest a molecular mass of 124 ± 15 kDa and a unique αα′β2 subunit composition, with α and α′ representing isoforms of the flavin adenine dinucleotide (FAD)-containing subunit and β representing a c-type cytochrome. Fluorescence and mass spectrometric analysis suggested that one FAD was covalently linked to Tyr394 of the α subunit. In contrast, the α′ subunit did not contain any FAD cofactor and is therefore considered to be catalytically inactive. The UV/visible spectrum was typical for a flavocytochrome with two heme c cofactors and one FAD cofactor. p-Cresol reduced the FAD but only one of the two heme cofactors. PCMH catalyzed both the hydroxylation of p-cresol to p-hydroxybenzyl alcohol and the subsequent oxidation of the latter to p-hydroxybenzaldehyde in the presence of artificial electron acceptors. The very low Km values (1.7 and 2.7 μM, respectively) suggest that the in vivo function of PCMH is to oxidize both p-cresol and p-hydroxybenzyl alcohol. The latter was a mixed inhibitor of p-cresol oxidation, with inhibition constants of a Kic (competitive inhibition) value of 18 ± 9 μM and a Kiu (uncompetitive inhibition) value of 235 ± 20 μM. A putative functional model for an unusual PCMH enzyme is presented. PMID:18658262

  6. Redox cofactors insertion in prokaryotic molybdoenzymes occurs via a conserved folding mechanism

    PubMed Central

    Arias-Cartin, Rodrigo; Ceccaldi, Pierre; Schoepp-Cothenet, Barbara; Frick, Klaudia; Blanc, Jean-Michel; Guigliarelli, Bruno; Walburger, Anne; Grimaldi, Stéphane; Friedrich, Thorsten; Receveur-Brechot, Véronique; Magalon, Axel

    2016-01-01

    A major gap of knowledge in metalloproteins is the identity of the prefolded state of the protein before cofactor insertion. This holds for molybdoenzymes serving multiple purposes for life, especially in energy harvesting. This large group of prokaryotic enzymes allows for coordination of molybdenum or tungsten cofactors (Mo/W-bisPGD) and Fe/S clusters. Here we report the structural data on a cofactor-less enzyme, the nitrate reductase respiratory complex and characterize the conformational changes accompanying Mo/W-bisPGD and Fe/S cofactors insertion. Identified conformational changes are shown to be essential for recognition of the dedicated chaperone involved in cofactors insertion. A solvent-exposed salt bridge is shown to play a key role in enzyme folding after cofactors insertion. Furthermore, this salt bridge is shown to be strictly conserved within this prokaryotic molybdoenzyme family as deduced from a phylogenetic analysis issued from 3D structure-guided multiple sequence alignment. A biochemical analysis with a distantly-related member of the family, respiratory complex I, confirmed the critical importance of the salt bridge for folding. Overall, our results point to a conserved cofactors insertion mechanism within the Mo/W-bisPGD family. PMID:27886223

  7. The family of berberine bridge enzyme-like enzymes: A treasure-trove of oxidative reactions.

    PubMed

    Daniel, Bastian; Konrad, Barbara; Toplak, Marina; Lahham, Majd; Messenlehner, Julia; Winkler, Andreas; Macheroux, Peter

    2017-10-15

    Biological oxidations form the basis of life on earth by utilizing organic compounds as electron donors to drive the generation of metabolic energy carriers, such as ATP. Oxidative reactions are also important for the biosynthesis of complex compounds, i.e. natural products such as alkaloids that provide vital benefits for organisms in all kingdoms of life. The vitamin B 2 -derived cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) enable an astonishingly diverse array of oxidative reactions that is based on the versatility of the redox-active isoalloxazine ring. The family of FAD-linked oxidases can be divided into subgroups depending on specific sequence features in an otherwise very similar structural context. The sub-family of berberine bridge enzyme (BBE)-like enzymes has recently attracted a lot of attention due to the challenging chemistry catalyzed by its members and the unique and unusual bi-covalent attachment of the FAD cofactor. This family is the focus of the present review highlighting recent advancements into the structural and functional aspects of members from bacteria, fungi and plants. In view of the unprecedented reaction catalyzed by the family's namesake, BBE from the California poppy, recent studies have provided further insights into nature's treasure chest of oxidative reactions. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  8. A General Tool for Engineering the NAD/NADP Cofactor Preference of Oxidoreductases.

    PubMed

    Cahn, Jackson K B; Werlang, Caroline A; Baumschlager, Armin; Brinkmann-Chen, Sabine; Mayo, Stephen L; Arnold, Frances H

    2017-02-17

    The ability to control enzymatic nicotinamide cofactor utilization is critical for engineering efficient metabolic pathways. However, the complex interactions that determine cofactor-binding preference render this engineering particularly challenging. Physics-based models have been insufficiently accurate and blind directed evolution methods too inefficient to be widely adopted. Building on a comprehensive survey of previous studies and our own prior engineering successes, we present a structure-guided, semirational strategy for reversing enzymatic nicotinamide cofactor specificity. This heuristic-based approach leverages the diversity and sensitivity of catalytically productive cofactor binding geometries to limit the problem to an experimentally tractable scale. We demonstrate the efficacy of this strategy by inverting the cofactor specificity of four structurally diverse NADP-dependent enzymes: glyoxylate reductase, cinnamyl alcohol dehydrogenase, xylose reductase, and iron-containing alcohol dehydrogenase. The analytical components of this approach have been fully automated and are available in the form of an easy-to-use web tool: Cofactor Specificity Reversal-Structural Analysis and Library Design (CSR-SALAD).

  9. Studies on free radicals, antioxidants, and co-factors

    PubMed Central

    Rahman, Khalid

    2007-01-01

    The interplay between free radicals, antioxidants, and co-factors is important in maintaining health, aging and age-related diseases. Free radicals induce oxidative stress, which is balanced by the body’s endogenous antioxidant systems with an input from co-factors, and by the ingestion of exogenous antioxidants. If the generation of free radicals exceeds the protective effects of antioxidants, and some co-factors, this can cause oxidative damage which accumulates during the life cycle, and has been implicated in aging, and age dependent diseases such as cardiovascular disease, cancer, neurodegenerative disorders, and other chronic conditions. The life expectancy of the world population is increasing, and it is estimated that by 2025, 29% of the world population will be aged ≥60 years, and this will lead to an increase in the number of older people acquiring age-related chronic diseases. This will place greater financial burden on health services and high social cost for individuals and society. In order to acheive healthy aging the older people should be encouraged to acquire healthy life styles which should include diets rich in antioxidants. The aim of this review is to highlight the main themes from studies on free radicals, antioxidants and co-factors, and to propose an evidence-based strategy for healthy aging. PMID:18044138

  10. Electron transfer driven decomposition of adenine and selected analogs as probed by experimental and theoretical methods

    NASA Astrophysics Data System (ADS)

    Cunha, T.; Mendes, M.; Ferreira da Silva, F.; Eden, S.; García, G.; Bacchus-Montabonel, M.-C.; Limão-Vieira, P.

    2018-04-01

    We report on a combined experimental and theoretical study of electron-transfer-induced decomposition of adenine (Ad) and a selection of analog molecules in collisions with potassium (K) atoms. Time-of-flight negative ion mass spectra have been obtained in a wide collision energy range (6-68 eV in the centre-of-mass frame), providing a comprehensive investigation of the fragmentation patterns of purine (Pu), adenine (Ad), 9-methyl adenine (9-mAd), 6-dimethyl adenine (6-dimAd), and 2-D adenine (2-DAd). Following our recent communication about selective hydrogen loss from the transient negative ions (TNIs) produced in these collisions [T. Cunha et al., J. Chem. Phys. 148, 021101 (2018)], this work focuses on the production of smaller fragment anions. In the low-energy part of the present range, several dissociation channels that are accessible in free electron attachment experiments are absent from the present mass spectra, notably NH2 loss from adenine and 9-methyl adenine. This can be understood in terms of a relatively long transit time of the K+ cation in the vicinity of the TNI tending to enhance the likelihood of intramolecular electron transfer. In this case, the excess energy can be redistributed through the available degrees of freedom inhibiting fragmentation pathways. Ab initio theoretical calculations were performed for 9-methyl adenine (9-mAd) and adenine (Ad) in the presence of a potassium atom and provided a strong basis for the assignment of the lowest unoccupied molecular orbitals accessed in the collision process.

  11. Which Came First, Proteins or Cofactors? Recreating Metabolic Reactions of the Early Earth

    NASA Astrophysics Data System (ADS)

    Maltais, T. R.; VanderVelde, D.; LaRowe, D.; Goldman, A. D.; Barge, L. M.

    2017-07-01

    We test whether cofactors can promote parts of core metabolic pathways by examining Coenzyeme A (CoA), the cofactor central to citrate synthesis in the citric acid cycle, as a target for examining cofactor activity without its protein enzyme.

  12. Transformation by Complementation of an Adenine Auxotroph of the Lignin-Degrading Basidiomycete Phanerochaete chrysosporium

    PubMed Central

    Alic, Margaret; Kornegay, Janet R.; Pribnow, David; Gold, Michael H.

    1989-01-01

    Swollen basidiospores of an adenine auxotroph of Phanerochaete chrysosporium were protoplasted with Novozyme 234 and transformed to prototrophy by using a plasmid containing the gene for an adenine biosynthetic enzyme from Schizophyllum commune. Transformation frequencies of 100 transformants per μg of DNA were obtained. Southern blot analysis of DNA extracted from transformants demonstrated that plasmid DNA was integrated into the chromosomal DNA in multiple tandem copies. Analysis of conidia and basidiospores from transformants demonstrated that the transforming character was mitotically and meiotically stable on both selective and nonselective media. Genetic crosses between double mutants transformed for adenine prototrophy and other auxotrophic strains yielded Ade− progeny, which indicated that integration occurred at a site(s) other than the resident adenine biosynthetic gene. Images PMID:16347848

  13. A mutational analysis of U12-dependent splice site dinucleotides

    PubMed Central

    DIETRICH, ROSEMARY C.; FULLER, JOHN D.; PADGETT, RICHARD A.

    2005-01-01

    Introns spliced by the U12-dependent minor spliceosome are divided into two classes based on their splice site dinucleotides. The /AU-AC/ class accounts for about one-third of U12-dependent introns in humans, while the /GU-AG/ class accounts for the other two-thirds. We have investigated the in vivo and in vitro splicing phenotypes of mutations in these dinucleotide sequences. A 5′ A residue can splice to any 3′ residue, although C is preferred. A 5′ G residue can splice to 3′ G or U residues with a preference for G. Little or no splicing was observed to 3′ A or C residues. A 5′ U or C residue is highly deleterious for U12-dependent splicing, although some combinations, notably 5′ U to 3′ U produced detectable spliced products. The dependence of 3′ splice site activity on the identity of the 5′ residue provides evidence for communication between the first and last nucleotides of the intron. Most mutants in the second position of the 5′ splice site and the next to last position of the 3′ splice site were defective for splicing. Double mutants of these residues showed no evidence of communication between these nucleotides. Varying the distance between the branch site and the 3′ splice site dinucleotide in the /GU-AG/ class showed that a somewhat larger range of distances was functional than for the /AU-AC/ class. The optimum branch site to 3′ splice site distance of 11–12 nucleotides appears to be the same for both classes. PMID:16043500

  14. TRAF6-Mediated SM22α K21 Ubiquitination Promotes G6PD Activation and NADPH Production, Contributing to GSH Homeostasis and VSMC Survival In Vitro and In Vivo.

    PubMed

    Dong, Li-Hua; Li, Liang; Song, Yu; Duan, Zhi-Li; Sun, Shao-Guang; Lin, Yan-Ling; Miao, Sui-Bing; Yin, Ya-Juan; Shu, Ya-Nan; Li, Huan; Chen, Peng; Zhao, Li-Li; Han, Mei

    2015-09-25

    Vascular smooth muscle cell (VSMC) survival under stressful conditions is integral to promoting vascular repair, but facilitates plaque stability during the development of atherosclerosis. The cytoskeleton-associated smooth muscle (SM) 22α protein is involved in the regulation of VSMC phenotypes, whereas the pentose phosphate pathway plays an essential role in cell proliferation through the production of dihydronicotinamide adenine dinucleotide phosphate. To identify the relationship between dihydronicotinamide adenine dinucleotide phosphate production and SM22α activity in the development and progression of vascular diseases. We showed that the expression and activity of glucose-6-phosphate dehydrogenase (G6PD) are promoted in platelet-derived growth factor (PDGF)-BB-induced proliferative VSMCs. PDGF-BB induced G6PD membrane translocation and activation in an SM22α K21 ubiquitination-dependent manner. Specifically, the ubiquitinated SM22α interacted with G6PD and mediated G6PD membrane translocation. Furthermore, we found that tumor necrosis factor receptor-associated factor (TRAF) 6 mediated SM22α K21 ubiquitination in a K63-linked manner on PDGF-BB stimulation. Knockdown of TRAF6 decreased the membrane translocation and activity of G6PD, in parallel with reduced SM22α K21 ubiquitination. Elevated levels of activated G6PD consequent to PDGF-BB induction led to increased dihydronicotinamide adenine dinucleotide phosphate generation through stimulation of the pentose phosphate pathway, which enhanced VSMC viability and reduced apoptosis in vivo and in vitro via glutathione homeostasis. We provide evidence that TRAF6-induced SM22α ubiquitination maintains VSMC survival through increased G6PD activity and dihydronicotinamide adenine dinucleotide phosphate production. The TRAF6-SM22α-G6PD pathway is a novel mechanism underlying the association between glucose metabolism and VSMC survival, which is beneficial for vascular repair after injury but facilitates

  15. Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase*

    PubMed Central

    Hu, Yilin; Ribbe, Markus W.

    2013-01-01

    The iron-molybdenum cofactor (the M-cluster) serves as the active site of molybdenum nitrogenase. Arguably one of the most complex metal cofactors in biological systems, the M-cluster is assembled through the formation of an 8Fe core prior to the insertion of molybdenum and homocitrate into this core. Here, we review the recent progress in the research area of M-cluster assembly, with an emphasis on our work that provides useful insights into the mechanistic details of this process. PMID:23539617

  16. Underlying mechanisms for syntrophic metabolism of essential enzyme cofactors in microbial communities

    PubMed Central

    Romine, Margaret F; Rodionov, Dmitry A; Maezato, Yukari; Osterman, Andrei L; Nelson, William C

    2017-01-01

    Many microorganisms are unable to synthesize essential B vitamin-related enzyme cofactors de novo. The underlying mechanisms by which such microbes survive in multi-species communities are largely unknown. We previously reported the near-complete genome sequence of two ~18-member unicyanobacterial microbial consortia that maintain stable membership on defined medium lacking vitamins. Here we have used genome analysis and growth studies on isolates derived from the consortia to reconstruct pathways for biogenesis of eight essential cofactors and predict cofactor usage and precursor exchange in these communities. Our analyses revealed that all but the two Halomonas and cyanobacterial community members were auxotrophic for at least one cofactor. We also observed a mosaic distribution of salvage routes for a variety of cofactor precursors, including those produced by photolysis. Potentially bidirectional transporters were observed to be preferentially in prototrophs, suggesting a mechanism for controlled precursor release. Furthermore, we found that Halomonas sp. do not require cobalamin nor control its synthesis, supporting the hypothesis that they overproduce and export vitamins. Collectively, these observations suggest that the consortia rely on syntrophic metabolism of cofactors as a survival strategy for optimization of metabolic exchange within a shared pool of micronutrients. PMID:28186498

  17. Transformation by complementation of an adenine auxotroph of the lignin-degrading basidiomycete Phanerochaete chrysosporium

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Alic, M.; Kornegay, J.R.; Pribnow, D.

    1989-02-01

    Swollen basiodiospores of an adenine auxotroph of Phanerochaete chrysosporium were protoplasted with Novozyme 234 and transformed to prototrophy by using a plasmid containing the gene for an adenine biosynthetic enzyme from Schizophyllum commune. Transformation frequencies of 100 transformants per {mu}g of DNA were obtained. Southern blot analysis of DNA extracted from transformants demonstrated that plasmid DNA was integrated into the chromosomal DNA in multiple tandem copies. Analysis of conidia and basiodiospores from transformants demonstrated that the transforming character was mitotically and meiotically stable on both selective and nonselective media. Genetic crosses between double mutants transformed for adenine prototrophy and othermore » auxotrophic strains yielded Ade{sup {minus}} progeny, which indicated that integration occurred at a site(s) other than the resident adenine biosynthetic gene.« less

  18. Bleaching herbicide norflurazon inhibits phytoene desaturase by competition with the cofactors.

    PubMed

    Breitenbach, J; Zhu, C; Sandmann, G

    2001-11-01

    Cofactor requirement was determined for the heterologous expressed phytoene desaturases from the cyanobacterium Synechococcus and the higher plant Gentiana lutea. The cyanobacterial enzyme is dependent on either NAD(P) or plastoquinone, whereas only quinones such as plastoquinone can function as a cofactor for the phytoene desaturase from G. lutea. Enzyme kinetic studies were carried out to determine a possible competition between the cofactors and the bleaching herbicide norflurazon. For the Synechococcus enzyme, competition between norflurazon and NADP, as well as plastoquinone, could be demonstrated. The K(m) values for these cofactors were 6.6 mM and 0.23 microM, respectively. Inhibition of the phytoene desaturase from G. lutea by norflurazon was also competitive with respect to plastoquinone. The K(m) values of both enzymes for plastoquinone were very close.

  19. Structural and functional analysis of betaine aldehyde dehydrogenase from Staphylococcus aureus.

    PubMed

    Halavaty, Andrei S; Rich, Rebecca L; Chen, Chao; Joo, Jeong Chan; Minasov, George; Dubrovska, Ievgeniia; Winsor, James R; Myszka, David G; Duban, Mark; Shuvalova, Ludmilla; Yakunin, Alexander F; Anderson, Wayne F

    2015-05-01

    When exposed to high osmolarity, methicillin-resistant Staphylococcus aureus (MRSA) restores its growth and establishes a new steady state by accumulating the osmoprotectant metabolite betaine. Effective osmoregulation has also been implicated in the acquirement of a profound antibiotic resistance by MRSA. Betaine can be obtained from the bacterial habitat or produced intracellularly from choline via the toxic betaine aldehyde (BA) employing the choline dehydrogenase and betaine aldehyde dehydrogenase (BADH) enzymes. Here, it is shown that the putative betaine aldehyde dehydrogenase SACOL2628 from the early MRSA isolate COL (SaBADH) utilizes betaine aldehyde as the primary substrate and nicotinamide adenine dinucleotide (NAD(+)) as the cofactor. Surface plasmon resonance experiments revealed that the affinity of NAD(+), NADH and BA for SaBADH is affected by temperature, pH and buffer composition. Five crystal structures of the wild type and three structures of the Gly234Ser mutant of SaBADH in the apo and holo forms provide details of the molecular mechanisms of activity and substrate specificity/inhibition of this enzyme.

  20. A Disposable paper breathalyzer with an alcohol sensing organic electrochemical transistor

    NASA Astrophysics Data System (ADS)

    Bihar, Eloїse; Deng, Yingxin; Miyake, Takeo; Saadaoui, Mohamed; Malliaras, George G.; Rolandi, Marco

    2016-06-01

    Breathalyzers estimate Blood Alcohol Content (BAC) from the concentration of ethanol in the breath. Breathalyzers are easy to use but are limited either by their high price and by environmental concerns, or by a short lifetime and the need for continuous recalibration. Here, we demonstrate a proof-of-concept disposable breathalyzer using an organic electrochemical transistor (OECT) modified with alcohol dehydrogenase (ADH) as the sensor. The OECT is made with the conducting polymer poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS), and is printed on paper. ADH and its cofactor nicotinamide adenine dinucleotide (NAD+) are immobilized onto the OECT with an electrolyte gel. When the OECT-breathalyzer is exposed to ethanol vapor, the enzymatic reaction of ADH and ethanol transforms NAD+ into NADH, which causes a decrease in the OECT source drain current. In this fashion, the OECT-breathalyzer easily detects ethanol in the breath equivalent to BAC from 0.01% to 0.2%. The use of a printed OECT may contribute to the development of breathalyzers that are disposable, ecofriendly, and integrated with wearable devices for real-time BAC monitoring.

  1. Discovery of antimicrobial compounds targeting bacterial type FAD synthetases.

    PubMed

    Sebastián, María; Anoz-Carbonell, Ernesto; Gracia, Begoña; Cossio, Pilar; Aínsa, José Antonio; Lans, Isaías; Medina, Milagros

    2018-12-01

    The increase of bacterial strains resistant to most of the available antibiotics shows a need to explore novel antibacterial targets to discover antimicrobial drugs. Bifunctional bacterial FAD synthetases (FADSs) synthesise the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These cofactors act in vital processes as part of flavoproteins, making FADS an essential enzyme. Bacterial FADSs are potential antibacterial targets because of differences to mammalian enzymes, particularly at the FAD producing site. We have optimised an activity-based high throughput screening assay targeting Corynebacterium ammoniagenes FADS (CaFADS) that identifies inhibitors of its different activities. We selected the three best high-performing inhibitors of the FMN:adenylyltransferase activity (FMNAT) and studied their inhibition mechanisms and binding properties. The specificity of the CaFADS hits was evaluated by studying also their effect on the Streptococcus pneumoniae FADS activities, envisaging differences that can be used to discover species-specific antibacterial drugs. The antimicrobial effect of these compounds was also evaluated on C. ammoniagenes, S. pneumoniae, and Mycobacterium tuberculosis cultures, finding hits with favourable antimicrobial properties.

  2. A Disposable paper breathalyzer with an alcohol sensing organic electrochemical transistor

    PubMed Central

    Bihar, Eloїse; Deng, Yingxin; Miyake, Takeo; Saadaoui, Mohamed; Malliaras, George G.; Rolandi, Marco

    2016-01-01

    Breathalyzers estimate Blood Alcohol Content (BAC) from the concentration of ethanol in the breath. Breathalyzers are easy to use but are limited either by their high price and by environmental concerns, or by a short lifetime and the need for continuous recalibration. Here, we demonstrate a proof-of-concept disposable breathalyzer using an organic electrochemical transistor (OECT) modified with alcohol dehydrogenase (ADH) as the sensor. The OECT is made with the conducting polymer poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS), and is printed on paper. ADH and its cofactor nicotinamide adenine dinucleotide (NAD+) are immobilized onto the OECT with an electrolyte gel. When the OECT-breathalyzer is exposed to ethanol vapor, the enzymatic reaction of ADH and ethanol transforms NAD+ into NADH, which causes a decrease in the OECT source drain current. In this fashion, the OECT-breathalyzer easily detects ethanol in the breath equivalent to BAC from 0.01% to 0.2%. The use of a printed OECT may contribute to the development of breathalyzers that are disposable, ecofriendly, and integrated with wearable devices for real-time BAC monitoring. PMID:27291059

  3. Study of Proton Transfer in E. Coli Photolyase

    NASA Astrophysics Data System (ADS)

    Zhang, Meng; Liu, Zheyun; Li, Jiang; Wang, Lijuan; Zhong, Dongping

    2013-06-01

    Photolyase is a flavoprotein which utilizes blue-light energy to repair UV-light damaged DNA. The catalytic cofactor of photolyase, flavin adenine dinucleotide (FAD), has five redox states. Conversions between these redox states involve intraprotein electron transfer and proton transfer, which play important role in protein function. Here we systematically studied proton transfer in E. coli photolyase in vitro by site-directed mutagenesis and steady-state UV-vis spectroscopy, and proposed the proton channel in photolyase. We found that in the mutant N378C/E363L, proton channel was completely eliminated when DNA substrate was bound to the protein. Proton is suggested to be transported from protein surface to FAD by two pathways: the proton relay pathway through E363 and surface water to N378 and then to FAD; and the proton diffusion pathway through the substrate binding pocket. In addition, reaction kinetics of conversions between the redox states was then solved and redox potentials of the redox states were determined. These results described a complete picture of FAD redox changes, which are fundamental to the functions of all flavoenzymes.

  4. Structural and functional analysis of betaine aldehyde dehydrogenase from Staphylococcus aureus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Halavaty, Andrei S.; Rich, Rebecca L.; Chen, Chao

    When exposed to high osmolarity, methicillin-resistant Staphylococcus aureus (MRSA) restores its growth and establishes a new steady state by accumulating the osmoprotectant metabolite betaine. Effective osmoregulation has also been implicated in the acquirement of a profound antibiotic resistance by MRSA. Betaine can be obtained from the bacterial habitat or produced intracellularly from choline via the toxic betaine aldehyde (BA) employing the choline dehydrogenase and betaine aldehyde dehydrogenase (BADH) enzymes. Here, it is shown that the putative betaine aldehyde dehydrogenase SACOL2628 from the early MRSA isolate COL ( SaBADH) utilizes betaine aldehyde as the primary substrate and nicotinamide adenine dinucleotide (NADmore » +) as the cofactor. Surface plasmon resonance experiments revealed that the affinity of NAD +, NADH and BA for SaBADH is affected by temperature, pH and buffer composition. Finally, five crystal structures of the wild type and three structures of the Gly234Ser mutant of SaBADH in the apo and holo forms provide details of the molecular mechanisms of activity and substrate specificity/inhibition of this enzyme.« less

  5. Free energy landscape of the Michaelis complex of lactate dehydrogenase: A network analysis of atomistic simulations

    NASA Astrophysics Data System (ADS)

    Pan, Xiaoliang; Schwartz, Steven

    2015-03-01

    It has long been recognized that the structure of a protein is a hierarchy of conformations interconverting on multiple time scales. However, the conformational heterogeneity is rarely considered in the context of enzymatic catalysis in which the reactant is usually represented by a single conformation of the enzyme/substrate complex. Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of two forms of the cofactor nicotinamide adenine dinucleotide (NADH and NAD+). Recent experimental results suggest that multiple substates exist within the Michaelis complex of LDH, and they are catalytic competent at different reaction rates. In this study, millisecond-scale all-atom molecular dynamics simulations were performed on LDH to explore the free energy landscape of the Michaelis complex, and network analysis was used to characterize the distribution of the conformations. Our results provide a detailed view of the kinetic network the Michaelis complex and the structures of the substates at atomistic scale. It also shed some light on understanding the complete picture of the catalytic mechanism of LDH.

  6. Genetic Ablation of CD38 Protects against Western Diet-Induced Exercise Intolerance and Metabolic Inflexibility.

    PubMed

    Chiang, Shian-Huey; Harrington, W Wallace; Luo, Guizhen; Milliken, Naphtali O; Ulrich, John C; Chen, Jing; Rajpal, Deepak K; Qian, Ying; Carpenter, Tiffany; Murray, Rusty; Geske, Robert S; Stimpson, Stephen A; Kramer, Henning F; Haffner, Curt D; Becherer, J David; Preugschat, Frank; Billin, Andrew N

    2015-01-01

    Nicotinamide adenine dinucleotide (NAD+) is a key cofactor required for essential metabolic oxidation-reduction reactions. It also regulates various cellular activities, including gene expression, signaling, DNA repair and calcium homeostasis. Intracellular NAD+ levels are tightly regulated and often respond rapidly to nutritional and environmental changes. Numerous studies indicate that elevating NAD+ may be therapeutically beneficial in the context of numerous diseases. However, the role of NAD+ on skeletal muscle exercise performance is poorly understood. CD38, a multi-functional membrane receptor and enzyme, consumes NAD+ to generate products such as cyclic-ADP-ribose. CD38 knockout mice show elevated tissue and blood NAD+ level. Chronic feeding of high-fat, high-sucrose diet to wild type mice leads to exercise intolerance and reduced metabolic flexibility. Loss of CD38 by genetic mutation protects mice from diet-induced metabolic deficit. These animal model results suggest that elevation of tissue NAD+ through genetic ablation of CD38 can profoundly alter energy homeostasis in animals that are maintained on a calorically-excessive Western diet.

  7. Free energy surface of the Michaelis complex of lactate dehydrogenase: a network analysis of microsecond simulations.

    PubMed

    Pan, Xiaoliang; Schwartz, Steven D

    2015-04-30

    It has long been recognized that the structure of a protein creates a hierarchy of conformations interconverting on multiple time scales. The conformational heterogeneity of the Michaelis complex is of particular interest in the context of enzymatic catalysis in which the reactant is usually represented by a single conformation of the enzyme/substrate complex. Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of two forms of the cofactor nicotinamide adenine dinucleotide (NADH and NAD(+)). Recent experimental results suggest that multiple substates exist within the Michaelis complex of LDH, and they show a strong variance in their propensity toward the on-enzyme chemical step. In this study, microsecond-scale all-atom molecular dynamics simulations were performed on LDH to explore the free energy landscape of the Michaelis complex, and network analysis was used to characterize the distribution of the conformations. Our results provide a detailed view of the kinetic network of the Michaelis complex and the structures of the substates at atomistic scales. They also shed light on the complete picture of the catalytic mechanism of LDH.

  8. Structural and functional analysis of betaine aldehyde dehydrogenase from Staphylococcus aureus

    DOE PAGES

    Halavaty, Andrei S.; Rich, Rebecca L.; Chen, Chao; ...

    2015-04-25

    When exposed to high osmolarity, methicillin-resistant Staphylococcus aureus (MRSA) restores its growth and establishes a new steady state by accumulating the osmoprotectant metabolite betaine. Effective osmoregulation has also been implicated in the acquirement of a profound antibiotic resistance by MRSA. Betaine can be obtained from the bacterial habitat or produced intracellularly from choline via the toxic betaine aldehyde (BA) employing the choline dehydrogenase and betaine aldehyde dehydrogenase (BADH) enzymes. Here, it is shown that the putative betaine aldehyde dehydrogenase SACOL2628 from the early MRSA isolate COL ( SaBADH) utilizes betaine aldehyde as the primary substrate and nicotinamide adenine dinucleotide (NADmore » +) as the cofactor. Surface plasmon resonance experiments revealed that the affinity of NAD +, NADH and BA for SaBADH is affected by temperature, pH and buffer composition. Finally, five crystal structures of the wild type and three structures of the Gly234Ser mutant of SaBADH in the apo and holo forms provide details of the molecular mechanisms of activity and substrate specificity/inhibition of this enzyme.« less

  9. Characterization of wheat endoplasmic reticulum oxidoreductin 1 and its application in Chinese steamed bread.

    PubMed

    Liu, Guang; Wang, JingJing; Hou, Yi; Huang, Yan-Bo; Wang, JiaJia; Li, Cunzhi; Guo, ShiJun; Li, Lin; Hu, Song-Qing

    2018-08-01

    This study investigated characteristics of recombinant wheat Endoplasmic Reticulum Oxidoreductin 1 (wEro1) and its influence on Chinese steamed bread (CSB) qualities. The purified wEro1 monomer, which contained two conserved redox active motif sites, bound to flavin adenine dinucleotide (FAD) cofactor with a molecular weight of ∼47 kDa. wEro1 catalyzed the reduction of both bound and free FAD, and its reduction activity of free FAD reached 7.8 U/mg. Moreover, wEro1 catalyzed the oxidation of dithiothreitol and wheat protein disulfide isomerase (wPDI). Both glutathione and the reduced ribonuclease could work as electron donors for wEro1 in catalyzing the oxidation of wPDI. Additionally, wEro1 supplementation improved the CSB qualities with an increased specific volume of CSB and decreased crumb hardness, which was attributed to water-insoluble wheat proteins increasing and gluten network strengthening. The results give an understanding of the properties and function of wEro1 to facilitate its application especially in the flour-processing industry. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. Orofacial inflammatory pain affects the expression of MT1 and NADPH-d in rat caudal spinal trigeminal nucleus and trigeminal ganglion

    PubMed Central

    Huang, Fang; He, Hongwen; Fan, Wenguo; Liu, Yongliang; Zhou, Hongyu; Cheng, Bin

    2013-01-01

    Very little is known about the role of melatonin in the trigeminal system, including the function of melatonin receptor 1. In the present study, adult rats were injected with formaldehyde into the right vibrissae pad to establish a model of orofacial inflammatory pain. The distribution of melatonin receptor 1 and nicotinamide adenine dinucleotide phosphate diaphorase in the caudal spinal trigeminal nucleus and trigeminal ganglion was determined with immunohistochemistry and histochemistry. The results show that there are significant differences in melatonin receptor 1 expression and nicotinamide adenine dinucleotide phosphate diaphorase expression in the trigeminal ganglia and caudal spinal nucleus during the early stage of orofacial inflammatory pain. Our findings suggest that when melatonin receptor 1 expression in the caudal spinal nucleus is significantly reduced, melatonin's regulatory effect on pain is attenuated. PMID:25206619

  11. Novel electrochemical sensor based on functionalized graphene for simultaneous determination of adenine and guanine in DNA.

    PubMed

    Huang, Ke-Jing; Niu, De-Jun; Sun, Jun-Yong; Han, Cong-Hui; Wu, Zhi-Wei; Li, Yan-Li; Xiong, Xiao-Qin

    2011-02-01

    A nano-material carboxylic acid functionalized graphene (graphene-COOH) was prepared and used to construct a novel biosensor for the simultaneous detection of adenine and guanine. The direct electrooxidation behaviors of adenine and guanine on the graphene-COOH modified glassy carbon electrode (graphene-COOH/GCE) were carefully investigated by cyclic voltammetry and differential pulse voltammetry. The results indicated that both adenine and guanine showed the increase of the oxidation peak currents with the negative shift of the oxidation peak potentials in contrast to that on the bare glassy carbon electrode. The electrochemical parameters of adenine and guanine on the graphene-COOH/GCE were calculated and a simple and reliable electroanalytical method was developed for the detection of adenine and guanine, respectively. The modified electrode exhibited good behaviors in the simultaneous detection of adenine and guanine with the peak separation as 0.334V. The detection limit for individual determination of guanine and adenine was 5.0×10(-8)M and 2.5×10(-8)M (S/N=3), respectively. Furthermore, the measurements of thermally denatured single-stranded DNA were carried out and the value of (G+C)/(A+T) of single-stranded DNA was calculated as 0.80. The biosensor exhibited some advantages, such as simplicity, rapidity, high sensitivity, good reproducibility and long-term stability. Copyright © 2010 Elsevier B.V. All rights reserved.

  12. Generation of reactive oxygen species in the reaction catalyzed by alpha-ketoglutarate dehydrogenase.

    PubMed

    Tretter, Laszlo; Adam-Vizi, Vera

    2004-09-08

    Alpha-ketoglutarate dehydrogenase (alpha-KGDH), a key enzyme in the Krebs' cycle, is a crucial early target of oxidative stress (Tretter and Adam-Vizi, 2000). The present study demonstrates that alpha-KGDH is able to generate H(2)O(2) and, thus, could also be a source of reactive oxygen species (ROS) in mitochondria. Isolated alpha-KGDH with coenzyme A (HS-CoA) and thiamine pyrophosphate started to produce H(2)O(2) after addition of alpha-ketoglutarate in the absence of nicotinamide adenine dinucleotide-oxidized (NAD(+)). NAD(+), which proved to be a powerful inhibitor of alpha-KGDH-mediated H(2)O(2) formation, switched the H(2)O(2) forming mode of the enzyme to the catalytic [nicotinamide adenine dinucleotide-reduced (NADH) forming] mode. In contrast, NADH stimulated H(2)O(2) formation by alpha-KGDH, and for this, neither alpha-ketoglutarate nor HS-CoA were required. When all of the substrates and cofactors of the enzyme were present, the NADH/NAD(+) ratio determined the rate of H(2)O(2) production. The higher the NADH/NAD(+) ratio the higher the rate of H(2)O(2) production. H(2)O(2) production as well as the catalytic function of the enzyme was activated by Ca(2+). In synaptosomes, using alpha-ketoglutarate as respiratory substrate, the rate of H(2)O(2) production increased by 2.5-fold, and aconitase activity decreased, indicating that alpha-KGDH can generate H(2)O(2) in in situ mitochondria. Given the NADH/NAD(+) ratio as a key regulator of H(2)O(2) production by alpha-KGDH, it is suggested that production of ROS could be significant not only in the respiratory chain but also in the Krebs' cycle when oxidation of NADH is impaired. Thus alpha-KGDH is not only a target of ROS but could significantly contribute to generation of oxidative stress in the mitochondria.

  13. Characterization of Plant Carotenoid Cyclases as Members of the Flavoprotein Family Functioning with No Net Redox Change1[W][OA

    PubMed Central

    Mialoundama, Alexis Samba; Heintz, Dimitri; Jadid, Nurul; Nkeng, Paul; Rahier, Alain; Deli, Jozsef; Camara, Bilal; Bouvier, Florence

    2010-01-01

    The later steps of carotenoid biosynthesis involve the formation of cyclic carotenoids. The reaction is catalyzed by lycopene β-cyclase (LCY-B), which converts lycopene into β-carotene, and by capsanthin-capsorubin synthase (CCS), which is mainly dedicated to the synthesis of κ-cyclic carotenoids (capsanthin and capsorubin) but also has LCY-B activity. Although the peptide sequences of plant LCY-Bs and CCS contain a putative dinucleotide-binding motif, it is believed that these two carotenoid cyclases proceed via protic activation and stabilization of resulting carbocation intermediates. Using pepper (Capsicum annuum) CCS as a prototypic carotenoid cyclase, we show that the monomeric protein contains one noncovalently bound flavin adenine dinucleotide (FAD) that is essential for enzyme activity only in the presence of NADPH, which functions as the FAD reductant. The reaction proceeds without transfer of hydrogen from the dinucleotide cofactors to β-carotene or capsanthin. Using site-directed mutagenesis, amino acids potentially involved in the protic activation were identified. Substitutions of alanine, lysine, and arginine for glutamate-295 in the conserved 293-FLEET-297 motif of pepper CCS or LCY-B abolish the formation of β-carotene and κ-cyclic carotenoids. We also found that mutations of the equivalent glutamate-196 located in the 194-LIEDT-198 domain of structurally divergent bacterial LCY-B abolish the formation of β-carotene. The data herein reveal plant carotenoid cyclases to be novel enzymes that combine characteristics of non-metal-assisted terpene cyclases with those attributes typically found in flavoenzymes that catalyze reactions, with no net redox, such as type 2 isopentenyl diphosphate isomerase. Thus, FAD in its reduced form could be implicated in the stabilization of the carbocation intermediate. PMID:20460582

  14. 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

    2016-01-05

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Preparation and storage of isotopically labeled reduced nicotinamide adenine dinucleotide

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Northrop, D.B.; Duggleby, R.G.

    1987-09-01

    A method for obtaining highly purified NADH in a dry, solid, and stable form is described. The method involves improvements of the ion-exchange and reversed-phase chromatographic procedures of C. J. Newton and S. M. Faynor, and D. B. Northrop. The necessary time to prepare pure NADH has been reduced to a few hours. The final product, obtained by drying the nucleotide from absolute ethanol, shows no detectable decomposition either during the drying procedure or during storage under nitrogen gas at -20 degrees C for several months. Using dry product prepared from fixed volumes of ethanolic solution, standardized solutions of knownmore » amounts of the highly purified and stored NADH can be obtained in a few seconds.« less

  16. Expression and localization of tubulin cofactors TBCD and TBCE in human gametes.

    PubMed

    Jiménez-Moreno, Victoria; Agirregoitia, Ekaitz

    2017-06-01

    The tubulin cofactors TBCD and TBCE play an essential role in regulation of the microtubule dynamics in a wide variety of somatic cells, but little information is known about the expression of these cofactors in human sperm and oocytes. In this study, we focused on the investigation of the presence of, and the differential distribution of, the tubulin cofactors TBCD and TBCE in human sperm and during human oocyte maturation. We performed expression assays for TBCD and TBCE by reverse transcription-polymerase chain reaction (RT-PCR), western blot and immunofluorescence and verified the presence of both cofactors in human gametes. TBCD and TBCE were located mainly in the middle region and in the tail of the sperm while in the oocyte the localization was cytosolic. The mRNA of both tubulin cofactors were present in the human oocytes but not in sperm cells. This finding gives a first insight into where TBCD and TBCE could carry out their function in the continuous changes that the cytoskeleton experiences during gametogenesis and also prior to fertilization.

  17. Enzymatic regeneration of adenosine triphosphate cofactor

    NASA Technical Reports Server (NTRS)

    Marshall, D. L.

    1974-01-01

    Regenerating adenosine triphosphate (ATP) from adenosine diphosphate (ADP) by enzymatic process which utilizes carbamyl phosphate as phosphoryl donor is technique used to regenerate expensive cofactors. Process allows complex enzymatic reactions to be considered as candidates for large-scale continuous processes.

  18. Salicylate Treatment Improves Age-Associated Vascular Endothelial Dysfunction: Potential Role of Nuclear Factor κB and Forkhead Box O Phosphorylation

    PubMed Central

    Durrant, Jessica R.; Connell, Melanie L.; Folian, Brian J.; Donato, Anthony J.; Seals, Douglas R.

    2011-01-01

    We hypothesized that I kappa B kinase (IKK)-mediated nuclear factor kappa B and forkhead BoxO3a phosphorylation will be associated with age-related endothelial dysfunction. Endothelium-dependent dilation and aortic protein expression/phosphorylation were determined in young and old male B6D2F1 mice and old mice treated with the IKK inhibitor, salicylate. IKK activation was greater in old mice and was associated with greater nitrotyrosine and cytokines. Endothelium-dependent dilation, nitric oxide (NO), and endothelial NO synthase phosphorylation were lower in old mice. Endothelium-dependent dilation and NO bioavailability were restored by a superoxide dismutase mimetic. Nuclear factor kappa B and forkhead BoxO3a phosphorylation were greater in old and were associated with increased expression/activity of nicotinamide adenine dinucleotide phosphate oxidase and lower manganese superoxide dismutase expression. Salicylate lowered IKK phosphorylation and reversed age-associated changes in nitrotyrosine, endothelium-dependent dilation, NO bioavailability, endothelial NO synthase, nuclear factor kappa B and forkhead BoxO3a phosphorylation, nicotinamide adenine dinucleotide phosphate oxidase, and manganese superoxide dismutase. Increased activation of IKK with advancing age stimulates nuclear factor kappa B and inactivates forkhead BoxO3a. This altered transcription factor activation contributes to a pro-inflammatory/pro-oxidative arterial phenotype that is characterized by increased cytokines and nicotinamide adenine dinucleotide phosphate oxidase and decreased manganese superoxide dismutase leading to oxidative stress-mediated endothelial dysfunction. PMID:21303813

  19. Characterization of the type 2 NADH:menaquinone oxidoreductases from Staphylococcus aureus and the bactericidal action of phenothiazines.

    PubMed

    Schurig-Briccio, Lici A; Yano, Takahiro; Rubin, Harvey; Gennis, Robert B

    2014-07-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is currently one of the principal multiple drug resistant bacterial pathogens causing serious infections, many of which are life-threatening. Consequently, new therapeutic targets are required to combat such infections. In the current work, we explore the type 2 Nicotinamide adenine dinucleotide reduced form (NADH) dehydrogenases (NDH-2s) as possible drug targets and look at the effects of phenothiazines, known to inhibit NDH-2 from Mycobacterium tuberculosis. NDH-2s are monotopic membrane proteins that catalyze the transfer of electrons from NADH via flavin adenine dinucleotide (FAD) to the quinone pool. They are required for maintaining the NADH/Nicotinamide adenine dinucleotide (NAD(+)) redox balance and contribute indirectly to the generation of proton motive force. NDH-2s are not present in mammals, but are the only form of respiratory NADH dehydrogenase in several pathogens, including S. aureus. In this work, the two putative ndh genes present in the S. aureus genome were identified, cloned and expressed, and the proteins were purified and characterized. Phenothiazines were shown to inhibit both of the S. aureus NDH-2s with half maximal inhibitory concentration (IC50) values as low as 8μM. However, evaluating the effects of phenothiazines on whole cells of S. aureus was complicated by the fact that they are also acting as uncouplers of oxidative phosphorylation. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Mannitol and Mannitol Dehydrogenases in Conidia of Aspergillus oryzae

    PubMed Central

    Horikoshi, Koki; Iida, Shigeji; Ikeda, Yonosuke

    1965-01-01

    Horikoshi, Koki (The Institute of Physical and Chemical Research, Tokyo, Japan), Shigeji Iida, and Yonosuke Ikeda. Mannitol and mannitol dehydrogenases in conidia of Aspergillus oryzae. J. Bacteriol. 89:326–330. 1965.—A sugar alcohol was isolated from the conidia of Aspergillus oryzae and identified as d-mannitol. Two types of d-mannitol dehydrogenases, nicotinamide adenine dinucleotide phosphate-linked and nicotinamide adenine dinucleotide-linked, were found in the conidia. Substrate specificities, pH optima, Michaelis-Menton constants, and the effects of inhibitors were studied. d-Mannitol was converted to fructose by the dehydrogenases. Synthesis of d-mannitol dehydrogenases was not observed during germination; the content of d-mannitol decreased at an early stage of germination. It was assumed, therefore, that d-mannitol might be used as the source of endogenous respiration and provide energy for the germination. PMID:14255698

  1. A dinucleotide motif in oligonucleotides shows potent immunomodulatory activity and overrides species-specific recognition observed with CpG motif.

    PubMed

    Kandimalla, Ekambar R; Bhagat, Lakshmi; Zhu, Fu-Gang; Yu, Dong; Cong, Yan-Ping; Wang, Daqing; Tang, Jimmy X; Tang, Jin-Yan; Knetter, Cathrine F; Lien, Egil; Agrawal, Sudhir

    2003-11-25

    Bacterial and synthetic DNAs containing CpG dinucleotides in specific sequence contexts activate the vertebrate immune system through Toll-like receptor 9 (TLR9). In the present study, we used a synthetic nucleoside with a bicyclic heterobase [1-(2'-deoxy-beta-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; R] to replace the C in CpG, resulting in an RpG dinucleotide. The RpG dinucleotide was incorporated in mouse- and human-specific motifs in oligodeoxynucleotides (oligos) and 3'-3-linked oligos, referred to as immunomers. Oligos containing the RpG motif induced cytokine secretion in mouse spleen-cell cultures. Immunomers containing RpG dinucleotides showed activity in transfected-HEK293 cells stably expressing mouse TLR9, suggesting direct involvement of TLR9 in the recognition of RpG motif. In J774 macrophages, RpG motifs activated NF-kappa B and mitogen-activated protein kinase pathways. Immunomers containing the RpG dinucleotide induced high levels of IL-12 and IFN-gamma, but lower IL-6 in time- and concentration-dependent fashion in mouse spleen-cell cultures costimulated with IL-2. Importantly, immunomers containing GTRGTT and GARGTT motifs were recognized to a similar extent by both mouse and human immune systems. Additionally, both mouse- and human-specific RpG immunomers potently stimulated proliferation of peripheral blood mononuclear cells obtained from diverse vertebrate species, including monkey, pig, horse, sheep, goat, rat, and chicken. An immunomer containing GTRGTT motif prevented conalbumin-induced and ragweed allergen-induced allergic inflammation in mice. We show that a synthetic bicyclic nucleotide is recognized in the C position of a CpG dinucleotide by immune cells from diverse vertebrate species without bias for flanking sequences, suggesting a divergent nucleotide motif recognition pattern of TLR9.

  2. Alteration of adenyl dinucleotide metabolism by environmental stress.

    PubMed Central

    Baker, J C; Jacobson, M K

    1986-01-01

    Exposure of cultured mammalian cells to a variety of conditions that induce the synthesis of stress proteins, including hyperthermia, ethanol, cadmium, and arsenite resulted in an increased cellular content of adenyl dinucleotides including diadenosine tetraphosphate (Ap4A). Exposure to other agents that cause metabolic perturbations not known to induce the synthesis of stress proteins, such as cyclohexamide, cytosine arabinoside, hydroxyurea, and ultraviolet irradiation did not alter the content of these nucleotides. It is proposed that these unique nucleotides may mediate adaptive responses of mammalian cells to environmental stress. PMID:3458199

  3. Prolonged Pulmonary Exposure to Diesel Exhaust Particles Exacerbates Renal Oxidative Stress, Inflammation and DNA Damage in Mice with Adenine-Induced Chronic Renal Failure.

    PubMed

    Nemmar, Abderrahim; Karaca, Turan; Beegam, Sumaya; Yuvaraju, Priya; Yasin, Javed; Hamadi, Naserddine Kamel; Ali, Badreldin H

    2016-01-01

    Epidemiological evidence indicates that patients with chronic kidney diseases have increased susceptibility to adverse outcomes related to long-term exposure to particulate air pollution. However, mechanisms underlying these effects are not fully understood. Presently, we assessed the effect of prolonged exposure to diesel exhaust particles (DEP) on chronic renal failure induced by adenine (0.25% w/w in feed for 4 weeks), which is known to involve inflammation and oxidative stress. DEP (0.5m/kg) was intratracheally (i.t.) instilled every 4th day for 4 weeks (7 i.t. instillation). Four days following the last exposure to either DEP or saline (control), various renal endpoints were measured. While body weight was decreased, kidney weight increased in DEP+adenine versus saline+adenine or DEP. Water intake, urine volume, relative kidney weight were significantly increased in adenine+DEP versus DEP and adenine+saline versus saline. Plasma creatinine and urea increased and creatinine clearance decreased in adenine+DEP versus DEP and adenine+saline versus saline. Tumor necrosis factor α, lipid peroxidation and reactive oxygen species were significantly increased in adenine+DEP compared with either DEP or adenine+saline. The antioxidant calase was significantly decreased in adenine+DEP compared with either adenine+saline or DEP. Notably, renal DNA damage was significantly potentiated in adenine+DEP compared with either adenine+saline or DEP. Similarly, systolic blood pressure was increased in adenine+DEP versus adenine+saline or DEP, and in DEP versus saline. Histological evaluation revealed more collagen deposition, higher number of necrotic cell counts and dilated tubules, cast formation and collapsing glomeruli in adenine+DEP versus adenine+saline or DEP. Prolonged pulmonary exposure to diesel exhaust particles worsen renal oxidative stress, inflammation and DNA damage in mice with adenine-induced chronic renal failure. Our data provide biological plausibility that air

  4. Communication: Site-selective bond excision of adenine upon electron transfer

    NASA Astrophysics Data System (ADS)

    Cunha, T.; Mendes, M.; Ferreira da Silva, F.; Eden, S.; García, G.; Limão-Vieira, P.

    2018-01-01

    This work demonstrates that selective excision of hydrogen atoms at a particular site of the DNA base adenine can be achieved in collisions with electronegative atoms by controlling the impact energy. The result is based on analysing the time-of-flight mass spectra yields of potassium collisions with a series of labeled adenine derivatives. The production of dehydrogenated parent anions is consistent with neutral H loss either from selective breaking of C-H or N-H bonds. These unprecedented results open up a new methodology in charge transfer collisions that can initiate selective reactivity as a key process in chemical reactions that are dominant in different areas of science and technology.

  5. Enzyme cofactors: Double-edged sword for catalysis

    NASA Astrophysics Data System (ADS)

    Ivanov, Ivaylo

    2013-01-01

    The metal cofactors responsible for the activity of CDK2 -- a representative member of the kinase superfamily of enzymes -- have now been shown to also have inhibitory effects during the catalytic cycle.

  6. Two-dimensional network stability of nucleobases and amino acids on graphite under ambient conditions: adenine, L-serine and L-tyrosine.

    PubMed

    Bald, Ilko; Weigelt, Sigrid; Ma, Xiaojing; Xie, Pengyang; Subramani, Ramesh; Dong, Mingdong; Wang, Chen; Mamdouh, Wael; Wang, Jianguo; Besenbacher, Flemming

    2010-04-14

    We have investigated the stability of two-dimensional self-assembled molecular networks formed upon co-adsorption of the DNA base, adenine, with each of the amino acids, L-serine and L-tyrosine, on a highly oriented pyrolytic graphite (HOPG) surface by drop-casting from a water solution. L-serine and L-tyrosine were chosen as model systems due to their different interaction with the solvent molecules and the graphite substrate, which is reflected in a high and low solubility in water, respectively, compared with adenine. Combined scanning tunneling microscopy (STM) measurements and density functional theory (DFT) calculations show that the self-assembly process is mainly driven by the formation of strong adenine-adenine hydrogen bonds. We find that pure adenine networks are energetically more stable than networks built up of either pure L-serine, pure L-tyrosine or combinations of adenine with L-serine or L-tyrosine, and that only pure adenine networks are stable enough to be observable by STM under ambient conditions.

  7. ON THE INTERACTION OF ADENINE WITH IONIZING RADIATION: MECHANISTICAL STUDIES AND ASTROBIOLOGICAL IMPLICATIONS

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Evans, Nicholas L.; Ullrich, Susanne; Bennett, Chris J.

    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 expectedmore » 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).« less

  8. Pleiotropic effects of the sirtuin inhibitor sirtinol involves concentration-dependent modulation of multiple nuclear receptor-mediated pathways in the androgen-responsive prostate cancer cell LNCaP

    USDA-ARS?s Scientific Manuscript database

    Sirtinol, a purported specific inhibitor of the nicotinamide adenine dinucleotide (NAD)-dependent type III histone deacetylase (also known as sirtuin), has been used extensively to identify chemopreventive/chemotherapeutic agents that modulate the activity of this group of enzymes. However, the mole...

  9. Molybdenum Enzymes, Cofactors, and Model Systems.

    ERIC Educational Resources Information Center

    Burgmayer, S. J. N; Stiefel, E. I.

    1985-01-01

    Discusses: (l) molybdoenzymes (examining their distribution and metabolic role, composition and redox strategy, cofactors, substrate reactions, and mechanistic possibilities); (2) structural information on molybdenum (Mo) centers; (3) modeling studies (Mo-co models, nitrogenase models, and the MO-S duo); and (4) the copper-molybdenum antagonism.…

  10. Radical S-Adenosyl-l-methionine Chemistry in the Synthesis of Hydrogenase and Nitrogenase Metal Cofactors*

    PubMed Central

    Byer, Amanda S.; Shepard, Eric M.; Peters, John W.; Broderick, Joan B.

    2015-01-01

    Nitrogenase, [FeFe]-hydrogenase, and [Fe]-hydrogenase enzymes perform catalysis at metal cofactors with biologically unusual non-protein ligands. The FeMo cofactor of nitrogenase has a MoFe7S9 cluster with a central carbon, whereas the H-cluster of [FeFe]-hydrogenase contains a 2Fe subcluster coordinated by cyanide and CO ligands as well as dithiomethylamine; the [Fe]-hydrogenase cofactor has CO and guanylylpyridinol ligands at a mononuclear iron site. Intriguingly, radical S-adenosyl-l-methionine enzymes are vital for the assembly of all three of these diverse cofactors. This minireview presents and discusses the current state of knowledge of the radical S-adenosylmethionine enzymes required for synthesis of these remarkable metal cofactors. PMID:25477518

  11. Adenine Inhibits TNF-α Signaling in Intestinal Epithelial Cells and Reduces Mucosal Inflammation in a Dextran Sodium Sulfate-Induced Colitis Mouse Model.

    PubMed

    Fukuda, Toshihiko; Majumder, Kaustav; Zhang, Hua; Turner, Patricia V; Matsui, Toshiro; Mine, Yoshinori

    2016-06-01

    Adenine (6-amino-6H-purine), found in molokheiya (Corchorus olitorius L.), has exerted vasorelaxation effects in the thoracic aorta. However, the mode of action of the anti-inflammatory effect of adenine is unclear. Thus, we investigated to clarify the effect of adenine on chronic inflammation of the gastrointestinal tract. In intestinal epithelial cells, adenine significantly inhibited tumor necrosis factor-α-induced interleukin-8 secretion. The inhibition of adenine was abolished under the treatment of inhibitors of adenyl cyclase (AC) and protein kinase A (PKA), indicating the effect of adenine was mediated through the AC/PKA pathway. Adenine (5, 10, and 50 mg/kg BW/day) was administered orally for 14 days to female BALB/c mice, and then 5% dextran sodium sulfate (DSS) was given to induce colitis. Adenine (5 mg/kg BW/day) significantly prevented DSS-induced colon shortening, expression of pro-inflammatory cytokines, and histological damage in the colon. These results suggest that adenine can be a promising nutraceutical for the prevention of intestinal inflammation.

  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.

  13. Redox cofactor engineering in industrial microorganisms: strategies, recent applications and future directions.

    PubMed

    Liu, Jiaheng; Li, Huiling; Zhao, Guangrong; Caiyin, Qinggele; Qiao, Jianjun

    2018-05-01

    NAD and NADP, a pivotal class of cofactors, which function as essential electron donors or acceptors in all biological organisms, drive considerable catabolic and anabolic reactions. Furthermore, they play critical roles in maintaining intracellular redox homeostasis. However, many metabolic engineering efforts in industrial microorganisms towards modification or introduction of metabolic pathways, especially those involving consumption, generation or transformation of NAD/NADP, often induce fluctuations in redox state, which dramatically impede cellular metabolism, resulting in decreased growth performance and biosynthetic capacity. Here, we comprehensively review the cofactor engineering strategies for solving the problematic redox imbalance in metabolism modification, as well as their features, suitabilities and recent applications. Some representative examples of in vitro biocatalysis are also described. In addition, we briefly discuss how tools and methods from the field of synthetic biology can be applied for cofactor engineering. Finally, future directions and challenges for development of cofactor redox engineering are presented.

  14. Dietary L-lysine prevents arterial calcification in adenine-induced uremic rats.

    PubMed

    Shimomura, Akihiro; Matsui, Isao; Hamano, Takayuki; Ishimoto, Takuya; Katou, Yumiko; Takehana, Kenji; Inoue, Kazunori; Kusunoki, Yasuo; Mori, Daisuke; Nakano, Chikako; Obi, Yoshitsugu; Fujii, Naohiko; Takabatake, Yoshitsugu; Nakano, Takayoshi; Tsubakihara, Yoshiharu; Isaka, Yoshitaka; Rakugi, Hiromi

    2014-09-01

    Vascular calcification (VC) is a life-threatening complication of CKD. Severe protein restriction causes a shortage of essential amino acids, and exacerbates VC in rats. Therefore, we investigated the effects of dietary l-lysine, the first-limiting amino acid of cereal grains, on VC. Male Sprague-Dawley rats at age 13 weeks were divided randomly into four groups: low-protein (LP) diet (group LP), LP diet+adenine (group Ade), LP diet+adenine+glycine (group Gly) as a control amino acid group, and LP diet+adenine+l-lysine·HCl (group Lys). At age 18 weeks, group LP had no VC, whereas groups Ade and Gly had comparable levels of severe VC. l-Lysine supplementation almost completely ameliorated VC. Physical parameters and serum creatinine, urea nitrogen, and phosphate did not differ among groups Ade, Gly, and Lys. Notably, serum calcium in group Lys was slightly but significantly higher than in groups Ade and Gly. Dietary l-lysine strongly suppressed plasma intact parathyroid hormone in adenine rats and supported a proper bone-vascular axis. The conserved orientation of the femoral apatite in group Lys also evidenced the bone-protective effects of l-lysine. Dietary l-lysine elevated plasma alanine, proline, arginine, and homoarginine but not lysine. Analyses in vitro demonstrated that alanine and proline inhibit apoptosis of cultured vascular smooth muscle cells, and that arginine and homoarginine attenuate mineral precipitations in a supersaturated calcium/phosphate solution. In conclusion, dietary supplementation of l-lysine ameliorated VC by modifying key pathways that exacerbate VC. Copyright © 2014 by the American Society of Nephrology.

  15. Dietary l-Lysine Prevents Arterial Calcification in Adenine-Induced Uremic Rats

    PubMed Central

    Shimomura, Akihiro; Matsui, Isao; Hamano, Takayuki; Ishimoto, Takuya; Katou, Yumiko; Takehana, Kenji; Inoue, Kazunori; Kusunoki, Yasuo; Mori, Daisuke; Nakano, Chikako; Obi, Yoshitsugu; Fujii, Naohiko; Takabatake, Yoshitsugu; Nakano, Takayoshi; Tsubakihara, Yoshiharu; Rakugi, Hiromi

    2014-01-01

    Vascular calcification (VC) is a life-threatening complication of CKD. Severe protein restriction causes a shortage of essential amino acids, and exacerbates VC in rats. Therefore, we investigated the effects of dietary l-lysine, the first-limiting amino acid of cereal grains, on VC. Male Sprague-Dawley rats at age 13 weeks were divided randomly into four groups: low-protein (LP) diet (group LP), LP diet+adenine (group Ade), LP diet+adenine+glycine (group Gly) as a control amino acid group, and LP diet+adenine+l-lysine·HCl (group Lys). At age 18 weeks, group LP had no VC, whereas groups Ade and Gly had comparable levels of severe VC. l-Lysine supplementation almost completely ameliorated VC. Physical parameters and serum creatinine, urea nitrogen, and phosphate did not differ among groups Ade, Gly, and Lys. Notably, serum calcium in group Lys was slightly but significantly higher than in groups Ade and Gly. Dietary l-lysine strongly suppressed plasma intact parathyroid hormone in adenine rats and supported a proper bone-vascular axis. The conserved orientation of the femoral apatite in group Lys also evidenced the bone-protective effects of l-lysine. Dietary l-lysine elevated plasma alanine, proline, arginine, and homoarginine but not lysine. Analyses in vitro demonstrated that alanine and proline inhibit apoptosis of cultured vascular smooth muscle cells, and that arginine and homoarginine attenuate mineral precipitations in a supersaturated calcium/phosphate solution. In conclusion, dietary supplementation of l-lysine ameliorated VC by modifying key pathways that exacerbate VC. PMID:24652795

  16. The GlcN6P cofactor serves multiple catalytic roles in the glmS ribozyme

    PubMed Central

    Bingaman, Jamie L.; Zhang, Sixue; Stevens, David R.; Yennawar, Neela H.; Hammes-Schiffer, Sharon; Bevilacqua, Philip C.

    2017-01-01

    RNA enzymes have remarkably diverse biological roles despite having limited chemical diversity. Protein enzymes enhance their reactivity through recruitment of cofactors. The naturally occurring glmS ribozyme uses the glucosamine-6-phosphate (GlcN6P) organic cofactor for phosphodiester bond cleavage. Prior structural and biochemical studies implicated GlcN6P as the general acid. Here we describe new catalytic roles for GlcN6P through experiments and calculations. Large stereospecific normal thio effects and lack of metal ion rescue in the holoribozyme show that nucleobases and the cofactor play direct chemical roles and align the active site for self-cleavage. Large stereospecific inverse thio effects in the aporibozyme suggest that the GlcN6P cofactor disrupts an inhibitory interaction of the nucleophile. Strong metal ion rescue in the aporibozyme reveals this cofactor also provides electrostatic stabilization. Ribozyme organic cofactors thus perform myriad catalytic roles, allowing RNA to compensate for its limited functional diversity. PMID:28192411

  17. Raman Spectroscopy of the Interferon-Induced 2’,5’-Oligoadenylates

    DTIC Science & Technology

    1987-06-25

    generation of the Raman spectrum of triethyl ammonium ion ••••••••••••••••••••••••••••••• 41 12. structures of purine, adenine, purine riboside , adenosine...ribose 5 1-phosphate, AMP, and ATP........ 48 13. Raman spectra of adenine and purine •••••••.••••••••• 49 14. Raman spectra of purine riboside and... nicotinamide adenine dinucleotide; TFAB, triethyl anunonium bicarbonate; TFA, triethyl amm::mium. ion; CD circular _dichroism; NMR, nuclear magnetic

  18. Radical S-Adenosyl-L-methionine Chemistry in the Synthesis of Hydrogenase and Nitrogenase Metal Cofactors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Byer, Amanda S.; Shepard, Eric M.; Peters, John W.

    Nitrogenase, [FeFe]-hydrogenase, and [Fe]-hydrogenase enzymes perform catalysis at metal cofactors with biologically unusual non-protein ligands. Furthermore, the FeMo cofactor of nitrogenase has a MoFe 7S 9 cluster with a central carbon, whereas the H-cluster of [FeFe]-hydrogenase contains a 2Fe subcluster coordinated by cyanide and CO ligands as well as dithiomethylamine; the [Fe]-hydrogenase cofactor has CO and guanylylpyridinol ligands at a mononuclear iron site. Intriguingly, radical S-adenosyl-L-methionine enzymes are vital for the assembly of all three of these diverse cofactors. Here, in this minireview, we present and discuss the current state of knowledge of the radical S-adenosylmethionine enzymes required for synthesismore » of these remarkable metal cofactors.« less

  19. Radical S-Adenosyl-L-methionine Chemistry in the Synthesis of Hydrogenase and Nitrogenase Metal Cofactors

    DOE PAGES

    Byer, Amanda S.; Shepard, Eric M.; Peters, John W.; ...

    2014-12-04

    Nitrogenase, [FeFe]-hydrogenase, and [Fe]-hydrogenase enzymes perform catalysis at metal cofactors with biologically unusual non-protein ligands. Furthermore, the FeMo cofactor of nitrogenase has a MoFe 7S 9 cluster with a central carbon, whereas the H-cluster of [FeFe]-hydrogenase contains a 2Fe subcluster coordinated by cyanide and CO ligands as well as dithiomethylamine; the [Fe]-hydrogenase cofactor has CO and guanylylpyridinol ligands at a mononuclear iron site. Intriguingly, radical S-adenosyl-L-methionine enzymes are vital for the assembly of all three of these diverse cofactors. Here, in this minireview, we present and discuss the current state of knowledge of the radical S-adenosylmethionine enzymes required for synthesismore » of these remarkable metal cofactors.« less

  20. Selective intra-dinucleotide interactions and periodicities of bases separated by K sites: a new vision and tool for phylogeny analyses.

    PubMed

    Valenzuela, Carlos Y

    2017-02-13

    Direct tests of the random or non-random distribution of nucleotides on genomes have been devised to test the hypothesis of neutral, nearly-neutral or selective evolution. These tests are based on the direct base distribution and are independent of the functional (coding or non-coding) or structural (repeated or unique sequences) properties of the DNA. The first approach described the longitudinal distribution of bases in tandem repeats under the Bose-Einstein statistics. A huge deviation from randomness was found. A second approach was the study of the base distribution within dinucleotides whose bases were separated by 0, 1, 2… K nucleotides. Again an enormous difference from the random distribution was found with significances out of tables and programs. These test values were periodical and included the 16 dinucleotides. For example a high "positive" (more observed than expected dinucleotides) value, found in dinucleotides whose bases were separated by (3K + 2) sites, was preceded by two smaller "negative" (less observed than expected dinucleotides) values, whose bases were separated by (3K) or (3K + 1) sites. We examined mtDNAs, prokaryote genomes and some eukaryote chromosomes and found that the significant non-random interactions and periodicities were present up to 1000 or more sites of base separation and in human chromosome 21 until separations of more than 10 millions sites. Each nucleotide has its own significant value of its distance to neutrality; this yields 16 hierarchical significances. A three dimensional table with the number of sites of separation between the bases and the 16 significances (the third dimension is the dinucleotide, individual or taxon involved) gives directly an evolutionary state of the analyzed genome that can be used to obtain phylogenies. An example is provided.

  1. Lactate Racemase Nickel-Pincer Cofactor Operates by a Proton-Coupled Hydride Transfer Mechanism.

    PubMed

    Rankin, Joel A; Mauban, Robert C; Fellner, Matthias; Desguin, Benoît; McCracken, John; Hu, Jian; Varganov, Sergey A; Hausinger, Robert P

    2018-03-09

    Lactate racemase (LarA) of Lactobacillus plantarum contains a novel organometallic cofactor with nickel coordinated to a covalently tethered pincer ligand, pyridinium-3-thioamide-5-thiocarboxylic acid mononucleotide, but its function in the enzyme mechanism has not been elucidated. This study presents direct evidence that the nickel-pincer cofactor facilitates a proton-coupled hydride transfer (PCHT) mechanism during LarA-catalyzed lactate racemization. No signal was detected by electron paramagnetic resonance spectroscopy for LarA in the absence or presence of substrate, consistent with a +2 metal oxidation state and inconsistent with a previously proposed proton-coupled electron transfer mechanism. Pyruvate, the predicted intermediate for a PCHT mechanism, was observed in quenched solutions of LarA. A normal substrate kinetic isotope effect ( k H / k D of 3.11 ± 0.17) was established using 2-α- 2 H-lactate, further supporting a PCHT mechanism. UV-visible spectroscopy revealed a lactate-induced perturbation of the cofactor spectrum, notably increasing the absorbance at 340 nm, and demonstrated an interaction of the cofactor with the inhibitor sulfite. A crystal structure of LarA provided greater resolution (2.4 Å) than previously reported and revealed sulfite binding to the pyridinium C4 atom of the reduced pincer cofactor, mimicking hydride reduction during a PCHT catalytic cycle. Finally, computational modeling supports hydride transfer to the cofactor at the C4 position or to the nickel atom, but with formation of a nickel-hydride species requiring dissociation of the His200 metal ligand. In aggregate, these studies provide compelling evidence that the nickel-pincer cofactor acts by a PCHT mechanism.

  2. Malic enzyme: Tritium isotope effects with alternative dinucleotide substrates and divalent metal ions

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Karsten, W.E.; Harris, B.G.; Cook, P.F.

    1992-01-01

    The NAD-malic enzyme from Ascaris suum catalyzes the divalent metal ion dependent oxidative decarboxylation of L-malate to yield pyruvate, carbon dioxide and NADH. Multiple isotope effect studies suggest a stepwise chemical mechanism with hydride transfer from L-malate to NAD occurring first to form oxalacetate, followed by decarboxylation. Utilizing L-malate-2-T, tritium V/K isotope effects have been determined for the hydride transfer step using a variety of alternative dinucleotide substrates and divalent metal ions. Combination of these data with deuterium isotope effects data and previously determined [sup 13]C isotope effects has allowed the calculation of intrinsic isotope effects for the malic enzymemore » catalyzed reaction. The identity of both the dinucleotide substrate and divalent metal ion has an effect of the size of the intrinsic isotope effect for hydride transfer.« less

  3. Methods for detection of methyl-CpG dinucleotides

    DOEpatents

    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.

  4. Methods for detection of methyl-CpG dinucleotides

    DOEpatents

    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.

  5. Methods for detection of methyl-CpG dinucleotides

    DOEpatents

    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.

  6. Binding of adenine to Stx2, the protein toxin from Escherichia coli O157:H7

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fraser, Marie E., E-mail: frasm@ucalgary.ca; Cherney, Maia M.; Marcato, Paola

    2006-07-01

    Crystals of Stx2 were grown in the presence of adenosine and adenine. In both cases, the resulting electron density showed only adenine bound at the active site of the A subunit, proving that the holotoxin is an active N-glycosidase. Stx2 is a protein toxin whose catalytic subunit acts as an N-glycosidase to depurinate a specific adenine base from 28S rRNA. In the holotoxin, the catalytic portion, A1, is linked to the rest of the A subunit, A2, and A2 interacts with the pentameric ring formed by the five B subunits. In order to test whether the holotoxin is active asmore » an N-glycosidase, Stx2 was crystallized in the presence of adenosine and adenine. The crystals diffracted to ∼1.8 Å and showed clear electron density for adenine in the active site. Adenosine had been cleaved, proving that Stx2 is an active N-glycosidase. While the holotoxin is active against small substrates, it would be expected that the B subunits would interfere with the binding of the 28S rRNA.« less

  7. Detection of ATP and NADH: A Bioluminescent Experience.

    ERIC Educational Resources Information Center

    Selig, Ted C.; And Others

    1984-01-01

    Described is a bioluminescent assay for adenosine triphosphate (ATP) and reduced nicotineamide-adenine dinucleotide (NADH) that meets the requirements of an undergraduate biochemistry laboratory course. The 3-hour experiment provides students with experience in bioluminescence and analytical biochemistry yet requires limited instrumentation,…

  8. Design and synthesis of novel adenine fluorescence probe based on Eu(III) complexes with dtpa-bis(guanine) ligand

    NASA Astrophysics Data System (ADS)

    Tian, Fengyun; Jiang, Xiaoqing; Dou, Xuekai; Wu, Qiong; Wang, Jun; Song, Youtao

    2017-05-01

    A novel adenine (Ad) fluorescence probe (EuIII-dtpa-bis(guanine)) was designed and synthesized by improving experimental method based on the Eu(III) complex and dtpa-bis(guanine) ligand. The dtpa-bis(guanine) ligand was first synthesized by the acylation action between dtpaa and guanine (Gu), and the corresponding Eu(III) complex was successfully prepared through heat-refluxing method with dtpa-bis(guanine) ligand. As a novel fluorescence probe, the EuIII-dtpa-bis(guanine) complex can detect adenine (Ad) with characteristics of strong targeting, high specificity and high recognition ability. The detection mechanism of the adenine (Ad) using this probe in buffer solution was studied by ultraviolet-visible (UV-vis) and fluorescence spectroscopy. When the EuIII-dtpa-bis(guanine) was introduced to the adenine (Ad) solution, the fluorescence emission intensity was significantly enhanced. However, adding other bases such as guanine (Gu), xanthine (Xa), hypoxanthine (Hy) and uric acid (Ur) with similar composition and structure to that of adenine (Ad) to the EuIII-dtpa-bis(guanine) solution, the fluorescence emission intensities are nearly invariable. Meanwhile, the interference of guanine (Gu), xanthine (Xa), hypoxanthine (Hy) and uric acid (Ur) on the detection of the adenine using EuIII-dtpa-bis(guanine) probe was also studied. It was found that presence of these bases does not affect the detection of adenine (Ad). A linear response of fluorescence emission intensities of EuIII-dtpa-bis(guanine) at 570 nm as a function of adenine (Ad) concentration in the range of 0.00-5.00 × 10- 5 mol L- 1 was observed. The detection limit is about 4.70 × 10- 7 mol L- 1.

  9. Paper microfluidic-based enzyme catalyzed double microreactor.

    PubMed

    Ferrer, Ivonne M; Valadez, Hector; Estala, Lissette; Gomez, Frank A

    2014-08-01

    We describe a paper microfluidic-based enzyme catalyzed double microreactor assay using fluorescent detection. Here, solutions of lactate dehydrogenase (LDH) and diaphorase (DI) were directly spotted onto the microfluidic paper-based analytical device (μPAD). Samples containing lactic acid, resazurin, and nicotinamide adenine dinucleotide oxidized form (NAD(+) ), potassium chloride (KCl), and BSA, in MES buffer were separately spotted onto the μPAD and MES buffer flowed through the device. A cascade reaction occurs upon the sample spot overlapping with LDH to form pyruvate and nicotinamide adenine dinucleotide reduced form (NADH). Subsequently, NADH is used in the conversion of resazurin to fluorescent resorufin by DI. The μPAD avoids the need of surface functionalization or enzyme immobilization steps. These microreactor devices are low cost and easy to fabricate and effect reaction based solely on buffer capillary action. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Broadening the cofactor specificity of a thermostable alcohol dehydrogenase using rational protein design introduces novel kinetic transient behavior.

    PubMed

    Campbell, Elliot; Wheeldon, Ian R; Banta, Scott

    2010-12-01

    Cofactor specificity in the aldo-keto reductase (AKR) superfamily has been well studied, and several groups have reported the rational alteration of cofactor specificity in these enzymes. Although most efforts have focused on mesostable AKRs, several putative AKRs have recently been identified from hyperthermophiles. The few that have been characterized exhibit a strong preference for NAD(H) as a cofactor, in contrast to the NADP(H) preference of the mesophilic AKRs. Using the design rules elucidated from mesostable AKRs, we introduced two site-directed mutations in the cofactor binding pocket to investigate cofactor specificity in a thermostable AKR, AdhD, which is an alcohol dehydrogenase from Pyrococcus furiosus. The resulting double mutant exhibited significantly improved activity and broadened cofactor specificity as compared to the wild-type. Results of previous pre-steady-state kinetic experiments suggest that the high affinity of the mesostable AKRs for NADP(H) stems from a conformational change upon cofactor binding which is mediated by interactions between a canonical arginine and the 2'-phosphate of the cofactor. Pre-steady-state kinetics with AdhD and the new mutants show a rich conformational behavior that is independent of the canonical arginine or the 2'-phosphate. Additionally, experiments with the highly active double mutant using NADPH as a cofactor demonstrate an unprecedented transient behavior where the binding mechanism appears to be dependent on cofactor concentration. These results suggest that the structural features involved in cofactor specificity in the AKRs are conserved within the superfamily, but the dynamic interactions of the enzyme with cofactors are unexpectedly complex. © 2010 Wiley Periodicals, Inc.

  11. Quercetin Attenuates Vascular Calcification through Suppressed Oxidative Stress in Adenine-Induced Chronic Renal Failure Rats.

    PubMed

    Chang, Xue-Ying; Cui, Lei; Wang, Xing-Zhi; Zhang, Lei; Zhu, Dan; Zhou, Xiao-Rong; Hao, Li-Rong

    2017-01-01

    This study investigated whether quercetin could alleviate vascular calcification in experimental chronic renal failure rats induced by adenine. 32 adult male Wistar rats were randomly divided into 4 groups fed normal diet, normal diet with quercetin supplementation (25 mg/kg·BW/d), 0.75% adenine diet, or adenine diet with quercetin supplementation. All rats were sacrificed after 6 weeks of intervention. Serum renal functions biomarkers and oxidative stress biomarkers were measured and status of vascular calcification in aorta was assessed. Furthermore, the induced nitric oxide synthase (iNOS)/p38 mitogen activated protein kinase (p38MAPK) pathway was determined to explore the potential mechanism. Adenine successfully induced renal failure and vascular calcification in rat model. Quercetin supplementation reversed unfavorable changes of phosphorous, uric acid (UA) and creatinine levels, malonaldehyde (MDA) content, and superoxide dismutase (SOD) activity in serum and the increases of calcium and alkaline phosphatase (ALP) activity in the aorta ( P < 0.05) and attenuated calcification and calcium accumulation in the medial layer of vasculature in histopathology. Western blot analysis showed that iNOS/p38MAPK pathway was normalized by the quercetin supplementation. Quercetin exerted a protective effect on vascular calcification in adenine-induced chronic renal failure rats, possibly through the modulation of oxidative stress and iNOs/p38MAPK pathway.

  12. Quercetin Attenuates Vascular Calcification through Suppressed Oxidative Stress in Adenine-Induced Chronic Renal Failure Rats

    PubMed Central

    Chang, Xue-ying; Cui, Lei; Wang, Xing-zhi; Zhang, Lei; Zhu, Dan

    2017-01-01

    Background This study investigated whether quercetin could alleviate vascular calcification in experimental chronic renal failure rats induced by adenine. Methods 32 adult male Wistar rats were randomly divided into 4 groups fed normal diet, normal diet with quercetin supplementation (25 mg/kg·BW/d), 0.75% adenine diet, or adenine diet with quercetin supplementation. All rats were sacrificed after 6 weeks of intervention. Serum renal functions biomarkers and oxidative stress biomarkers were measured and status of vascular calcification in aorta was assessed. Furthermore, the induced nitric oxide synthase (iNOS)/p38 mitogen activated protein kinase (p38MAPK) pathway was determined to explore the potential mechanism. Results Adenine successfully induced renal failure and vascular calcification in rat model. Quercetin supplementation reversed unfavorable changes of phosphorous, uric acid (UA) and creatinine levels, malonaldehyde (MDA) content, and superoxide dismutase (SOD) activity in serum and the increases of calcium and alkaline phosphatase (ALP) activity in the aorta (P < 0.05) and attenuated calcification and calcium accumulation in the medial layer of vasculature in histopathology. Western blot analysis showed that iNOS/p38MAPK pathway was normalized by the quercetin supplementation. Conclusions Quercetin exerted a protective effect on vascular calcification in adenine-induced chronic renal failure rats, possibly through the modulation of oxidative stress and iNOs/p38MAPK pathway. PMID:28691026

  13. 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.

  14. The in vivo effects of adenine-induced chronic kidney disease on some renal and hepatic function and CYP450 metabolizing enzymes.

    PubMed

    Al Za'abi, M; Shalaby, A; Manoj, P; Ali, B H

    2017-05-04

    Adenine-induced model of chronic kidney disease (CKD) is a widely used model especially in studies testing novel nephroprotective agents. We investigated the effects of adenine-induced CKD in rats on the activities of some xenobiotic metabolizing enzymes in liver and kidneys, and on some in vivo indicators of drug metabolism (viz pentobarbitone sleeping time, and plasma concentration of theophylline 90 min post administration). CKD was induced by orally feeding adenine (0.25 % w/w) for 35 days. Adenine induced all the characteristics of CKD, which was confirmed by biochemical and histological findings. Glutathione concentration and activities of some enzymes involved in its metabolism were reduced in kidneys and livers of rats with CKD. Renal CYP450 1A1 activity was significantly inhibited by adenine, but other measured isoenzymes (1A2, 3A4 and 2E1) were not significantly affected. Adenine significantly prolonged pentobarbitone-sleeping time and increased plasma theophylline concentration 90 min post administration. Adenine also induced a moderate degree of hepatic damages as indicated histologically and by significant elevations in some plasma enzymes. The results suggest that adenine-induced CKD is associated with significant in vivo inhibitory activities on some drug-metabolizing enzymes, with most of the effect on the kidneys rather than the liver.

  15. Spectroscopic investigation on cocrystal formation between adenine and fumaric acid based on infrared and Raman techniques

    NASA Astrophysics Data System (ADS)

    Du, Yong; Fang, Hong Xia; Zhang, Qi; Zhang, Hui Li; Hong, Zhi

    2016-01-01

    As an important component of double-stranded DNA, adenine has powerful hydrogen-bond capability, due to rich hydrogen bond donors and acceptors existing within its molecular structure. Therefore, it is easy to form cocrystal between adenine and other small molecules with intermolecular hydrogen-bond effect. In this work, cocrystal of adenine and fumaric acid has been characterized as model system by FT-IR and FT-Raman spectral techniques. The experimental results show that the cocrystal formed between adenine and fumaric acid possesses unique spectroscopical characteristic compared with that of starting materials. Density functional theory (DFT) calculation has been performed to optimize the molecular structures and simulate vibrational modes of adenine, fumaric acid and the corresponding cocrystal. Combining the theoretical and experimental vibrational results, the characteristic bands corresponding to bending and stretching vibrations of amino and carbonyl groups within cocrystal are shifted into lower frequencies upon cocrystal formation, and the corresponding bond lengths show some increase due to the effect of intermolecular hydrogen bonding. Different vibrational modes shown in the experimental spectra have been assigned based on the simulation DFT results. The study could provide experimental and theoretical benchmarks to characterize cocrystal formed between active ingredients and cocrystal formers and also the intermolecular hydrogen-bond effect within cocrystal formation process by vibrational spectroscopic techniques.

  16. 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.

  17. Effect of atracylodes rhizome polysaccharide in rats with adenine-induced chronic renal failure.

    PubMed

    Yang, C; Liu, C; Zhou, Q; Xie, Y C; Qiu, X M; Feng, X

    2015-01-01

    The aim of the study was to elucidate the therapeutic effects of Atracylodes rhizome polysaccharide on adenine-induced chronic renal failure in rats. Fifty male Sprague Dawley rats were selected and randomly divided in to 5 groups (n=10 rats per group): The normal control group, the chronic renal failure pathological control group, the dexamethasone treatment group and two Atracylodes rhizome polysaccharide treatment groups, treated with two different concentrations of the polysaccharide, the Atracylodes rhizome polysaccharide high group and the Atracylodes rhizome polysaccharide low group. All the rats, except those in the normal control group were fed adenine-enriched diets, containing 10 g adenine per kg food for 3 weeks. After being fed with adenine, the dexamethasone treatment group, Atracylodes rhizome polysaccharide high group and Atracylodes rhizome polysaccharide low group rats were administered the drug orally for 2 weeks. On day 35, the kidney coefficient of the rats and the serum levels of creatinine, blood urea nitrogen, total protein and hemalbumin were determined. Subsequent to experimentation on a model of chronic renal failure in rats, the preparation was proven to be able to reduce serum levels of creatinine, blood urea nitrogen and hemalbumin levels (P<0.05) and improve renal function. Atracylodes rhizome polysaccharide had reversed the majority of the indices of chronic renal failure in rats.

  18. Role of Electron-Driven Proton-Transfer Processes in the Ultrafast Deactivation of Photoexcited Anionic 8-oxoGuanine-Adenine and 8-oxoGuanine-Cytosine Base Pairs.

    PubMed

    Wu, Xiuxiu; Karsili, Tolga N V; Domcke, Wolfgang

    2017-01-14

    It has been reported that 8-oxo-7,8-dihydro-guanosine (8-oxo-G), which is the main product of oxidative damage of DNA, can repair cyclobutane pyrimidine dimer (CPD) lesions when incorporated into DNA or RNA strands in proximity to such lesions. It has therefore been suggested that the 8-oxo-G nucleoside may have been a primordial precursor of present-day flavins in DNA or RNA repair. Because the electron transfer leading to the splitting of a thymine-thymine pair in a CPD lesion occurs in the photoexcited state, a reasonably long excited-state lifetime of 8-oxo-G is required. The neutral (protonated) form of 8-oxo-G exhibits a very short (sub-picosecond) intrinsic excited-state lifetime which is unfavorable for repair. It has therefore been argued that the anionic (deprotonated) form of 8-oxo-G, which exhibits a much longer excited-state lifetime, is more likely to be a suitable cofactor for DNA repair. Herein, we have investigated the exited-state quenching mechanisms in the hydrogen-bonded complexes of deprotonated 8-oxo-G - with adenine (A) and cytosine (C) using ab initio wave-function-based electronic-structure calculations. The calculated reaction paths and potential-energy profiles reveal the existence of barrierless electron-driven inter-base proton-transfer reactions which lead to low-lying S₁/S₀ conical intersections. The latter can promote ultrafast excited-state deactivation of the anionic base pairs. While the isolated deprotonated 8-oxo-G - nucleoside may have been an efficient primordial repair cofactor, the excited states of the 8-oxo-G - -A and 8-oxo-G - -C base pairs are likely too short-lived to be efficient electron-transfer repair agents.

  19. Kinetic Isotope Effects as a Probe of Hydrogen Transfers to and from Common Enzymatic Cofactors

    PubMed Central

    Roston, Daniel; Islam, Zahidul; Kohen, Amnon

    2013-01-01

    Enzymes use a number of common cofactors as sources of hydrogen to drive biological processes, but the physics of the hydrogen transfers to and from these cofactors is not fully understood. Researchers study the mechanistically important contributions from quantum tunneling and enzyme dynamics and connect those processes to the catalytic power of enzymes that use these cofactors. Here we describe some progress that has been made in studying these reactions, particularly through the use of kinetic isotope effects (KIEs). We first discuss the general theoretical framework necessary to interpret experimental KIEs, and then describe practical uses for KIEs in the context of two case studies. The first example is alcohol dehydrogenase, which uses a nicotinamide cofactor to catalyze a hydride transfer, and the second example is thymidylate synthase, which uses a folate cofactor to catalyze both a hydride and a proton transfer. PMID:24161942

  20. The phylogenomic roots of modern biochemistry: origins of proteins, cofactors and protein biosynthesis.

    PubMed

    Caetano-Anollés, Gustavo; Kim, Kyung Mo; Caetano-Anollés, Derek

    2012-02-01

    The complexity of modern biochemistry developed gradually on early Earth as new molecules and structures populated the emerging cellular systems. Here, we generate a historical account of the gradual discovery of primordial proteins, cofactors, and molecular functions using phylogenomic information in the sequence of 420 genomes. We focus on structural and functional annotations of the 54 most ancient protein domains. We show how primordial functions are linked to folded structures and how their interaction with cofactors expanded the functional repertoire. We also reveal protocell membranes played a crucial role in early protein evolution and show translation started with RNA and thioester cofactor-mediated aminoacylation. Our findings allow elaboration of an evolutionary model of early biochemistry that is firmly grounded in phylogenomic information and biochemical, biophysical, and structural knowledge. The model describes how primordial α-helical bundles stabilized membranes, how these were decorated by layered arrangements of β-sheets and α-helices, and how these arrangements became globular. Ancient forms of aminoacyl-tRNA synthetase (aaRS) catalytic domains and ancient non-ribosomal protein synthetase (NRPS) modules gave rise to primordial protein synthesis and the ability to generate a code for specificity in their active sites. These structures diversified producing cofactor-binding molecular switches and barrel structures. Accretion of domains and molecules gave rise to modern aaRSs, NRPS, and ribosomal ensembles, first organized around novel emerging cofactors (tRNA and carrier proteins) and then more complex cofactor structures (rRNA). The model explains how the generation of protein structures acted as scaffold for nucleic acids and resulted in crystallization of modern translation.

  1. A comparative study of metabolic state of stem cells during osteogenic and adipogenic differentiations via fluorescence lifetime imaging microscopy

    NASA Astrophysics Data System (ADS)

    Chakraborty, Sandeep; Ou, Meng-Hsin; Kuo, Jean-Cheng; Chiou, Arthur

    2016-10-01

    Cellular metabolic state can serve as a biomarker to indicate the differentiation potential of stem cells into other specialized cell lineages. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was applied to determine the fluorescence lifetime and the amounts of the auto-fluorescent metabolic co-factor reduced nicotinamide adenine dinucleotide (NADH) to elucidate the cellular metabolism of human mesenchymal stem cells (hMSCs) in osteogenic and adipogenic differentiation processes. 2P-FLIM provides the free to protein-bound NADH ratio which can serve as the indicator of cellular metabolic state. We measured NADH fluorescence lifetime at 0, 7, and 14 days after hMSCs were induced for either osteogenesis or adipogenesis. In both cases, the average fluorescence lifetime increased significantly at day 14 (P < 0.001), while the ratio of free to protein-bound NADH ratio decreased significantly in 7- days (P < 0.001) and 14-days (P < 0.001). Thus, our results indicated a higher metabolic rate in both osteogenic and adipogenic differentiation processes when compared with undifferentiated hMSCs. This approach may be further utilized to study proliferation efficiency and differentiation potential of stem cells into other specialized cell lineages.

  2. Direct electron transfer of glucose oxidase on carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Guiseppi-Elie, Anthony; Lei, Chenghong; Baughman, Ray H.

    2002-10-01

    In this report, exploitation of the unique properties of single-walled carbon nanotubes (SWNT) leads to the achievement of direct electron transfer with the redox active centres of adsorbed oxidoreductase enzymes. Flavin adenine dinucleotide (FAD), the redox active prosthetic group of flavoenzymes that catalyses important biological redox reactions and the flavoenzyme glucose oxidase (GOx), were both found to spontaneously adsorb onto carbon nanotube bundles. Both FAD and GOx were found to spontaneously adsorb to unannealed carbon nanotubes that were cast onto glassy carbon electrodes and to display quasi-reversible one-electron transfer. Similarly, GOx was found to spontaneously adsorb to annealed, single-walled carbon nanotube paper and to display quasi-reversible one-electron transfer. In particular, GOx immobilized in this way was shown, in the presence of glucose, to maintain its substrate-specific enzyme activity. It is believed that the tubular fibrils become positioned within tunnelling distance of the cofactors with little consequence to denaturation. The combination of SWNT with redox active enzymes would appear to offer an excellent and convenient platform for a fundamental understanding of biological redox reactions as well as the development of reagentless biosensors and nanobiosensors.

  3. New Therapeutic Concept of NAD Redox Balance for Cisplatin Nephrotoxicity

    PubMed Central

    Oh, Gi-Su; Kim, Hyung-Jin; Shen, AiHua; Lee, Su-Bin; Yang, Sei-Hoon; Shim, Hyeok; Cho, Eun-Young; Kwon, Kang-Beom; Kwak, Tae Hwan; So, Hong-Seob

    2016-01-01

    Cisplatin is a widely used chemotherapeutic agent for the treatment of various tumors. In addition to its antitumor activity, cisplatin affects normal cells and may induce adverse effects such as ototoxicity, nephrotoxicity, and peripheral neuropathy. Various mechanisms such as DNA adduct formation, mitochondrial dysfunction, oxidative stress, and inflammatory responses are closely associated with cisplatin-induced nephrotoxicity; however, the precise mechanism remains unclear. The cofactor nicotinamide adenine dinucleotide (NAD+) has emerged as a key regulator of cellular energy metabolism and homeostasis. Recent studies have demonstrated associations between disturbance in intracellular NAD+ levels and clinical progression of various diseases through the production of reactive oxygen species and inflammation. Furthermore, we demonstrated that reduction of the intracellular NAD+/NADH ratio is critically involved in cisplatin-induced kidney damage through inflammation and oxidative stress and that increase of the cellular NAD+/NADH ratio suppresses cisplatin-induced kidney damage by modulation of potential damage mediators such as oxidative stress and inflammatory responses. In this review, we describe the role of NAD+ metabolism in cisplatin-induced nephrotoxicity and discuss a potential strategy for the prevention or treatment of cisplatin-induced adverse effects with a particular focus on NAD+-dependent cellular pathways. PMID:26881219

  4. Development of quantum dots-based biosensor towards on-farm detection of subclinical ketosis.

    PubMed

    Weng, Xuan; Chen, Longyan; Neethirajan, Suresh; Duffield, Todd

    2015-10-15

    Early detection of dairy animal health issues allows the producer or veterinarian to intervene before the animals' production levels, or even survival, is threatened. An increased concentration of β-hydroxybutyrate (βHBA) is a key biomarker for diagnosis of subclinical ketosis (SCK), and provides information on the health stress in cows well before any external symptoms are observable. In this study, quantum dots (QDs) modified with cofactor nicotinamide adenine dinucleotide (NAD(+)) were prepared for the sensing event, by which the βHBA concentration in the cow's blood and milk samples was determined via fluorescence analysis of the functionalized QDs. The detection was performed on a custom designed microfluidic platform combining with a low cost and miniaturized optical sensor. The sensing mechanism was first validated by a microplate reader method and then applied to the microfluidic platform. Standard βHBA solution, βHBA in blood and milk samples from cows were successfully measured by this novel technology with a detection limit at a level of 35 µM. Side by side comparison of the developed microfluidic biosensor with a commercial kit presented its good performance. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Aluminum and its effect in the equilibrium between folded/unfolded conformation of NADH.

    PubMed

    Formoso, Elena; Mujika, Jon I; Grabowski, Slawomir J; Lopez, Xabier

    2015-11-01

    Nicotinamide adenine dinucleotide (NADH) is one of the most abundant cofactor employed by proteins and enzymes. The molecule is formed by two nucleotides that can lead to two main conformations: folded/closed and unfolded/open. Experimentally, it has been determined that the closed form is about 2 kcal/mol more stable than the open formed. Computationally, a correct description of the NADH unfolding process is challenging due to different reasons: 1) The unfolding process shows a very low energy difference between the two conformations 2) The molecule can form a high number of internal hydrogen bond interactions 3) Subtle effects such as dispersion may be important. In order to tackle all these effects, we have employed a number of different state of the art computational techniques, including: a) well-tempered metadynamics, b) geometry optimizations, and c) Quantum Theory of Atoms in Molecules (QTAIM) calculations, to investigate the conformational change of NADH in solution and interacting with aluminum. All the results indicate that aluminum indeed favors the closed conformation of NADH, due mainly to the formation of a more rigid structure through key hydrogen bond interactions. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Dunnione ameliorates cisplatin ototoxicity through modulation of NAD(+) metabolism.

    PubMed

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

    2016-03-01

    Ototoxicity is an important issue in patients receiving cisplatin chemotherapy. Numerous studies have demonstrated that cisplatin-induced ototoxicity is related to oxidative stress and DNA damage. However, the precise mechanism underlying cisplatin-associated ototoxicity is still unclear. The cofactor nicotinamide adenine dinucleotide (NAD(+)) has emerged as an important regulator of energy metabolism and cellular homeostasis. Here, we demonstrate that the levels and activities of sirtuin-1 (SIRT1) are suppressed by the reduction of intracellular NAD(+) levels in cisplatin-mediated ototoxicity. We provide evidence that the decreases in SIRT1 activity and expression facilitated by increasing poly(ADP-ribose) polymerase-1 (PARP-1) activation and microRNA-34a levels through cisplatin-mediated p53 activation aggravate the associated ototoxicity. Furthermore, we show that the induction of cellular NAD(+) levels using dunnione, which targets intracellular NQO1, prevents the toxic effects of cisplatin through the regulation of PARP-1 and SIRT1 activity. These results suggest that direct modulation of cellular NAD(+) levels by pharmacological agents could be a promising therapeutic approach for protection from cisplatin-induced ototoxicity. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Unique Aspects of Cryptochrome in Chronobiology and Metabolism, Pancreatic β-Cell Dysfunction, and Regeneration: Research into Cysteine414-Alanine Mutant CRY1.

    PubMed

    Okano, Satoshi

    2016-01-01

    Cryptochrome proteins (CRYs), which can bind noncovalently to cofactor (chromophore) flavin adenine dinucleotide (FAD), occur widely among organisms. CRYs play indispensable roles in the generation of circadian rhythm in mammals. Transgenic mice (Tg mice), ubiquitously expressing mouse CRY1 having a mutation in which cysteine414 (the zinc-binding site of CRY1) being replaced with alanine, display unique phenotypes in their circadian rhythms. Moreover, male Tg mice exhibit symptoms of diabetes characterized by beta-cell dysfunction, resembling human maturity onset diabetes of the young (MODY). The lowered proliferation of β -cells is a primary cause of age-dependent β -cell loss. Furthermore, unusually enlarged duct-like structures developed prominently in the Tg mice pancreases. The duct-like structures contained insulin-positive cells, suggesting neogenesis of β -cells in the Tg mice. This review, based mainly on the author's investigation of the unique features of Tg mice, presents reported results and recent findings related to molecular processes associated with mammalian cryptochromes, especially their involvement in the regulation of metabolism. New information is described with emphasis on the aspects of islet architecture, pancreatic β -cell dysfunction, and regeneration.

  8. Facilitation of NADH Electrooxidation at Treated Carbon Nanotubes

    PubMed Central

    Wooten, Marilyn; Gorski, Waldemar

    2010-01-01

    The relationship between the state of the surface of carbon nanotubes (CNT) and their electrochemical activity was investigated using the enzyme cofactor dihydronicotinamide adenine dinucleotide (NADH) as a redox probe. The boiling of CNT in water, while nondestructive, activated them toward the oxidation of NADH as indicated by a shift in the anodic peak potential of NADH (ENADH) from 0.4 to 0.0 V. The shift in ENADH was due to the redox mediation of NADH oxidation by traces of quinone species that were formed on the surface of treated CNT. The harsher treatment that comprised of microwaving of CNT in concentrated nitric acid had a similar effect on the ENADH and, additionally, it increased the anodic peak current of NADH. The latter correlated with the formation of defects on the surface of acid-microwaved CNT as indicated by their Raman spectra. The increase in current was discussed considering a role of surface mediators on the buckled graphene sheets of acid-microwaved CNT. The other carbon allotropes including the edge plane pyrolytic graphite, graphite powder, and glassy carbon did not display a comparable activation toward the oxidation of NADH. PMID:20088562

  9. DEER distance measurement between a spin label and a native FAD semiquinone in electron transfer flavoprotein.

    PubMed

    Swanson, Michael A; Kathirvelu, Velavan; Majtan, Tomas; Frerman, Frank E; Eaton, Gareth R; Eaton, Sandra S

    2009-11-11

    The human mitochondrial electron transfer flavoprotein (ETF) accepts electrons from at least 10 different flavoprotein dehydrogenases and transfers electrons to a single electron acceptor in the inner membrane. Paracoccus denitrificans ETF has the identical function, shares the same three-dimensional structure and functional domains, and exhibits the same conformational mobility. It has been proposed that the mobility of the alphaII domain permits the promiscuous behavior of ETF with respect to a variety of redox partners. Double electron-electron resonance (DEER) measurements between a spin label and an enzymatically reduced flavin adenine dinucleotide (FAD) cofactor in P. denitrificans ETF gave two distributions of distances: a major component centered at 4.2 +/- 0.1 nm and a minor component centered at 5.1 +/- 0.2 nm. Both components had widths of approximately 0.3 nm. A distance of 4.1 nm was calculated using the crystal structure of P. denitrificans ETF, which agrees with the major component obtained from the DEER measurement. The observation of a second distribution suggests that ETF, in the absence of substrate, adopts some conformations that are intermediate between the predominant free and substrate-bound states.

  10. DEER Distance Measurement Between a Spin Label and a Native FAD Semiquinone in Electron Transfer Flavoprotein

    PubMed Central

    Swanson, Michael A.; Kathirvelu, Velavan; Majtan, Tomas; Frerman, Frank E.; Eaton, Gareth R.; Eaton, Sandra S.

    2009-01-01

    The human mitochondrial electron transfer flavoprotein (ETF) accepts electrons from at least 10 different flavoprotein dehydrogenases and transfers electrons to a single electron acceptor in the inner membrane. Paracoccus denitrificans ETF has the identical function, shares the same three dimensional structure and functional domains, and exhibits the same conformational mobility. It has been proposed that the mobility of the αII domain permits the promiscuous behavior of ETF with respect to a variety of redox partners. Double electron-electron resonance (DEER) measurements between a spin label and an enzymatically reduced flavin adenine dinucleotide (FAD) cofactor in P. denitrificans ETF gave two distributions of distances: a major component centered at 4.2 ± 0.1 nm and a minor component centered at 5.1 ± 0.2 nm. Both components had widths of approximately 0.3 nm. A distance of 4.1 nm was calculated using the crystal structure of P. denitrificans ETF, which agrees with the major component obtained from the DEER measurement. The observation of a second distribution suggests that ETF, in the absence of substrate, adopts some conformations that are intermediate between the predominant free and substrate-bound states. PMID:19886689

  11. Gene Transfers Shaped the Evolution of De Novo NAD+ Biosynthesis in Eukaryotes

    PubMed Central

    Ternes, Chad M.; Schönknecht, Gerald

    2014-01-01

    NAD+ is an essential molecule for life, present in each living cell. It can function as an electron carrier or cofactor in redox biochemistry and energetics, and serves as substrate to generate the secondary messenger cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate. Although de novo NAD+ biosynthesis is essential, different metabolic pathways exist in different eukaryotic clades. The kynurenine pathway starting with tryptophan was most likely present in the last common ancestor of all eukaryotes, and is active in fungi and animals. The aspartate pathway, detected in most photosynthetic eukaryotes, was probably acquired from the cyanobacterial endosymbiont that gave rise to chloroplasts. An evolutionary analysis of enzymes catalyzing de novo NAD+ biosynthesis resulted in evolutionary trees incongruent with established organismal phylogeny, indicating numerous gene transfers. Endosymbiotic gene transfers probably introduced the aspartate pathway into eukaryotes and may have distributed it among different photosynthetic clades. In addition, several horizontal gene transfers substituted eukaryotic genes with bacterial orthologs. Although horizontal gene transfer is accepted as a key mechanism in prokaryotic evolution, it is supposed to be rare in eukaryotic evolution. The essential metabolic pathway of de novo NAD+ biosynthesis in eukaryotes was shaped by numerous gene transfers. PMID:25169983

  12. Supramolecular polymeric chemosensor for biomedical applications: design and synthesis of a luminescent zinc metallopolymer as a chemosensor for adenine detection.

    PubMed

    Chow, Cheuk-Fai

    2012-11-01

    Adenine is an important bio-molecule that plays many crucial roles in food safety and biomedical diagnostics. Differentiating adenine from a mixture of adenosine and other nucleic bases (guanine, thymine, cytosine, and uracil) is particularly important for both biological and clinical applications. A neutral Zn(II) metallosupramolecular polymer based on acyl hydrazone derived coordination centres (P1) were generated through self-assembly polymerization. It is a linear coordination polymer that behaves like self-standing film. The synthesis, (1)H-NMR characterization, and spectroscopic properties of this supramolecular material are reported. P1 was found to be a chemosensor specific to adenine, with a luminescent enhancement. The binding properties of P1 with common nucleic bases and nucleosides reveal that this supramolecular polymer is very selective to adenine molecules (~20 to 420 times more selectivity than other nucleic bases). The formation constant (K) of P1 to adenine was found to be log K = 4.10 ± 0.02. This polymeric chemosensor produces a specific response to adenine down to 90 ppb. Spectrofluorimetric and (1)H-NMR titration studies showed that the P1 polymer allows each Zn(II) coordination centre to bind to two adenine molecules through hydrogen bonding with their imine and hydrazone protons.

  13. Cofactor-dependent specificity of a DEAD-box protein.

    PubMed

    Young, Crystal L; Khoshnevis, Sohail; Karbstein, Katrin

    2013-07-16

    DEAD-box proteins, a large class of RNA-dependent ATPases, regulate all aspects of gene expression and RNA metabolism. They can facilitate dissociation of RNA duplexes and remodeling of RNA-protein complexes, serve as ATP-dependent RNA-binding proteins, or even anneal duplexes. These proteins have highly conserved sequence elements that are contained within two RecA-like domains; consequently, their structures are nearly identical. Furthermore, crystal structures of DEAD-box proteins with bound RNA reveal interactions exclusively between the protein and the RNA backbone. Together, these findings suggest that DEAD-box proteins interact with their substrates in a nonspecific manner, which is confirmed in biochemical experiments. Nevertheless, this contrasts with the need to target these enzymes to specific substrates in vivo. Using the DEAD-box protein Rok1 and its cofactor Rrp5, which both function during maturation of the small ribosomal subunit, we show here that Rrp5 provides specificity to the otherwise nonspecific biochemical activities of the Rok1 DEAD-domain. This finding could reconcile the need for specific substrate binding of some DEAD-box proteins with their nonspecific binding surface and expands the potential roles of cofactors to specificity factors. Identification of helicase cofactors and their RNA substrates could therefore help define the undescribed roles of the 19 DEAD-box proteins that function in ribosome assembly.

  14. Heptacopper(II) and dicopper(II)-adenine complexes: synthesis, structural characterization, and magnetic properties

    DOE PAGES

    Leite Ferreira, B. J. M.; Brandão, Paula; Dos Santos, A. M.; ...

    2015-07-13

    The syntheses, crystal structures, and magnetic properties of two new copper(II) complexes with molecular formulas [Cu 7(μ 2-OH 2) 6(μ 3-O) 6(adenine) 6(NO 3) 26H 2O (1) and [Cu 2(μ 2-H 2O) 2(adenine) 2(H 2O) 4](NO 3) 42H 2O (2) are reported. We composed the heptanuclear compound of a central octahedral CuO 6 core sharing edges with six adjacent copper octahedra. In 2, the copper octahedra shares one equatorial edge. In both compounds, these basic copper cluster units are further linked by water bridges and bridging adenine ligands through N3 and N9 donors. All copper(II) centers exhibit Jahn-Teller distorted octahedralmore » coordination characteristic of a d 9 center. Our study of the magnetic properties of the heptacopper complex revealed a dominant ferromagnetic intra-cluster interaction, while the dicopper complex exhibits antiferromagnetic intra-dimer interactions with weakly ferromagnetic inter-dimer interaction.« less

  15. 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

    USDA-ARS?s Scientific Manuscript database

    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...

  16. Nicotinamide Adenine Dinucleotide (NAD+) and Nicotinamide: Sex Differences in Cerebral Ischemia

    PubMed Central

    Siegel, Chad S.; McCullough, Louise D.

    2013-01-01

    Background Previous literature suggests that cell death pathways activated after cerebral ischemia differ between the sexes. While caspase-dependent mechanisms predominate in the female brain, caspase-independent cell death induced by activation of Poly (ADP-ribose) polymerase (PARP) predominates in the male brain. PARP-1 gene deletion decreases infarction volume in the male brain, but paradoxically increases damage in PARP-1 knockout females. Purpose This study examined stroke induced changes in NAD+, a key energy molecule involved in PARP-1 activation in both sexes. Methods Mice were subjected to Middle Cerebral Artery Occlusion and NAD+ levels were assessed. Caspase-3 activity and nuclear translocation was assessed 6 hours after ischemia. In additional cohorts, Nicotinamide (500mg/kg i.p.) a precursor of NAD+ or vehicle was administered and infarction volume was measured 24 hours after ischemia. Results Males have higher baseline NAD+ levels than females. Significant stroke-induced NAD+ depletion occurred in males and ovariectomized females but not in intact females. PARP-1 deletion prevented the stroke induced loss in NAD+ in males, but worsened NAD+ loss in PARP-1 deficient females. Preventing NAD+ loss with nicotinamide reduced infarct in wild-type males and PARP-1 knockout mice of both sexes, with no effect in WT females. Caspase-3 activity was significantly increased in PARP-1 knockout females compared to males and wild-type females, this was reversed with nicotinamide. Conclusions Sex differences exist in baseline and stroke-induced NAD+ levels. Nicotinamide protected males and PARP knockout mice, but had minimal effects in the wild-type female brain. This may be secondary to differences in energy metabolism between the sexes. PMID:23403179

  17. Improved Ethanol Production from Xylose by Candida shehatae Induced by Dielectric Barrier Discharge Air Plasma

    NASA Astrophysics Data System (ADS)

    Chen, Huixia; Xiu, Zhilong; Bai, Fengwu

    2014-06-01

    Xylose fermentation is essential for ethanol production from lignocellulosic biomass. Exposure of the xylose-fermenting yeast Candida shehatae (C. shehatae) CICC1766 to atmospheric pressure dielectric barrier discharge (DBD) air plasma yields a clone (designated as C81015) with stability, which exhibits a higher ethanol fermentation rate from xylose, giving a maximal enhancement in ethanol production of 36.2% compared to the control (untreated). However, the biomass production of C81015 is lower than that of the control. Analysis of the NADH (nicotinamide adenine dinucleotide)- and NADPH (nicotinamide adenine dinucleotide phosphate)-linked xylose reductases and NAD+-linked xylitol dehydrogenase indicates that their activities are enhanced by 34.1%, 61.5% and 66.3%, respectively, suggesting that the activities of these three enzymes are responsible for improving ethanol fermentation in C81015 with xylose as a substrate. The results of this study show that DBD air plasma could serve as a novel and effective means of generating microbial strains that can better use xylose for ethanol fermentation.

  18. Application of Negative Curvature Hollow-Core Fiber in an Optical Fiber Sensor Setup for Multiphoton Spectroscopy

    PubMed Central

    Stawska, Hanna Izabela; Mazur, Leszek Mateusz; Kosolapov, Alexey; Kolyadin, Anton; Bereś-Pawlik, Elżbieta

    2017-01-01

    In this paper, an application of negative curvature hollow core fiber (NCHCF) in an all-fiber, multiphoton fluorescence sensor setup is presented. The dispersion parameter (D) of this fiber does not exceed the value of 5 ps/nm × km across the optical spectrum of (680–750) nm, making it well suited for the purpose of multiphoton excitation of biological fluorophores. Employing 1.5 m of this fiber in a simple, all-fiber sensor setup allows us to perform multiphoton experiments without any dispersion compensation methods. Multiphoton excitation of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) with this fiber shows a 6- and 9-fold increase, respectively, in the total fluorescence signal collected when compared with the commercial solution in the form of a hollow-core photonic band gap fiber (HCPBF). To the author’s best knowledge, this is the first time an NCHCF was used in an optical-fiber sensor setup for multiphoton fluorescence experiments. PMID:28984838

  19. Application of Negative Curvature Hollow-Core Fiber in an Optical Fiber Sensor Setup for Multiphoton Spectroscopy.

    PubMed

    Popenda, Maciej Andrzej; Stawska, Hanna Izabela; Mazur, Leszek Mateusz; Jakubowski, Konrad; Kosolapov, Alexey; Kolyadin, Anton; Bereś-Pawlik, Elżbieta

    2017-10-06

    In this paper, an application of negative curvature hollow core fiber (NCHCF) in an all-fiber, multiphoton fluorescence sensor setup is presented. The dispersion parameter (D) of this fiber does not exceed the value of 5 ps/nm × km across the optical spectrum of (680-750) nm, making it well suited for the purpose of multiphoton excitation of biological fluorophores. Employing 1.5 m of this fiber in a simple, all-fiber sensor setup allows us to perform multiphoton experiments without any dispersion compensation methods. Multiphoton excitation of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) with this fiber shows a 6- and 9-fold increase, respectively, in the total fluorescence signal collected when compared with the commercial solution in the form of a hollow-core photonic band gap fiber (HCPBF). To the author's best knowledge, this is the first time an NCHCF was used in an optical-fiber sensor setup for multiphoton fluorescence experiments.

  20. 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.

  1. Spectroscopic investigation on cocrystal formation between adenine and fumaric acid based on infrared and Raman techniques.

    PubMed

    Du, Yong; Fang, Hong Xia; Zhang, Qi; Zhang, Hui Li; Hong, Zhi

    2016-01-15

    As an important component of double-stranded DNA, adenine has powerful hydrogen-bond capability, due to rich hydrogen bond donors and acceptors existing within its molecular structure. Therefore, it is easy to form cocrystal between adenine and other small molecules with intermolecular hydrogen-bond effect. In this work, cocrystal of adenine and fumaric acid has been characterized as model system by FT-IR and FT-Raman spectral techniques. The experimental results show that the cocrystal formed between adenine and fumaric acid possesses unique spectroscopical characteristic compared with that of starting materials. Density functional theory (DFT) calculation has been performed to optimize the molecular structures and simulate vibrational modes of adenine, fumaric acid and the corresponding cocrystal. Combining the theoretical and experimental vibrational results, the characteristic bands corresponding to bending and stretching vibrations of amino and carbonyl groups within cocrystal are shifted into lower frequencies upon cocrystal formation, and the corresponding bond lengths show some increase due to the effect of intermolecular hydrogen bonding. Different vibrational modes shown in the experimental spectra have been assigned based on the simulation DFT results. The study could provide experimental and theoretical benchmarks to characterize cocrystal formed between active ingredients and cocrystal formers and also the intermolecular hydrogen-bond effect within cocrystal formation process by vibrational spectroscopic techniques. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Improved Model for Predicting the Free Energy Contribution of Dinucleotide Bulges to RNA Duplex Stability.

    PubMed

    Tomcho, Jeremy C; Tillman, Magdalena R; Znosko, Brent M

    2015-09-01

    Predicting the secondary structure of RNA is an intermediate in predicting RNA three-dimensional structure. Commonly, determining RNA secondary structure from sequence uses free energy minimization and nearest neighbor parameters. Current algorithms utilize a sequence-independent model to predict free energy contributions of dinucleotide bulges. To determine if a sequence-dependent model would be more accurate, short RNA duplexes containing dinucleotide bulges with different sequences and nearest neighbor combinations were optically melted to derive thermodynamic parameters. These data suggested energy contributions of dinucleotide bulges were sequence-dependent, and a sequence-dependent model was derived. This model assigns free energy penalties based on the identity of nucleotides in the bulge (3.06 kcal/mol for two purines, 2.93 kcal/mol for two pyrimidines, 2.71 kcal/mol for 5'-purine-pyrimidine-3', and 2.41 kcal/mol for 5'-pyrimidine-purine-3'). The predictive model also includes a 0.45 kcal/mol penalty for an A-U pair adjacent to the bulge and a -0.28 kcal/mol bonus for a G-U pair adjacent to the bulge. The new sequence-dependent model results in predicted values within, on average, 0.17 kcal/mol of experimental values, a significant improvement over the sequence-independent model. This model and new experimental values can be incorporated into algorithms that predict RNA stability and secondary structure from sequence.

  3. Development of an Amperometric Biosensor Platform for the Combined Determination of L-Malic, Fumaric, and L-Aspartic Acid.

    PubMed

    Röhlen, Désirée L; Pilas, Johanna; Schöning, Michael J; Selmer, Thorsten

    2017-10-01

    Three amperometric biosensors have been developed for the detection of L-malic acid, fumaric acid, and L -aspartic acid, all based on the combination of a malate-specific dehydrogenase (MDH, EC 1.1.1.37) and diaphorase (DIA, EC 1.8.1.4). The stepwise expansion of the malate platform with the enzymes fumarate hydratase (FH, EC 4.2.1.2) and aspartate ammonia-lyase (ASPA, EC 4.3.1.1) resulted in multi-enzyme reaction cascades and, thus, augmentation of the substrate spectrum of the sensors. Electrochemical measurements were carried out in presence of the cofactor β-nicotinamide adenine dinucleotide (NAD + ) and the redox mediator hexacyanoferrate (III) (HCFIII). The amperometric detection is mediated by oxidation of hexacyanoferrate (II) (HCFII) at an applied potential of + 0.3 V vs. Ag/AgCl. For each biosensor, optimum working conditions were defined by adjustment of cofactor concentrations, buffer pH, and immobilization procedure. Under these improved conditions, amperometric responses were linear up to 3.0 mM for L-malate and fumarate, respectively, with a corresponding sensitivity of 0.7 μA mM -1 (L-malate biosensor) and 0.4 μA mM -1 (fumarate biosensor). The L-aspartate detection system displayed a linear range of 1.0-10.0 mM with a sensitivity of 0.09 μA mM -1 . The sensor characteristics suggest that the developed platform provides a promising method for the detection and differentiation of the three substrates.

  4. Resonance Raman spectroscopic investigation of the light-harvesting chromophore in escherichia coli photolyase and Vibrio cholerae cryptochrome-1.

    PubMed

    Sokolova, Olga; Cecala, Christine; Gopal, Anand; Cortazar, Frank; McDowell-Buchanan, Carla; Sancar, Aziz; Gindt, Yvonne M; Schelvis, Johannes P M

    2007-03-27

    Photolyases and cryptochromes are flavoproteins that belong to the class of blue-light photoreceptors. They usually bind two chromophores: flavin adenine dinucleotide (FAD), which forms the active site, and a light-harvesting pigment, which is a 5,10-methenyltetrahydrofolate polyglutamate (MTHF) in most cases. In Escherichia coli photolyase (EcPhr), the MTHF cofactor is present in substoichiometric amounts after purification, while in Vibrio cholerae cryptochrome-1 (VcCry1) the MTHF cofactor is bound more strongly and is present at stoichiometric levels after purification. In this paper, we have used resonance Raman spectroscopy to monitor the effect of loss of MTHF on the protein-FAD interactions in EcPhr and to probe the protein-MTHF interactions in both EcPhr and VcCry1. We find that removal of MTHF does not perturb protein-FAD interactions, suggesting that it may not affect the physicochemical properties of FAD in EcPhr. Our data demonstrate that the pteridine ring of MTHF in EcPhr has different interactions with the protein matrix than that of MTHF in VcCry1. Comparison to solution resonance Raman spectra of MTHF suggests that the carbonyl of its pteridine ring in EcPhr experiences stronger hydrogen bonding and a more polar environment than in VcCry1, but that hydrogen bonding to the pteridine ring amine hydrogens is stronger in VcCry-1. These differences in hydrogen bonding may account for the higher binding affinity of MTHF in VcCry1 compared to EcPhr.

  5. Organic cofactors participated more frequently than transition metals in redox reactions of primitive proteins.

    PubMed

    Ji, Hong-Fang; Chen, Lei; Zhang, Hong-Yu

    2008-08-01

    Protein redox reactions are one of the most basic and important biochemical actions. As amino acids are weak redox mediators, most protein redox functions are undertaken by protein cofactors, which include organic ligands and transition metal ions. Since both kinds of redox cofactors were available in the pre-protein RNA world, it is challenging to explore which one was more involved in redox processes of primitive proteins? In this paper, using an examination of the redox cofactor usage of putative ancient proteins, we infer that organic ligands participated more frequently than transition metals in redox reactions of primitive proteins, at least as protein cofactors. This is further supported by the relative abundance of amino acids in the primordial world. Supplementary material for this article can be found on the BioEssays website. (c) 2008 Wiley Periodicals, Inc.

  6. Some reactions of the hydroxyl adduct of adenine

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Vanhemmen, J.J.

    1975-01-01

    The chemical reactions of purine derivatives resulting from pulse radiolysis were studied. Some reactions of the hydroxyl adduct of adenine are described and one of these reactions was compared with similar reactions of hydroxyl adducts of other purine derivatives. Evidence is given that in various purines opening of the imidazole ring is due to unimolecular rearrangements of the hydroxyl adducts. (GRA)

  7. Insight into Coenzyme A cofactor binding and the mechanism of acyl-transfer in an acylating aldehyde dehydrogenase from Clostridium phytofermentans

    PubMed Central

    Tuck, Laura R.; Altenbach, Kirsten; Ang, Thiau Fu; Crawshaw, Adam D.; Campopiano, Dominic J.; Clarke, David J.; Marles-Wright, Jon

    2016-01-01

    The breakdown of fucose and rhamnose released from plant cell walls by the cellulolytic soil bacterium Clostridium phytofermentans produces toxic aldehyde intermediates. To enable growth on these carbon sources, the pathway for the breakdown of fucose and rhamnose is encapsulated within a bacterial microcompartment (BMC). These proteinaceous organelles sequester the toxic aldehyde intermediates and allow the efficient action of acylating aldehyde dehydrogenase enzymes to produce an acyl-CoA that is ultimately used in substrate-level phosphorylation to produce ATP. Here we analyse the kinetics of the aldehyde dehydrogenase enzyme from the fucose/rhamnose utilisation BMC with different short-chain fatty aldehydes and show that it has activity against substrates with up to six carbon atoms, with optimal activity against propionaldehyde. We have also determined the X-ray crystal structure of this enzyme in complex with CoA and show that the adenine nucleotide of this cofactor is bound in a distinct pocket to the same group in NAD+. This work is the first report of the structure of CoA bound to an aldehyde dehydrogenase enzyme and our crystallographic model provides important insight into the differences within the active site that distinguish the acylating from non-acylating aldehyde dehydrogenase enzymes. PMID:26899032

  8. Insight into Coenzyme A cofactor binding and the mechanism of acyl-transfer in an acylating aldehyde dehydrogenase from Clostridium phytofermentans.

    PubMed

    Tuck, Laura R; Altenbach, Kirsten; Ang, Thiau Fu; Crawshaw, Adam D; Campopiano, Dominic J; Clarke, David J; Marles-Wright, Jon

    2016-02-22

    The breakdown of fucose and rhamnose released from plant cell walls by the cellulolytic soil bacterium Clostridium phytofermentans produces toxic aldehyde intermediates. To enable growth on these carbon sources, the pathway for the breakdown of fucose and rhamnose is encapsulated within a bacterial microcompartment (BMC). These proteinaceous organelles sequester the toxic aldehyde intermediates and allow the efficient action of acylating aldehyde dehydrogenase enzymes to produce an acyl-CoA that is ultimately used in substrate-level phosphorylation to produce ATP. Here we analyse the kinetics of the aldehyde dehydrogenase enzyme from the fucose/rhamnose utilisation BMC with different short-chain fatty aldehydes and show that it has activity against substrates with up to six carbon atoms, with optimal activity against propionaldehyde. We have also determined the X-ray crystal structure of this enzyme in complex with CoA and show that the adenine nucleotide of this cofactor is bound in a distinct pocket to the same group in NAD(+). This work is the first report of the structure of CoA bound to an aldehyde dehydrogenase enzyme and our crystallographic model provides important insight into the differences within the active site that distinguish the acylating from non-acylating aldehyde dehydrogenase enzymes.

  9. TusA (YhhP) and IscS are required for molybdenum cofactor-dependent base-analog detoxification

    PubMed Central

    Kozmin, Stanislav G; Stepchenkova, Elena I; Schaaper, Roel M

    2013-01-01

    Lack of molybdenum cofactor (Moco) in Escherichia coli leads to hypersensitivity to the mutagenic and toxic effects of N-hydroxylated base analogs, such as 6-N-hydroxylaminopurine (HAP). This phenotype is due to the loss of two Moco-dependent activities, YcbX and YiiM, that are capable of reducing HAP to adenine. Here, we describe two novel HAP-sensitive mutants containing a defect in iscS or tusA (yhhP) gene. IscS is a major L-cysteine desulfurase involved in iron–sulfur cluster synthesis, thiamine synthesis, and tRNA thiomodification. TusA is a small sulfur-carrier protein that interacts with IscS. We show that both IscS and TusA operate within the Moco-dependent pathway. Like other Moco-deficient strains, tusA and iscS mutants are HAP sensitive and resistant to chlorate under anaerobic conditions. The base-analog sensitivity of iscS or tusA strains could be suppressed by supplying exogenous L-cysteine or sulfide or by an increase in endogenous sulfur donors (cysB constitutive mutant). The data suggest that iscS and tusA mutants have a defect in the mobilization of sulfur required for active YcbX/YiiM proteins as well as nitrate reductase, presumably due to lack of functional Moco. Overall, our data imply a novel and indispensable role of the IscS/TusA complex in the activity of several molybdoenzymes. PMID:23894086

  10. Nicotinamide Cofactors Suppress Active-Site Labeling of Aldehyde Dehydrogenases.

    PubMed

    Stiti, Naim; Chandrasekar, Balakumaran; Strubl, Laura; Mohammed, Shabaz; Bartels, Dorothea; van der Hoorn, Renier A L

    2016-06-17

    Active site labeling by (re)activity-based probes is a powerful chemical proteomic tool to globally map active sites in native proteomes without using substrates. Active site labeling is usually taken as a readout for the active state of the enzyme because labeling reflects the availability and reactivity of active sites, which are hallmarks for enzyme activities. Here, we show that this relationship holds tightly, but we also reveal an important exception to this rule. Labeling of Arabidopsis ALDH3H1 with a chloroacetamide probe occurs at the catalytic Cys, and labeling is suppressed upon nitrosylation and oxidation, and upon treatment with other Cys modifiers. These experiments display a consistent and strong correlation between active site labeling and enzymatic activity. Surprisingly, however, labeling is suppressed by the cofactor NAD(+), and this property is shared with other members of the ALDH superfamily and also detected for unrelated GAPDH enzymes with an unrelated hydantoin-based probe in crude extracts of plant cell cultures. Suppression requires cofactor binding to its binding pocket. Labeling is also suppressed by ALDH modulators that bind at the substrate entrance tunnel, confirming that labeling occurs through the substrate-binding cavity. Our data indicate that cofactor binding adjusts the catalytic Cys into a conformation that reduces the reactivity toward chloroacetamide probes.

  11. Curcumin and resveratrol rescue cortical-hippocampal system from chronic fluoride-induced neurodegeneration and enhance memory retrieval.

    PubMed

    Sharma, Chhavi; Suhalka, Pooja; Bhatnagar, Maheep

    2018-04-13

    The aim of this study was: (1) to evaluate the neuroprotective effect of resveratrol and curcumin on nicotinamide adenine dinucleotide phosphate diaphorase activity in neuronal cell in subregions of mice brain, (2) to evaluate the effects on antioxidant status and (3) to evaluate the protective effects of phytochemicals on learning and memory following fluoride exposure. Young mice (one month old, body weight (BW) 30 ± 5 mg) were provided with 120 ppm sodium fluoride dissolved in drinking water. They were given curcumin (30 mg/kg BW) or resveratrol (30 mg/kg BW) orally once in a day up to 30 days. Effects of resveratrol and curcumin on spatial learning and memory were studied using Morris water maze and classic maze test. Effects on brain antioxidants' (lactose dehydrogenase (LDH), malondialdehyde and reactive oxygen species) status were also studied in vitro. Histochemistry was done to assess the effect of treatments on nitric oxide neurotransmitter. Our study showed that in fluoride-treated animals, the number of nicotinamide adenine dinucleotide phosphate diaphorase positive neurons, intracellular Ca 2+ , reactive oxygen species level, LDH and malondialdehyde concentration increased significantly. Interestingly, after treatment with curcumin or resveratrol, a significant decrease in the number of nicotinamide adenine dinucleotide phosphate diaphorase positive neurons and antioxidant status was observed. This decrease was more considerable in resveratrol-treated group. Our study indicates that both antioxidants, curcumin and resveratrol, are useful in reducing neurodegeneration in selective areas of cornus ammonis 1 (CA1), CA3, dentate gyrus (DG) and the cortex of mice brain and in recuperating the loss of memory and learning caused due to fluoride exposure.

  12. Protective effect of dietary potassium against vascular injury in salt-sensitive hypertension.

    PubMed

    Kido, Makiko; Ando, Katsuyuki; Onozato, Maristela L; Tojo, Akihiro; Yoshikawa, Masahiro; Ogita, Teruhiko; Fujita, Toshiro

    2008-02-01

    Hypertensive cardiovascular damage is accelerated by salt loading but counteracted by dietary potassium supplementation. We suggested recently that antioxidant actions of potassium contribute to protection against salt-induced cardiac dysfunction. Therefore, we examined whether potassium supplementation ameliorated cuff-induced vascular injury in salt-sensitive hypertension via suppression of oxidative stress. Four-week-old Dahl salt-sensitive rats were fed a normal-salt (0.3% NaCl), high-salt (8% NaCl), or high-salt plus high-potassium (8% KCl) diet for 5 weeks, and some of the rats fed a high-salt diet were also given antioxidants. One week after the start of the treatments, a silicone cuff was implanted around the femoral artery. Examination revealed increased cuff-induced neointimal proliferation with adventitial macrophage infiltration in arteries from salt-loaded Dahl salt-sensitive rats compared with that in arteries from non-salt-loaded animals (intima/media ratio: 0.471+/-0.070 versus 0.302+/-0.037; P<0.05), associated with regional superoxide overproduction and reduced nicotinamide-adenine dinucleotide phosphate oxidase activation and mRNA overexpression. On the other hand, simultaneous potassium supplementation attenuated salt-induced neointimal hyperplasia (intima/media ratio: 0.205+/-0.012; P<0.001), adventitial macrophage infiltration, superoxide overproduction, and reduced nicotinamide-adenine dinucleotide phosphate oxidase activation and overexpression. Antioxidants, which decrease vascular oxidative stress, also reduced neointima formation induced by salt excess. In conclusion, high-potassium diets seems to have a protective effect against the development of vascular damage induced by salt loading mediated, at least in part, through suppression of the production of reactive oxygen species probably generated by reduced nicotinamide-adenine dinucleotide phosphate oxidase.

  13. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Synthesis, Delivery and Regulation of Eukaryotic Heme and Fe-S Cluster Cofactors

    PubMed Central

    Barupala, Dulmini P.; Dzul, Stephen P.; Riggs-Gelasco, Pamela Jo; Stemmler, Timothy L.

    2016-01-01

    In humans, the bulk of iron in the body (over 75%) is directed towards heme- or Fe-S cluster cofactor synthesis, and the complex, highly regulated pathways in place to accomplish biosynthesis have evolved to safely assemble and load these cofactors into apoprotein partners. In eukaryotes, heme biosynthesis is both initiated and finalized within the mitochondria, while cellular Fe-S cluster assembly is controlled by correlated pathways both within the mitochondria and within the cytosol. Iron plays a vital role in a wide array of metabolic processes and defects in iron cofactor assembly leads to human diseases. This review describes progress towards our molecular-level understanding of cellular heme and Fe-S cluster biosynthesis, focusing on the regulation and mechanistic details that are essential for understanding human disorders related to the breakdown in these essential pathways. PMID:26785297

  15. 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.

  16. Redox-dependent substrate-cofactor interactions in the Michaelis-complex of a flavin-dependent oxidoreductase

    NASA Astrophysics Data System (ADS)

    Werther, Tobias; Wahlefeld, Stefan; Salewski, Johannes; Kuhlmann, Uwe; Zebger, Ingo; Hildebrandt, Peter; Dobbek, Holger

    2017-07-01

    How an enzyme activates its substrate for turnover is fundamental for catalysis but incompletely understood on a structural level. With redox enzymes one typically analyses structures of enzyme-substrate complexes in the unreactive oxidation state of the cofactor, assuming that the interaction between enzyme and substrate is independent of the cofactors oxidation state. Here, we investigate the Michaelis complex of the flavoenzyme xenobiotic reductase A with the reactive reduced cofactor bound to its substrates by X-ray crystallography and resonance Raman spectroscopy and compare it to the non-reactive oxidized Michaelis complex mimics. We find that substrates bind in different orientations to the oxidized and reduced flavin, in both cases flattening its structure. But only authentic Michaelis complexes display an unexpected rich vibrational band pattern uncovering a strong donor-acceptor complex between reduced flavin and substrate. This interaction likely activates the catalytic ground state of the reduced flavin, accelerating the reaction within a compressed cofactor-substrate complex.

  17. Cytosolic iron chaperones: Proteins delivering iron cofactors in the cytosol of mammalian cells.

    PubMed

    Philpott, Caroline C; Ryu, Moon-Suhn; Frey, Avery; Patel, Sarju

    2017-08-04

    Eukaryotic cells contain hundreds of metalloproteins that are supported by intracellular systems coordinating the uptake and distribution of metal cofactors. Iron cofactors include heme, iron-sulfur clusters, and simple iron ions. Poly(rC)-binding proteins are multifunctional adaptors that serve as iron ion chaperones in the cytosolic/nuclear compartment, binding iron at import and delivering it to enzymes, for storage (ferritin) and export (ferroportin). Ferritin iron is mobilized by autophagy through the cargo receptor, nuclear co-activator 4. The monothiol glutaredoxin Glrx3 and BolA2 function as a [2Fe-2S] chaperone complex. These proteins form a core system of cytosolic iron cofactor chaperones in mammalian cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. What can molecular modelling bring to the design of artificial inorganic cofactors?

    PubMed

    Muñoz Robles, Victor; Ortega-Carrasco, Elisabeth; González Fuentes, Eric; Lledós, Agustí; Maréchal, Jean-Didier

    2011-01-01

    In recent years, the development of synthetic metalloenzymes based on the insertion of inorganic catalysts into biological macromolecules has become a vivid field of investigation. The success of the design of these composites is highly dependent on an atomic understanding of the recognition process between inorganic and biological entities. Despite facing several challenging complexities, molecular modelling techniques could be particularly useful in providing such knowledge. This study aims to discuss how the prediction of the structural and energetic properties of the host-cofactor interactions can be performed by computational means. To do so, we designed a protocol that combines several methodologies like protein-ligand dockings and QM/MM techniques. The overall approach considers fundamental bioinorganic questions like the participation of the amino acids of the receptor to the first coordination sphere of the metal, the impact of the receptor/cofactor flexibility on the structure of the complex, the cost of inserting the inorganic catalyst in place of the natural ligand/substrate into the host and how experimental knowledge can improve or invalidate a theoretical model. As a real case system, we studied an artificial metalloenzyme obtained by the insertion of a Fe(Schiff base) moiety into the heme oxygenase of Corynebacterium diphtheriae. The experimental structure of this species shows a distorted cofactor leading to an unusual octahedral configuration of the iron with two proximal residues chelating the metal and no external ligand. This geometry is far from the conformation adopted by similar cofactors in other hosts and shows that a fine tuning exists between the coordination environment of the metal, the deformability of its organic ligand and the conformational adaptability of the receptor. In a field where very little structural information is yet available, this work should help in building an initial molecular modelling framework for the discovery

  19. The spectrum of genomic signatures: from dinucleotides to chaos game representation.

    PubMed

    Wang, Yingwei; Hill, Kathleen; Singh, Shiva; Kari, Lila

    2005-02-14

    In the post genomic era, access to complete genome sequence data for numerous diverse species has opened multiple avenues for examining and comparing primary DNA sequence organization of entire genomes. Previously, the concept of a genomic signature was introduced with the observation of species-type specific Dinucleotide Relative Abundance Profiles (DRAPs); dinucleotides were identified as the subsequences with the greatest bias in representation in a majority of genomes. Herein, we demonstrate that DRAP is one particular genomic signature contained within a broader spectrum of signatures. Within this spectrum, an alternative genomic signature, Chaos Game Representation (CGR), provides a unique visualization of patterns in sequence organization. A genomic signature is associated with a particular integer order or subsequence length that represents a measure of the resolution or granularity in the analysis of primary DNA sequence organization. We quantitatively explore the organizational information provided by genomic signatures of different orders through different distance measures, including a novel Image Distance. The Image Distance and other existing distance measures are evaluated by comparing the phylogenetic trees they generate for 26 complete mitochondrial genomes from a diversity of species. The phylogenetic tree generated by the Image Distance is compatible with the known relatedness of species. Quantitative evaluation of the spectrum of genomic signatures may be used to ultimately gain insight into the determinants and biological relevance of the genome signatures.

  20. An experimental and theoretical vibrational study of interaction of adenine and thymine with artificial seawaters: A prebiotic chemistry experiment.

    PubMed

    Anizelli, Pedro R; Baú, João P T; Nabeshima, Henrique S; da Costa, Marcello F; de Santana, Henrique; Zaia, Dimas A M

    2014-05-21

    Nucleic acid bases play important roles in living beings. Thus, their interaction with salts the prebiotic Earth could be an important issue for the understanding of origin of life. In this study, the effect of pH and artificial seawaters on the structure of adenine and thymine was studied via parallel determinations using FT-IR, Raman spectroscopy and theoretical calculations. Thymine and adenine lyophilized in solutions at basic and acidic conditions showed characteristic bands of the enol-imino tautomer due to the deprotonation and the hydrochloride form due to protonation, respectively. The interaction of thymine and adenine with different seawaters representative of different geological periods on Earth was also studied. In the case of thymine a strong interaction with Sr(2+) promoted changes in the Raman and infrared spectra. For adenine changes in infrared and Raman spectra were observed in the presence of salts from all seawaters tested. The experimental results were compared to theoretical calculations, which showed structural changes due to the presence of ions Na(+), Mg(2+), Ca(2+) and Sr(2+) of artificial seawaters. For thymine the bands arising from C4=C5 and C6=O stretching were shifted to lower values, and for adenine, a new band at 1310cm(-1) was observed. The reactivity of adenine and thymine was studied by comparing changes in nucleophilicity and energy of the HOMO orbital. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Single-molecule analysis of steroid receptor and cofactor action in living cells

    PubMed Central

    Paakinaho, Ville; Presman, Diego M.; Ball, David A.; Johnson, Thomas A.; Schiltz, R. Louis; Levitt, Peter; Mazza, Davide; Morisaki, Tatsuya; Karpova, Tatiana S.; Hager, Gordon L.

    2017-01-01

    Population-based assays have been employed extensively to investigate the interactions of transcription factors (TFs) with chromatin and are often interpreted in terms of static and sequential binding. However, fluorescence microscopy techniques reveal a more dynamic binding behaviour of TFs in live cells. Here we analyse the strengths and limitations of in vivo single-molecule tracking and performed a comprehensive analysis on the intranuclear dwell times of four steroid receptors and a number of known cofactors. While the absolute residence times estimates can depend on imaging acquisition parameters due to sampling bias, our results indicate that only a small proportion of factors are specifically bound to chromatin at any given time. Interestingly, the glucocorticoid receptor and its cofactors affect each other’s dwell times in an asymmetric manner. Overall, our data indicate transient rather than stable TF-cofactors chromatin interactions at response elements at the single-molecule level. PMID:28635963

  2. Synthesis, delivery and regulation of eukaryotic heme and Fe-S cluster cofactors.

    PubMed

    Barupala, Dulmini P; Dzul, Stephen P; Riggs-Gelasco, Pamela Jo; Stemmler, Timothy L

    2016-02-15

    In humans, the bulk of iron in the body (over 75%) is directed towards heme- or Fe-S cluster cofactor synthesis, and the complex, highly regulated pathways in place to accomplish biosynthesis have evolved to safely assemble and load these cofactors into apoprotein partners. In eukaryotes, heme biosynthesis is both initiated and finalized within the mitochondria, while cellular Fe-S cluster assembly is controlled by correlated pathways both within the mitochondria and within the cytosol. Iron plays a vital role in a wide array of metabolic processes and defects in iron cofactor assembly leads to human diseases. This review describes progress towards our molecular-level understanding of cellular heme and Fe-S cluster biosynthesis, focusing on the regulation and mechanistic details that are essential for understanding human disorders related to the breakdown in these essential pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Hydrothermal stability of adenine under controlled fugacities of N2, CO2 and H2.

    PubMed

    Franiatte, Michael; Richard, Laurent; Elie, Marcel; Nguyen-Trung, Chinh; Perfetti, Erwan; LaRowe, Douglas E

    2008-04-01

    An experimental study has been carried out on the stability of adenine (one of the five nucleic acid bases) under hydrothermal conditions. The experiments were performed in sealed autoclaves at 300 degrees C under fugacities of CO(2), N(2) and H(2) supposedly representative of those in marine hydrothermal systems on the early Earth. The composition of the gas phase was obtained from the degradation of oxalic acid, sodium nitrite and ammonium chloride, and the oxidation of metallic iron. The results of the experiments indicate that after 200 h, adenine is still present in detectable concentration in the aqueous phase. In fact, the concentration of adenine does not seem to be decreasing after approximately 24 h, which suggests that an equilibrium state may have been established with the inorganic constituents of the hydrothermal fluid. Such a conclusion is corroborated by independent thermodynamic calculations.

  4. Randomised double-blind trial of acyclovir (Zovirax) and adenine arabinoside in herpes simplex amoeboid corneal ulceration.

    PubMed

    Collum, L M; Logan, P; McAuliffe-Curtin, D; Hung, S O; Patterson, A; Rees, P J

    1985-11-01

    Fifty-one patients were treated in a dual-centre, double-blind comparison of acyclovir and adenine arabinoside in herpetic amoeboid (geographic) corneal ulceration. Twenty-four of the 25 patients receiving acyclovir healed in a mean time of 12.2 days, while 24 of the 26 patients treated with adenine arabinoside healed in a mean time of 11.0 days. There was no statistically significant difference between the two groups in terms of healing. A second analysis, excluding any patients who had received antiviral treatment immediately prior to entry into the study, showed that 18 of the 19 who received acyclovir healed in an average of 11.7 days and 18 of the 19 recipients of adenine arabinoside healed in a mean time of 11.2 days. Again the difference was not statistically significant.

  5. Cofactor Editing by the G-protein Metallochaperone Domain Regulates the Radical B12 Enzyme IcmF.

    PubMed

    Li, Zhu; Kitanishi, Kenichi; Twahir, Umar T; Cracan, Valentin; Chapman, Derrell; Warncke, Kurt; Banerjee, Ruma

    2017-03-10

    IcmF is a 5'-deoxyadenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the carbon skeleton rearrangement of isobutyryl-CoA to butyryl-CoA. It is a bifunctional protein resulting from the fusion of a G-protein chaperone with GTPase activity and the cofactor- and substrate-binding mutase domains with isomerase activity. IcmF is prone to inactivation during catalytic turnover, thus setting up its dependence on a cofactor repair system. Herein, we demonstrate that the GTPase activity of IcmF powers the ejection of the inactive cob(II)alamin cofactor and requires the presence of an acceptor protein, adenosyltransferase, for receiving it. Adenosyltransferase in turn converts cob(II)alamin to AdoCbl in the presence of ATP and a reductant. The repaired cofactor is then reloaded onto IcmF in a GTPase-gated step. The mechanistic details of cofactor loading and offloading from the AdoCbl-dependent IcmF are distinct from those of the better characterized and homologous methylmalonyl-CoA mutase/G-protein chaperone system. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Deducing the temporal order of cofactor function in ligand-regulated gene transcription: theory and experimental verification.

    PubMed

    Dougherty, Edward J; Guo, Chunhua; Simons, S Stoney; Chow, Carson C

    2012-01-01

    Cofactors are intimately involved in steroid-regulated gene expression. Two critical questions are (1) the steps at which cofactors exert their biological activities and (2) the nature of that activity. Here we show that a new mathematical theory of steroid hormone action can be used to deduce the kinetic properties and reaction sequence position for the functioning of any two cofactors relative to a concentration limiting step (CLS) and to each other. The predictions of the theory, which can be applied using graphical methods similar to those of enzyme kinetics, are validated by obtaining internally consistent data for pair-wise analyses of three cofactors (TIF2, sSMRT, and NCoR) in U2OS cells. The analysis of TIF2 and sSMRT actions on GR-induction of an endogenous gene gave results identical to those with an exogenous reporter. Thus new tools to determine previously unobtainable information about the nature and position of cofactor action in any process displaying first-order Hill plot kinetics are now available.

  7. Deducing the Temporal Order of Cofactor Function in Ligand-Regulated Gene Transcription: Theory and Experimental Verification

    PubMed Central

    Dougherty, Edward J.; Guo, Chunhua; Simons, S. Stoney; Chow, Carson C.

    2012-01-01

    Cofactors are intimately involved in steroid-regulated gene expression. Two critical questions are (1) the steps at which cofactors exert their biological activities and (2) the nature of that activity. Here we show that a new mathematical theory of steroid hormone action can be used to deduce the kinetic properties and reaction sequence position for the functioning of any two cofactors relative to a concentration limiting step (CLS) and to each other. The predictions of the theory, which can be applied using graphical methods similar to those of enzyme kinetics, are validated by obtaining internally consistent data for pair-wise analyses of three cofactors (TIF2, sSMRT, and NCoR) in U2OS cells. The analysis of TIF2 and sSMRT actions on GR-induction of an endogenous gene gave results identical to those with an exogenous reporter. Thus new tools to determine previously unobtainable information about the nature and position of cofactor action in any process displaying first-order Hill plot kinetics are now available. PMID:22272313

  8. Pulmonary preservation studies: effects on endothelial function and pulmonary adenine nucleotides.

    PubMed

    Paik, Hyo Chae; Hoffmann, Steven C; Egan, Thomas M

    2003-02-27

    Lung transplantation is an effective therapy plagued by a high incidence of early graft dysfunction, in part because of reperfusion injury. The optimal preservation solution for lung transplantation is unknown. We performed experiments using an isolated perfused rat lung model to test the effect of lung preservation with three solutions commonly used in clinical practice. Lungs were retrieved from Sprague-Dawley rats and flushed with one of three solutions: modified Euro-Collins (MEC), University of Wisconsin (UW), or low potassium dextran and glucose (LPDG), then stored cold for varying periods before reperfusion with Earle's balanced salt solution using the isolated perfused rat lung model. Outcome measures were capillary filtration coefficient (Kfc), wet-to-dry weight ratio, and lung tissue levels of adenine nucleotides and cyclic AMP. All lungs functioned well after 4 hr of storage. By 6 hr, UW-flushed lungs had a lower Kfc than LPDG-flushed lungs. After 8 hr of storage, only UW-flushed lungs had a measurable Kfc. Adenine nucleotide levels were higher in UW-flushed lungs after prolonged storage. Cyclic AMP levels correlated with Kfc in all groups. Early changes in endothelial permeability seemed to be better attenuated in lungs flushed with UW compared with LPDG or MEC; this was associated with higher amounts of adenine nucleotides. MEC-flushed lungs failed earlier than LPDG-flushed or UW-flushed lungs. The content of the solution may be more important for lung preservation than whether the ionic composition is intracellular or extracellular.

  9. A water-forming NADH oxidase from Lactobacillus pentosus suitable for the regeneration of synthetic biomimetic cofactors

    PubMed Central

    Nowak, Claudia; Beer, Barbara; Pick, André; Roth, Teresa; Lommes, Petra; Sieber, Volker

    2015-01-01

    The cell-free biocatalytic production of fine chemicals by oxidoreductases has continuously grown over the past years. Since especially dehydrogenases depend on the stoichiometric use of nicotinamide pyridine cofactors, an integrated efficient recycling system is crucial to allow process operation under economic conditions. Lately, the variety of cofactors for biocatalysis was broadened by the utilization of totally synthetic and cheap biomimetics. Though, to date the regeneration has been limited to chemical or electrochemical methods. Here, we report an enzymatic recycling by the flavoprotein NADH-oxidase from Lactobacillus pentosus (LpNox). Since this enzyme has not been described before, we first characterized it in regard to its optimal reaction parameters. We found that the heterologously overexpressed enzyme only contained 13% FAD. In vitro loading of the enzyme with FAD, resulted in a higher specific activity towards its natural cofactor NADH as well as different nicotinamide derived biomimetics. Apart from the enzymatic recycling, which gives water as a by-product by transferring four electrons onto oxygen, unbound FAD can also catalyze the oxidation of biomimetic cofactors. Here a two electron process takes place yielding H2O2 instead. The enzymatic and chemical recycling was compared in regard to reaction kinetics for the natural and biomimetic cofactors. With LpNox and FAD, two recycling strategies for biomimetic cofactors are described with either water or hydrogen peroxide as by-product. PMID:26441891

  10. The Local Dinucleotide Preference of APOBEC3G Can Be Altered from 5′-CC to 5′-TC by a Single Amino Acid Substitution

    PubMed Central

    Rathore, Anurag; Carpenter, Michael A; Demir, Özlem; Ikeda, Terumasa; Li, Ming; Shaban, Nadine; Law, Emily K.; Anokhin, Dmitry; Brown, William L.; Amaro, Rommie E.; Harris, Reuben S.

    2013-01-01

    APOBEC3A and APOBEC3G are DNA cytosine deaminases with biological functions in foreign DNA and retrovirus restriction, respectively. APOBEC3A has an intrinsic preference for cytosine preceded by thymine (5′-TC) in single-stranded DNA substrates, whereas APOBEC3G prefers the target cytosine to be preceded by another cytosine (5′-CC). To determine the amino acids responsible for these strong dinucleotide preferences, we analyzed a series of chimeras in which putative DNA binding loop regions of APOBEC3G were replaced with the corresponding regions from APOBEC3A. Loop 3 replacement enhanced APOBEC3G catalytic activity but did not alter its intrinsic 5′-CC dinucleotide substrate preference. Loop 7 replacement caused APOBEC3G to become APOBEC3A-like and strongly prefer 5′-TC substrates. Simultaneous loop 3/7 replacement resulted in a hyperactive APOBEC3G variant that also preferred 5′-TC dinucleotides. Single amino acid exchanges revealed D317 as a critical determinant of dinucleotide substrate specificity. Multi-copy explicitly solvated all-atom molecular dynamics simulations suggested a model in which D317 acts as a helix-capping residue by constraining the mobility of loop 7, forming a novel binding pocket that favorably accommodates cytosine. All catalytically active APOBEC3G variants, regardless of dinucleotide preference, retained HIV-1 restriction activity. These data support a model in which the loop 7 region governs the selection of local dinucleotide substrates for deamination but is unlikely to be part of the higher level targeting mechanisms that direct these enzymes to biological substrates such as HIV-1 cDNA. PMID:23938202

  11. Human Tamm-Horsfall protein, a renal specific protein, serves as a cofactor in complement 3b degradation

    PubMed Central

    2017-01-01

    Tamm-Horsfall protein (THP) is an abundant urinary protein of renal origin. We hypothesize that THP can act as an inhibitor of complement since THP binds complement 1q (C1q) of the classical complement pathway, inhibits activation of this pathway, and is important in decreasing renal ischemia-reperfusion injury (a complement-mediated condition). In this study, we began to investigate whether THP interacted with the alternate complement pathway via complement factor H (CFH). THP was shown to bind CFH using ligand blots and in an ELISA (KD of 1 × 10−6 M). Next, the ability of THP to alter CFH’s normal action as it functioned as a cofactor in complement factor I (CFI)–mediated complement 3b (C3b) degradation was investigated. Unexpectedly, control experiments in these in vitro assays suggested that THP, without added CFH, could act as a cofactor in CFI-mediated C3b degradation. This cofactor activity was present equally in THP isolated from 10 different individuals. While an ELISA demonstrated small amounts of CFH contaminating THP samples, these CFH amounts were insufficient to explain the degree of cofactor activity present in THP. An ELISA demonstrated that THP directly bound C3b (KD ~ 5 × 10−8 m), a prerequisite for a protein acting as a C3b degradation cofactor. The cofactor activity of THP likely resides in the protein portion of THP since partially deglycosylated THP still retained cofactor activity. In conclusion, THP appears to participate directly in complement inactivation by its ability to act as a cofactor for C3b degradation, thus adding support to the hypothesis that THP might act as an endogenous urinary tract inhibitor of complement. PMID:28742158

  12. Absorption by DNA single strands of adenine isolated in vacuo: The role of multiple chromophores

    NASA Astrophysics Data System (ADS)

    Nielsen, Lisbeth Munksgaard; Pedersen, Sara Øvad; Kirketerp, Maj-Britt Suhr; Nielsen, Steen Brøndsted

    2012-02-01

    The degree of electronic coupling between DNA bases is a topic being up for much debate. Here we report on the intrinsic electronic properties of isolated DNA strands in vacuo free of solvent, which is a good starting point for high-level excited states calculations. Action spectra of DNA single strands of adenine reveal sign of exciton coupling between stacked bases from blueshifted absorption bands (˜3 nm) relative to that of the dAMP mononucleotide (one adenine base). The bands are blueshifted by about 10 nm compared to those of solvated strands, which is a shift similar to that for the adenine molecule and the dAMP mononucleotide. Desolvation has little effect on the bandwidth, which implies that inhomogenous broadening of the absorption bands in aqueous solution is of minor importance compared to, e.g., conformational disorder. Finally, at high photon energies, internal conversion competes with electron detachment since dissociation of the bare photoexcited ions on the microsecond time scale is measured.

  13. Enzymatic Basis for Differentiation of Rhizobium into Fast- and Slow-Growing Groups

    PubMed Central

    Drets, G. Martinez-De; Arias, A.

    1972-01-01

    Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and other enzymes related to carbohydrate metabolism were studied in rhizobia. A nicotinamide adenine dinucleotide phosphate-6-phosphogluconate dehydrogenase was detected in strains of the fast-growing group of Rhizobium but not in strains of the slow-growing group. An enzymatic differentiation of rhizobia was established. PMID:4400417

  14. Structural energetics of the adenine tract from an intrinsic transcription terminator.

    PubMed

    Huang, Yuegao; Weng, Xiaoli; Russu, Irina M

    2010-04-02

    Intrinsic transcription termination sites generally contain a tract of adenines in the DNA template that yields a tract of uracils at the 3' end of the nascent RNA. To understand how this base sequence contributes to termination of transcription, we have investigated two nucleic acid structures. The first is the RNA-DNA hybrid that contains the uracil tract 5'-rUUUUUAU-3' from the tR2 intrinsic terminator of bacteriophage lambda. The second is the homologous DNA-DNA duplex that contains the adenine tract 5'-dATAAAAA-3'. This duplex is present at the tR2 site when the DNA is not transcribed. The opening and the stability of each rU-dA/dT-dA base pair in the two structures are characterized by imino proton exchange and nuclear magnetic resonance spectroscopy. The results reveal concerted opening of the central rU-dA base pairs in the RNA-DNA hybrid. Furthermore, the stability profile of the adenine tract in the RNA-DNA hybrid is very different from that of the tract in the template DNA-DNA duplex. In the RNA-DNA hybrid, the stabilities of rU-dA base pairs range from 4.3 to 6.5 kcal/mol (at 10 degrees C). The sites of lowest stability are identified at the central positions of the tract. In the template DNA-DNA duplex, the dT-dA base pairs are more stable than the corresponding rU-dA base pairs in the hybrid by 0.9 to 4.6 kcal/mol and, in contrast to the RNA-DNA hybrid, the central base pairs have the highest stability. These results suggest that the central rU-dA/dT-dA base pairs in the adenine tract make the largest energetic contributions to transcription termination by promoting both the dissociation of the RNA transcript and the closing of the transcription bubble. The results also suggest that the high stability of dT-dA base pairs in the DNA provides a signal for the pausing of RNA polymerase at the termination site. Copyright 2010 Elsevier Ltd. All rights reserved.

  15. 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. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Fluorescence spectroscopy using excitation and emission matrix for quantification of tissue native fluorophores and cancer diagnosis

    NASA Astrophysics Data System (ADS)

    Wu, Binlin; Gayen, S. K.; Xu, M.

    2014-03-01

    Native fluorescence spectrum of normal and cancerous human prostate tissues is studied to distinguish between normal and cancerous tissues, and cancerous tissues at different cancer grade. The tissue samples were obtained from Cooperative Human Tissue Network (CHTN) and National Disease Research Interchange(NDRI). An excitation and emission matrix (EEM) was generated for each tissue sample by acquiring native fluorescence spectrum of the sample using multiple excitation wavelengths. The non-negative matrix factorization algorithm was used to generate fluorescence EEMs that correspond to the fluorophores in biological tissues, including tryptophan, collagen, elastin, nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD) and the background paraffin. We hypothesize that, as a consequence of metabolic changes associated with the development of cancer, the concentrations of NADH and FAD are different in normal and cancerous tissues, and also different for different cancer grades. We used the ratio of the abundances of FAD and NADH to distinguish between normal and cancerous tissues, and the tissue cancer grade. The FAD-to-NADH ratio was found to be the highest for normal tissue and decreased as the cancer grade increased.

  17. 3′-NADP and 3′-NAADP, Two Metabolites Formed by the Bacterial Type III Effector AvrRxo1*♦

    PubMed Central

    Schuebel, Felix; Rocker, Andrea; Edelmann, Daniel; Schessner, Julia; Brieke, Clara; Meinhart, Anton

    2016-01-01

    An arsenal of effector proteins is injected by bacterial pathogens into the host cell or its vicinity to increase virulence. The commonly used top-down approaches inferring the toxic mechanism of individual effector proteins from the host's phenotype are often impeded by multiple targets of different effectors as well as by their pleiotropic effects. Here we describe our bottom-up approach, showing that the bacterial type III effector AvrRxo1 of plant pathogens is an authentic phosphotransferase that produces two novel metabolites by phosphorylating nicotinamide/nicotinic acid adenine dinucleotide at the adenosine 3′-hydroxyl group. Both products of AvrRxo1, 3′-NADP and 3′-nicotinic acid adenine dinucleotide phosphate (3′-NAADP), are substantially different from the ubiquitous co-enzyme 2′-NADP and the calcium mobilizer 2′-NAADP. Interestingly, 3′-NADP and 3′-NAADP have previously been used as inhibitors or signaling molecules but were regarded as “artificial” compounds so far. Our findings now necessitate a shift in thinking about the biological importance of 3′-phosphorylated NAD derivatives. PMID:27621317

  18. Optical biopsy using fluorescence spectroscopy for prostate cancer diagnosis

    NASA Astrophysics Data System (ADS)

    Wu, Binlin; Gao, Xin; Smith, Jason; Bailin, Jacob

    2017-02-01

    Native fluorescence spectra are acquired from fresh normal and cancerous human prostate tissues. The fluorescence data are analyzed using a multivariate analysis algorithm such as non-negative matrix factorization. The nonnegative spectral components are retrieved and attributed to the native fluorophores such as collagen, reduced nicotinamide adenine dinucleotide (NADH), and flavin adenine dinucleotide (FAD) in tissue. The retrieved weights of the components, e.g. NADH and FAD are used to estimate the relative concentrations of the native fluorophores and the redox ratio. A machine learning algorithm such as support vector machine (SVM) is used for classification to distinguish normal and cancerous tissue samples based on either the relative concentrations of NADH and FAD or the redox ratio alone. The classification performance is shown based on statistical measures such as sensitivity, specificity, and accuracy, along with the area under receiver operating characteristic (ROC) curve. A cross validation method such as leave-one-out is used to evaluate the predictive performance of the SVM classifier to avoid bias due to overfitting.

  19. 3'-NADP and 3'-NAADP, Two Metabolites Formed by the Bacterial Type III Effector AvrRxo1.

    PubMed

    Schuebel, Felix; Rocker, Andrea; Edelmann, Daniel; Schessner, Julia; Brieke, Clara; Meinhart, Anton

    2016-10-28

    An arsenal of effector proteins is injected by bacterial pathogens into the host cell or its vicinity to increase virulence. The commonly used top-down approaches inferring the toxic mechanism of individual effector proteins from the host's phenotype are often impeded by multiple targets of different effectors as well as by their pleiotropic effects. Here we describe our bottom-up approach, showing that the bacterial type III effector AvrRxo1 of plant pathogens is an authentic phosphotransferase that produces two novel metabolites by phosphorylating nicotinamide/nicotinic acid adenine dinucleotide at the adenosine 3'-hydroxyl group. Both products of AvrRxo1, 3'-NADP and 3'-nicotinic acid adenine dinucleotide phosphate (3'-NAADP), are substantially different from the ubiquitous co-enzyme 2'-NADP and the calcium mobilizer 2'-NAADP. Interestingly, 3'-NADP and 3'-NAADP have previously been used as inhibitors or signaling molecules but were regarded as "artificial" compounds so far. Our findings now necessitate a shift in thinking about the biological importance of 3'-phosphorylated NAD derivatives. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Dunnione ameliorates cisplatin-induced small intestinal damage by modulating NAD{sup +} metabolism

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pandit, Arpana; Kim, Hyung-Jin; Oh, Gi-Su

    2015-11-27

    Although cisplatin is a widely used anticancer drug for the treatment of a variety of tumors, its use is critically limited because of adverse effects such as ototoxicity, nephrotoxicity, neuropathy, and gastrointestinal damage. Cisplatin treatment increases oxidative stress biomarkers in the small intestine, which may induce apoptosis of epithelial cells and thereby elicit damage to the small intestine. Nicotinamide adenine dinucleotide (NAD{sup +}) is a cofactor for various enzymes associated with cellular homeostasis. In the present study, we demonstrated that the hyper-activation of poly(ADP-ribose) polymerase-1 (PARP-1) is closely associated with the depletion of NAD{sup +} in the small intestine aftermore » cisplatin treatment, which results in downregulation of sirtuin1 (SIRT1) activity. Furthermore, a decrease in SIRT1 activity was found to play an important role in cisplatin-mediated small intestinal damage through nuclear factor (NF)-κB p65 activation, facilitated by its acetylation increase. However, use of dunnione as a strong substrate for the NADH:quinone oxidoreductase 1 (NQO1) enzyme led to an increase in intracellular NAD{sup +} levels and prevented the cisplatin-induced small intestinal damage correlating with the modulation of PARP-1, SIRT1, and NF-κB. These results suggest that direct modulation of cellular NAD{sup +} levels by pharmacological NQO1 substrates could be a promising therapeutic approach for protecting against cisplatin-induced small intestinal damage. - Highlights: • NAD{sup +} acts as a cofactor for numerous enzymes including Sirtuins and PARP. • Up-regulation of SIRT1 could attenuate the cisplatin-induced intestinal damage. • Modulation of the cellular NAD{sup +} could be a promising therapeutic approach.« less

  1. Insights into Hydrocarbon Formation by Nitrogenase Cofactor Homologs

    PubMed Central

    Lee, Chi Chung; Hu, Yilin

    2015-01-01

    ABSTRACT The L-cluster is an all-iron homolog of nitrogenase cofactors. Driven by europium(II) diethylenetriaminepentaacetate [Eu(II)-DTPA], the isolated L-cluster is capable of ATP-independent reduction of CO and CN− to C1 to C4 and C1 to C6 hydrocarbons, respectively. Compared to its cofactor homologs, the L-cluster generates considerably more CH4 from the reduction of CO and CN−, which could be explained by the presence of a “free” Fe atom that is “unmasked” by homocitrate as an additional site for methanation. Moreover, the elevated CH4 formation is accompanied by a decrease in the amount of longer hydrocarbons and/or the lengths of the hydrocarbon products, illustrating a competition between CH4 formation/release and C−C coupling/chain extension. These observations suggest the possibility of designing simpler synthetic clusters for hydrocarbon formation while establishing the L-cluster as a platform for mechanistic investigations of CO and CN− reduction without complications originating from the heterometal and homocitrate components. PMID:25873377

  2. Revealing Hidden Conformational Space of LOV Protein VIVID Through Rigid Residue Scan Simulations

    NASA Astrophysics Data System (ADS)

    Zhou, Hongyu; Zoltowski, Brian D.; Tao, Peng

    2017-04-01

    VIVID(VVD) protein is a Light-Oxygen-Voltage(LOV) domain in circadian clock system. Upon blue light activation, a covalent bond is formed between VVD residue Cys108 and its cofactor flavin adenine dinucleotide(FAD), and prompts VVD switching from Dark state to Light state with significant conformational deviation. However, the mechanism of this local environment initiated global protein conformational change remains elusive. We employed a recently developed computational approach, rigid residue scan(RRS), to systematically probe the impact of the internal degrees of freedom in each amino acid residue of VVD on its overall dynamics by applying rigid body constraint on each residue in molecular dynamics simulations. Key residues were identified with distinctive impacts on Dark and Light states, respectively. All the simulations display wide range of distribution on a two-dimensional(2D) plot upon structural root-mean-square deviations(RMSD) from either Dark or Light state. Clustering analysis of the 2D RMSD distribution leads to 15 representative structures with drastically different conformation of N-terminus, which is also a key difference between Dark and Light states of VVD. Further principle component analyses(PCA) of RRS simulations agree with the observation of distinctive impact from individual residues on Dark and Light states.

  3. Riboflavin accumulation and characterization of cDNAs encoding lumazine synthase and riboflavin synthase in bitter melon (Momordica charantia).

    PubMed

    Tuan, Pham Anh; Kim, Jae Kwang; Lee, Sanghyun; Chae, Soo Cheon; Park, Sang Un

    2012-12-05

    Riboflavin (vitamin B2) is the universal precursor of the coenzymes flavin mononucleotide and flavin adenine dinucleotide--cofactors that are essential for the activity of a wide variety of metabolic enzymes in animals, plants, and microbes. Using the RACE PCR approach, cDNAs encoding lumazine synthase (McLS) and riboflavin synthase (McRS), which catalyze the last two steps in the riboflavin biosynthetic pathway, were cloned from bitter melon (Momordica charantia), a popular vegetable crop in Asia. Amino acid sequence alignments indicated that McLS and McRS share high sequence identity with other orthologous genes and carry an N-terminal extension, which is reported to be a plastid-targeting sequence. Organ expression analysis using quantitative real-time RT PCR showed that McLS and McRS were constitutively expressed in M. charantia, with the strongest expression levels observed during the last stage of fruit ripening (stage 6). This correlated with the highest level of riboflavin content, which was detected during ripening stage 6 by HPLC analysis. McLS and McRS were highly expressed in the young leaves and flowers, whereas roots exhibited the highest accumulation of riboflavin. The cloning and characterization of McLS and McRS from M. charantia may aid the metabolic engineering of vitamin B2 in crops.

  4. The cyclic-di-GMP diguanylate cyclase CdgA has a role in biofilm formation and exopolysaccharide production in Azospirillum brasilense.

    PubMed

    Ramírez-Mata, Alberto; López-Lara, Lilia I; Xiqui-Vázquez, Ma Luisa; Jijón-Moreno, Saúl; Romero-Osorio, Angelica; Baca, Beatriz E

    2016-04-01

    In bacteria, proteins containing GGDEF domains are involved in production of the second messenger c-di-GMP. Here we report that the cdgA gene encoding diguanylate cyclase A (CdgA) is involved in biofilm formation and exopolysaccharide (EPS) production in Azospirillum brasilense Sp7. Biofilm quantification using crystal violet staining revealed that inactivation of cdgA decreased biofilm formation. In addition, confocal laser scanning microscopy analysis of green-fluorescent protein-labeled bacteria showed that, during static growth, the biofilms had differential levels of development: bacteria harboring a cdgA mutation exhibited biofilms with considerably reduced thickness compared with those of the wild-type Sp7 strain. Moreover, DNA-specific staining and treatment with DNase I, and epifluorescence studies demonstrated that extracellular DNA and EPS are components of the biofilm matrix in Azospirillum. After expression and purification of the CdgA protein, diguanylate cyclase activity was detected. The enzymatic activity of CdgA-producing cyclic c-di-GMP was determined using GTP as a substrate and flavin adenine dinucleotide (FAD(+)) and Mg(2)(+) as cofactors. Together, our results revealed that A. brasilense possesses a functional c-di-GMP biosynthesis pathway. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  5. An NAD+-dependent transcriptional program governs self-renewal and radiation resistance in glioblastoma.

    PubMed

    Gujar, Amit D; Le, Son; Mao, Diane D; Dadey, David Y A; Turski, Alice; Sasaki, Yo; Aum, Diane; Luo, Jingqin; Dahiya, Sonika; Yuan, Liya; Rich, Keith M; Milbrandt, Jeffrey; Hallahan, Dennis E; Yano, Hiroko; Tran, David D; Kim, Albert H

    2016-12-20

    Accumulating evidence suggests cancer cells exhibit a dependency on metabolic pathways regulated by nicotinamide adenine dinucleotide (NAD + ). Nevertheless, how the regulation of this metabolic cofactor interfaces with signal transduction networks remains poorly understood in glioblastoma. Here, we report nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting step in NAD + synthesis, is highly expressed in glioblastoma tumors and patient-derived glioblastoma stem-like cells (GSCs). High NAMPT expression in tumors correlates with decreased patient survival. Pharmacological and genetic inhibition of NAMPT decreased NAD + levels and GSC self-renewal capacity, and NAMPT knockdown inhibited the in vivo tumorigenicity of GSCs. Regulatory network analysis of RNA sequencing data using GSCs treated with NAMPT inhibitor identified transcription factor E2F2 as the center of a transcriptional hub in the NAD + -dependent network. Accordingly, we demonstrate E2F2 is required for GSC self-renewal. Downstream, E2F2 drives the transcription of members of the inhibitor of differentiation (ID) helix-loop-helix gene family. Finally, we find NAMPT mediates GSC radiation resistance. The identification of a NAMPT-E2F2-ID axis establishes a link between NAD + metabolism and a self-renewal transcriptional program in glioblastoma, with therapeutic implications for this formidable cancer.

  6. NAD+ Supplementation Attenuates Methylmercury Dopaminergic and Mitochondrial Toxicity in Caenorhabditis Elegans

    PubMed Central

    Caito, Samuel W.; Aschner, Michael

    2016-01-01

    Methylmercury (MeHg) is a neurotoxic contaminant of our fish supply that has been linked to dopaminergic (DAergic) dysfunction that characterizes Parkinson’s disease. We have previously shown that MeHg causes both morphological and behavioral changes in the Caenorhabditis elegans DAergic neurons that are associated with oxidative stress. We were therefore interested in whether the redox sensitive cofactor nicotinamide adenine dinucleotide (NAD+) may be affected by MeHg and whether supplementation of NAD + may prevent MeHg-induced toxicities. Worms treated with MeHg showed depletion in cellular NAD + levels, which was prevented by NAD + supplementation prior to MeHg treatment. NAD + supplementation also prevented DAergic neurodegeneration and deficits in DAergic-dependent behavior upon MeHg exposure. In a mutant worm line that cannot synthesize NAD + from nicotinamide, MeHg lethality and DAergic behavioral deficits were more sensitive to MeHg than wildtype worms, demonstrating the importance of NAD + in MeHg toxicity. In wildtype worms, NAD + supplementation provided protection from MeHg-induced oxidative stress and mitochondrial dysfunction. These data show the importance of NAD + levels in the response to MeHg exposure. NAD + supplementation may be beneficial for MeHg-induced toxicities and preventing cellular damage involved in Parkinson’s disease. PMID:26865665

  7. Assimilation of Endogenous Nicotinamide Riboside Is Essential for Calorie Restriction-mediated Life Span Extension in Saccharomyces cerevisiae*

    PubMed Central

    Lu, Shu-Ping; Kato, Michiko; Lin, Su-Ju

    2009-01-01

    NAD+ (nicotinamide adenine dinucleotide) is an essential cofactor involved in various biological processes including calorie restriction-mediated life span extension. Administration of nicotinamide riboside (NmR) has been shown to ameliorate deficiencies related to aberrant NAD+ metabolism in both yeast and mammalian cells. However, the biological role of endogenous NmR remains unclear. Here we demonstrate that salvaging endogenous NmR is an integral part of NAD+ metabolism. A balanced NmR salvage cycle is essential for calorie restriction-induced life span extension and stress resistance in yeast. Our results also suggest that partitioning of the pyridine nucleotide flux between the classical salvage cycle and the NmR salvage branch might be modulated by the NAD+-dependent Sir2 deacetylase. Furthermore, two novel deamidation steps leading to nicotinic acid mononucleotide and nicotinic acid riboside production are also uncovered that further underscore the complexity and flexibility of NAD+ metabolism. In addition, utilization of extracellular nicotinamide mononucleotide requires prior conversion to NmR mediated by a periplasmic phosphatase Pho5. Conversion to NmR may thus represent a strategy for the transport and assimilation of large nonpermeable NAD+ precursors. Together, our studies provide a molecular basis for how NAD+ homeostasis factors confer metabolic flexibility. PMID:19416965

  8. Assimilation of endogenous nicotinamide riboside is essential for calorie restriction-mediated life span extension in Saccharomyces cerevisiae.

    PubMed

    Lu, Shu-Ping; Kato, Michiko; Lin, Su-Ju

    2009-06-19

    NAD(+) (nicotinamide adenine dinucleotide) is an essential cofactor involved in various biological processes including calorie restriction-mediated life span extension. Administration of nicotinamide riboside (NmR) has been shown to ameliorate deficiencies related to aberrant NAD(+) metabolism in both yeast and mammalian cells. However, the biological role of endogenous NmR remains unclear. Here we demonstrate that salvaging endogenous NmR is an integral part of NAD(+) metabolism. A balanced NmR salvage cycle is essential for calorie restriction-induced life span extension and stress resistance in yeast. Our results also suggest that partitioning of the pyridine nucleotide flux between the classical salvage cycle and the NmR salvage branch might be modulated by the NAD(+)-dependent Sir2 deacetylase. Furthermore, two novel deamidation steps leading to nicotinic acid mononucleotide and nicotinic acid riboside production are also uncovered that further underscore the complexity and flexibility of NAD(+) metabolism. In addition, utilization of extracellular nicotinamide mononucleotide requires prior conversion to NmR mediated by a periplasmic phosphatase Pho5. Conversion to NmR may thus represent a strategy for the transport and assimilation of large nonpermeable NAD(+) precursors. Together, our studies provide a molecular basis for how NAD(+) homeostasis factors confer metabolic flexibility.

  9. Dimethyl sulfoxide reductase activity by anaerobically grown Escherichia coli HB101.

    PubMed Central

    Bilous, P T; Weiner, J H

    1985-01-01

    Escherichia coli grew anaerobically on a minimal medium with glycerol as the carbon and energy source and dimethyl sulfoxide (DMSO) as the terminal electron acceptor. DMSO reductase activity, measured with an artificial electron donor (reduced benzyl viologen), was preferentially associated with the membrane fraction (77 +/- 10% total cellular activity). A Km for DMSO reduction of 170 +/- 60 microM was determined for the membrane-bound activity. Methyl viologen, reduced flavin mononucleotide, and reduced flavin adenine dinucleotide also served as electron donors for DMSO reduction. Methionine sulfoxide, a DMSO analog, could substitute for DMSO in both the growth medium and in the benzyl viologen assay. DMSO reductase activity was present in cells grown anaerobically on DMSO but was repressed by the presence of nitrate or by aerobic growth. Anaerobic growth on DMSO coinduced nitrate, fumarate, and and trimethylamine-N-oxide reductase activities. The requirement of a molybdenum cofactor for DMSO reduction was suggested by the inhibition of growth and a 60% reduction in DMSO reductase activity in the presence of 10 mM sodium tungstate. Furthermore, chlorate-resistant mutants chlA, chlB, chlE, and chlG were unable to grow anaerobically on DMSO. DMSO reduction appears to be under the control of the fnr gene. PMID:3888958

  10. A Dedicated Type II NADPH Dehydrogenase Performs the Penultimate Step in the Biosynthesis of Vitamin K1 in Synechocystis and Arabidopsis

    PubMed Central

    Fatihi, Abdelhak; Latimer, Scott; Schmollinger, Stefan; Block, Anna; Dussault, Patrick H.; Vermaas, Wim F.J.; Merchant, Sabeeha S.; Basset, Gilles J.

    2015-01-01

    Mutation of Arabidopsis thaliana NAD(P)H DEHYDROGENASE C1 (NDC1; At5g08740) results in the accumulation of demethylphylloquinone, a late biosynthetic intermediate of vitamin K1. Gene coexpression and phylogenomics analyses showed that conserved functional associations occur between vitamin K biosynthesis and NDC1 homologs throughout the prokaryotic and eukaryotic lineages. Deletion of Synechocystis ndbB, which encodes for one such homolog, resulted in the same defects as those observed in the cyanobacterial demethylnaphthoquinone methyltransferase knockout. Chemical modeling and assay of purified demethylnaphthoquinone methyltransferase demonstrated that, by virtue of the strong electrophilic nature of S-adenosyl-l-methionine, the transmethylation of the demethylated precursor of vitamin K is strictly dependent on the reduced form of its naphthoquinone ring. NDC1 was shown to catalyze such a prerequisite reduction by using NADPH and demethylphylloquinone as substrates and flavine adenine dinucleotide as a cofactor. NDC1 displayed Michaelis-Menten kinetics and was markedly inhibited by dicumarol, a competitive inhibitor of naphthoquinone oxidoreductases. These data demonstrate that the reduction of the demethylnaphthoquinone ring represents an authentic step in the biosynthetic pathway of vitamin K, that this reaction is enzymatically driven, and that a selection pressure is operating to retain type II NAD(P)H dehydrogenases in this process. PMID:26023160

  11. Chromatographic analysis of tryptophan metabolites

    PubMed Central

    Sadok, Ilona; Gamian, Andrzej

    2017-01-01

    The kynurenine pathway generates multiple tryptophan metabolites called collectively kynurenines and leads to formation of the enzyme cofactor nicotinamide adenine dinucleotide. The first step in this pathway is tryptophan degradation, initiated by the rate‐limiting enzymes indoleamine 2,3‐dioxygenase, or tryptophan 2,3‐dioxygenase, depending on the tissue. The balanced kynurenine metabolism, which has been a subject of multiple studies in last decades, plays an important role in several physiological and pathological conditions such as infections, autoimmunity, neurological disorders, cancer, cataracts, as well as pregnancy. Understanding the regulation of tryptophan depletion provide novel diagnostic and treatment opportunities, however it requires reliable methods for quantification of kynurenines in biological samples with complex composition (body fluids, tissues, or cells). Trace concentrations, interference of sample components, and instability of some tryptophan metabolites need to be addressed using analytical methods. The novel separation approaches and optimized extraction protocols help to overcome difficulties in analyzing kynurenines within the complex tissue material. Recent developments in chromatography coupled with mass spectrometry provide new opportunity for quantification of tryptophan and its degradation products in various biological samples. In this review, we present current accomplishments in the chromatographic methodologies proposed for detection of tryptophan metabolites and provide a guide for choosing the optimal approach. PMID:28590049

  12. A thermostable L-aspartate oxidase: a new tool for biotechnological applications.

    PubMed

    Bifulco, Davide; Pollegioni, Loredano; Tessaro, Davide; Servi, Stefano; Molla, Gianluca

    2013-08-01

    L-Amino acid oxidases (LAAOs) are homodimeric flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the stereospecific oxidative deamination of L-amino acids to α-keto acids, ammonia, and hydrogen peroxide. Unlike the D-selective counterpart, the biotechnological application of LAAOs has not been thoroughly advanced because of the difficulties in their expression as recombinant protein in prokaryotic hosts. In this work, L-aspartate oxidase from the thermophilic archea Sulfolobus tokodaii (StLASPO, specific for L-aspartate and L-asparagine only) was efficiently produced as recombinant protein in E. coli in the active form as holoenzyme. This recombinant flavoenzyme shows the classical properties of FAD-containing oxidases. Indeed, StLASPO shows distinctive features that makes it attractive for biotechnological applications: high thermal stability (it is fully stable up to 80 °C) and high temperature optimum, stable activity in a broad range of pH (7.0-10.0), weak inhibition by the product oxaloacetate and by D-aspartate, and tight binding of the FAD cofactor. This latter property significantly distinguishes StLASPO from the E. coli counterpart. StLASPO represents an appropriate novel biocatalyst for the production of D-aspartate and a well-suited protein scaffold to evolve a LAAO activity by protein engineering.

  13. Revealing Hidden Conformational Space of LOV Protein VIVID Through Rigid Residue Scan Simulations

    PubMed Central

    Zhou, Hongyu; Zoltowski, Brian D.; Tao, Peng

    2017-01-01

    VIVID(VVD) protein is a Light-Oxygen-Voltage(LOV) domain in circadian clock system. Upon blue light activation, a covalent bond is formed between VVD residue Cys108 and its cofactor flavin adenine dinucleotide(FAD), and prompts VVD switching from Dark state to Light state with significant conformational deviation. However, the mechanism of this local environment initiated global protein conformational change remains elusive. We employed a recently developed computational approach, rigid residue scan(RRS), to systematically probe the impact of the internal degrees of freedom in each amino acid residue of VVD on its overall dynamics by applying rigid body constraint on each residue in molecular dynamics simulations. Key residues were identified with distinctive impacts on Dark and Light states, respectively. All the simulations display wide range of distribution on a two-dimensional(2D) plot upon structural root-mean-square deviations(RMSD) from either Dark or Light state. Clustering analysis of the 2D RMSD distribution leads to 15 representative structures with drastically different conformation of N-terminus, which is also a key difference between Dark and Light states of VVD. Further principle component analyses(PCA) of RRS simulations agree with the observation of distinctive impact from individual residues on Dark and Light states. PMID:28425502

  14. Cyclic Dinucleotides in the Scope of the Mammalian Immune System.

    PubMed

    Mankan, Arun K; Müller, Martina; Witte, Gregor; Hornung, Veit

    2017-01-01

    First discovered in prokaryotes and more recently in eukaryotes, cyclic dinucleotides (CDNs) constitute a unique branch of second messenger signaling systems. Within prokaryotes CDNs regulate a wide array of different biological processes, whereas in the vertebrate system CDN signaling is largely dedicated to activation of the innate immune system. In this book chapter we summarize the occurrence and signaling pathways of these small-molecule second messengers, most importantly in the scope of the mammalian immune system. In this regard, our main focus is the role of the cGAS-STING axis in the context of microbial infection and sterile inflammation and its implications for therapeutic applications.

  15. The Fe-V Cofactor of Vanadium Nitrogenase Contains an Interstitial Carbon Atom.

    PubMed

    Rees, Julian A; Bjornsson, Ragnar; Schlesier, Julia; Sippel, Daniel; Einsle, Oliver; DeBeer, Serena

    2015-11-02

    The first direct evidence is provided for the presence of an interstitial carbide in the Fe-V cofactor of Azotobacter vinelandii vanadium nitrogenase. As for our identification of the central carbide in the Fe-Mo cofactor, we employed Fe Kβ valence-to-core X-ray emission spectroscopy and density functional theory calculations, and herein report the highly similar spectra of both variants of the cofactor-containing protein. The identification of an analogous carbide, and thus an atomically homologous active site in vanadium nitrogenase, highlights the importance and influence of both the interstitial carbide and the identity of the heteroatom on the electronic structure and catalytic activity of the enzyme. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

  16. The Tail Wagging the Dog: Insights into Catalysis in R67 Dihydrofolate Reductase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kamath, Ganesh K; Agarwal, Pratul K

    2010-01-01

    Plasmid-encoded R67 dihydrofolate reductase (DHFR) catalyzes a hydride transfer reaction between substrate dihydrofolate (DHF) and its cofactor, nicotinamide adenine dinucleotide phosphate (NADPH). R67 DHFR is a homotetramer that exhibits numerous characteristics of a primitive enzyme, including promiscuity in binding of substrate and cofactor, formation of nonproductive complexes, and the absence of a conserved acid in its active site. Furthermore, R67's active site is a pore, which is mostly accessible by bulk solvent. This study uses a computational approach to characterize the mechanism of hydride transfer. Not surprisingly, NADPH remains fixed in one-half of the active site pore using numerous interactionsmore » with R67. Also, stacking between the nicotinamide ring of the cofactor and the pteridine ring of the substrate, DHF, at the hourglass center of the pore, holds the reactants in place. However, large movements of the p-aminobenzoylglutamate tail of DHF occur in the other half of the pore because of ion pair switching between symmetry-related K32 residues from two subunits. This computational result is supported by experimental results that the loss of these ion pair interactions (located >13 {angstrom} from the center of the pore) by addition of salt or in asymmetric K32M mutants leads to altered enzyme kinetics [Hicks, S. N., et al. (2003) Biochemistry 42, 10569-10578; Hicks, S. N., et al. (2004) J. Biol. Chem. 279, 46995?47002]. The tail movement at the edge of the active site, coupled with the fixed position of the pteridine ring in the center of the pore, leads to puckering of the pteridine ring and promotes formation of the transition state. Flexibility coupled to R67 function is unusual as it contrasts with the paradigm that enzymes use increased rigidity to facilitate attainment of their transition states. A comparison with chromosomal DHFR indicates a number of similarities, including puckering of the nicotinamide ring and changes in the DHF

  17. NADH/NADPH bi-cofactor-utilizing and thermoactive ketol-acid reductoisomerase from Sulfolobus acidocaldarius.

    PubMed

    Chen, Chin-Yu; Ko, Tzu-Ping; Lin, Kuan-Fu; Lin, Bo-Lin; Huang, Chun-Hsiang; Chiang, Cheng-Hung; Horng, Jia-Cherng

    2018-05-08

    Ketol-acid reductoisomerase (KARI) is a bifunctional enzyme in the second step of branched-chain amino acids biosynthetic pathway. Most KARIs prefer NADPH as a cofactor. However, KARI with a preference for NADH is desirable in industrial applications including anaerobic fermentation for the production of branched-chain amino acids or biofuels. Here, we characterize a thermoacidophilic archaeal Sac-KARI from Sulfolobus acidocaldarius and present its crystal structure at a 1.75-Å resolution. By comparison with other holo-KARI structures, one sulphate ion is observed in each binding site for the 2'-phosphate of NADPH, implicating its NADPH preference. Sac-KARI has very high affinity for NADPH and NADH, with K M values of 0.4 μM for NADPH and 6.0 μM for NADH, suggesting that both are good cofactors at low concentrations although NADPH is favoured over NADH. Furthermore, Sac-KARI can catalyze 2(S)-acetolactate (2S-AL) with either cofactor from 25 to 60 °C, but the enzyme has higher activity by using NADPH. In addition, the catalytic activity of Sac-KARI increases significantly with elevated temperatures and reaches an optimum at 60 °C. Bi-cofactor utilization and the thermoactivity of Sac-KARI make it a potential candidate for use in metabolic engineering or industrial applications under anaerobic or harsh conditions.

  18. Head-to-head right-handed cross-links of the antitumor-active bis(mu-N,N'-di-p-tolylformamidinato)dirhodium(II,II) unit with the dinucleotides d(GpA) and d(ApG).

    PubMed

    Chifotides, Helen T; Dunbar, Kim R

    2008-01-01

    Reactions of cis-[Rh(2)(DTolF)(2)(NCCH(3))(6)](BF(4))(2) with the dinucleotides d(GpA) and d(ApG) proceed to form [Rh(2)(DTolF)(2){d(GpA)}] and [Rh(2)(DTolF)(2){d(ApG)}], respectively, with bridging purine bases spanning the Rh-Rh unit in the equatorial positions. Both dirhodium adducts exhibit head-to-head (HH) arrangement of the bases, as indicated by the presence of H8/H8 NOE cross-peaks in the 2D ROESY NMR spectra. The guanine bases bind to the dirhodium core at positions N7 and O6, a conclusion that is supported by the absence of N7 protonation at low pH values and the notable increase in the acidity of the guanine N1H sites (pK(a) approximately 7.4 in 4:1 CD(3)CN/D(2)O), inferred from the pH-dependence titrations of the guanine H8 proton resonances. In both dirhodium adducts, the adenine bases coordinate to the metal atoms through N6 and N7, which induces stabilization of the rare imino tautomer of the bases with a concomitant substantial decrease in the basicity of the N1H adenine sites (pK(a) approximately 7.0-7.1 in 4:1 CD(3)CN/D(2)O), as compared to the imino form of free adenosine. The presence of the adenine bases in the rare imino form is further corroborated by the observation of DQF-COSY H2/N1H and ROE N1H/N6H cross-peaks in the 2D NMR spectra of [Rh(2)(DTolF)(2){d(GpA)}] and [Rh(2)(DTolF)(2){d(ApG)}] in CD(3)CN at -38 degrees C. The 2D NMR spectroscopic data and the molecular modeling results suggest the presence of right-handed variants, HH1R, in solution for both adducts (HH1R refers to the relative base canting and the direction of propagation of the phosphodiester backbone with respect to the 5' base). Complete characterization of [Rh(2)(DTolF)(2){d(GpA)}] and [Rh(2)(DTolF)(2){d(ApG)}] by 2D NMR spectroscopy and molecular modeling supports anti-orientation of the sugar residues for both adducts about the glycosyl bonds as well as N- and S-type conformations for the 5'- and 3'-deoxyribose residues, respectively.

  19. Developmental expression patterns of candidate co-factors for vertebrate Six family transcription factors

    PubMed Central

    Neilson, Karen M.; Pignoni, Francesca; Yan, Bo; Moody, Sally A.

    2010-01-01

    Six family transcription factors play important roles in craniofacial development. Their transcriptional activity can be modified by co-factor proteins. Two Six genes and one co-factor gene (Eya1) are involved in the human Branchio-otic (BO) and Branchio-otic-renal (BOR) syndromes. However, mutations in Six and Eya genes only account for about half of these patients. To discover potential new causative genes, we searched the Xenopus genome for orthologues of Drosophila co-factor proteins that interact with the fly Six-related factor, SO. We identified 33 Xenopus genes with high sequence identity to 20 of the 25 fly SO-interacting proteins. We provide the developmental expression patterns of the Xenopus orthologues for 11 of the fly genes, and demonstrate that all are expressed in developing craniofacial tissues with at least partial overlap with Six1/Six2. We speculate that these genes may function as Six-interacting partners with important roles in vertebrate craniofacial development and perhaps congenital syndromes. PMID:21089078

  20. Divergent assembly mechanisms of the manganese/iron cofactors in R2lox and R2c proteins.

    PubMed

    Kutin, Yuri; Srinivas, Vivek; Fritz, Matthieu; Kositzki, Ramona; Shafaat, Hannah S; Birrell, James; Bill, Eckhard; Haumann, Michael; Lubitz, Wolfgang; Högbom, Martin; Griese, Julia J; Cox, Nicholas

    2016-09-01

    A manganese/iron cofactor which performs multi-electron oxidative chemistry is found in two classes of ferritin-like proteins, the small subunit (R2) of class Ic ribonucleotide reductase (R2c) and the R2-like ligand-binding oxidase (R2lox). It is unclear how a heterodimeric Mn/Fe metallocofactor is assembled in these two related proteins as opposed to a homodimeric Fe/Fe cofactor, especially considering the structural similarity and proximity of the two metal-binding sites in both protein scaffolds and the similar first coordination sphere ligand preferences of Mn II and Fe II . Using EPR and Mössbauer spectroscopies as well as X-ray anomalous dispersion, we examined metal loading and cofactor activation of both proteins in vitro (in solution). We find divergent cofactor assembly mechanisms for the two systems. In both cases, excess Mn II promotes heterobimetallic cofactor assembly. In the absence of Fe II , R2c cooperatively binds Mn II at both metal sites, whereas R2lox does not readily bind Mn II at either site. Heterometallic cofactor assembly is favored at substoichiometric Fe II concentrations in R2lox. Fe II and Mn II likely bind to the protein in a stepwise fashion, with Fe II binding to site 2 initiating cofactor assembly. In R2c, however, heterometallic assembly is presumably achieved by the displacement of Mn II by Fe II at site 2. The divergent metal loading mechanisms are correlated with the putative in vivo functions of R2c and R2lox, and most likely with the intracellular Mn II /Fe II concentrations in the host organisms from which they were isolated. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Sulphur shuttling across a chaperone during molybdenum cofactor maturation.

    PubMed

    Arnoux, Pascal; Ruppelt, Christian; Oudouhou, Flore; Lavergne, Jérôme; Siponen, Marina I; Toci, René; Mendel, Ralf R; Bittner, Florian; Pignol, David; Magalon, Axel; Walburger, Anne

    2015-02-04

    Formate dehydrogenases (FDHs) are of interest as they are natural catalysts that sequester atmospheric CO2, generating reduced carbon compounds with possible uses as fuel. FDHs activity in Escherichia coli strictly requires the sulphurtransferase EcFdhD, which likely transfers sulphur from IscS to the molybdenum cofactor (Mo-bisPGD) of FDHs. Here we show that EcFdhD binds Mo-bisPGD in vivo and has submicromolar affinity for GDP-used as a surrogate of the molybdenum cofactor's nucleotide moieties. The crystal structure of EcFdhD in complex with GDP shows two symmetrical binding sites located on the same face of the dimer. These binding sites are connected via a tunnel-like cavity to the opposite face of the dimer where two dynamic loops, each harbouring two functionally important cysteine residues, are present. On the basis of structure-guided mutagenesis, we propose a model for the sulphuration mechanism of Mo-bisPGD where the sulphur atom shuttles across the chaperone dimer.

  2. Saccharomyces cerevisiae engineered for xylose metabolism requires gluconeogenesis and the oxidative branch of the pentose phosphate pathway for aerobic xylose assimilation

    USDA-ARS?s Scientific Manuscript database

    Saccharomyces strains engineered to ferment xylose using Scheffersomyces stipitis xylose reductase (XR) and xylitol dehydrogenase (XDH) genes appear to be limited by metabolic imbalances due to differing cofactor specificities of XR and XDH. The S. stipitis XR, which uses nicotinamide adenine dinucl...

  3. Chemomimetic biocatalysis: exploiting the synthetic potential of cofactor-dependent enzymes to create new catalysts.

    PubMed

    Prier, Christopher K; Arnold, Frances H

    2015-11-11

    Despite the astonishing breadth of enzymes in nature, no enzymes are known for many of the valuable catalytic transformations discovered by chemists. Recent work in enzyme design and evolution, however, gives us good reason to think that this will change. We describe a chemomimetic biocatalysis approach that draws from small-molecule catalysis and synthetic chemistry, enzymology, and molecular evolution to discover or create enzymes with non-natural reactivities. We illustrate how cofactor-dependent enzymes can be exploited to promote reactions first established with related chemical catalysts. The cofactors can be biological, or they can be non-biological to further expand catalytic possibilities. The ability of enzymes to amplify and precisely control the reactivity of their cofactors together with the ability to optimize non-natural reactivity by directed evolution promises to yield exceptional catalysts for challenging transformations that have no biological counterparts.

  4. Ameliorative effect of ursolic acid on renal fibrosis in adenine-induced chronic kidney disease in rats.

    PubMed

    Thakur, Richa; Sharma, Anshuk; Lingaraju, Madhu C; Begum, Jubeda; Kumar, Dhirendra; Mathesh, Karikalan; Kumar, Pawan; Singh, Thakur Uttam; Kumar, Dinesh

    2018-05-01

    Ursolic acid (UA), an ursane-type pentacyclic triterpenoid commonly found in apple peels and holy basil has been shown to possess many beneficial effects. Renal fibrosis is a complication of kidney injury and associated with increased risk of morbidity and mortality. In our previous investigation, a lupane-type pentacyclic triterpenoid, betulinic acid (BA) was found to have protective effect on chronic kidney disease (CKD) and renal fibrosis. This prompted us to explore the therapeutic value of UA, a chemically related compound to BA in CKD. CKD was induced by feeding adenine with the feed at a concentration of 0.75% for 28 days. UA at the dose rate of 30 mg/kg in 0.5% carboxy methyl cellulose (CMC) was administered by oral route, simultaneously with adenine feeding for 28 days. Adenine feeding increased the kidney weight to body weight index, decreased the kidney function due to injury as indicated by increased markers like serum urea, uric acid, creatinine, cystatin C and neutrophil gelatinase-associated lipocalin (NGAL) and initiated the fibrotic response in kidney by increasing the profibrotic proteins viz. transforming growth factor-beta (TGF-β), connective tissue growth factor (CTGF), fibronectin and collagen. However, treatment with UA reversed the damage induced by adenine as shown by reduced kidney injury and fibrosis markers which was further clearly evident in histological picture indicating the suitability of UA for use in CKD. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  5. Selective self-assembly of adenine-silver nanoparticles forms rings resembling the size of cells

    NASA Astrophysics Data System (ADS)

    Choi, Sungmoon; Park, Soonyoung; Yang, Seon-Ah; Jeong, Yujin; Yu, Junhua

    2015-12-01

    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.

  6. 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

  7. Metabolic Impact of Redox Cofactor Perturbations on the Formation of Aroma Compounds in Saccharomyces cerevisiae

    PubMed Central

    Sanchez, Isabelle; Dequin, Sylvie; Camarasa, Carole

    2015-01-01

    Redox homeostasis is a fundamental requirement for the maintenance of metabolism, energy generation, and growth in Saccharomyces cerevisiae. The redox cofactors NADH and NADPH are among the most highly connected metabolites in metabolic networks. Changes in their concentrations may induce widespread changes in metabolism. Redox imbalances were achieved with a dedicated biological tool overexpressing native NADH-dependent or engineered NADPH-dependent 2,3-butanediol dehydrogenase, in the presence of acetoin. We report that targeted perturbation of the balance of cofactors (NAD+/NADH or, to a lesser extent, NADP+/NADPH) significantly affected the production of volatile compounds. In most cases, variations in the redox state of yeasts modified the formation of all compounds from the same biochemical pathway (isobutanol, isoamyl alcohol, and their derivatives) or chemical class (ethyl esters), irrespective of the cofactors. These coordinated responses were found to be closely linked to the impact of redox status on the availability of intermediates of central carbon metabolism. This was the case for α-keto acids and acetyl coenzyme A (acetyl-CoA), which are precursors for the synthesis of many volatile compounds. We also demonstrated that changes in the availability of NADH selectively affected the synthesis of some volatile molecules (e.g., methionol, phenylethanol, and propanoic acid), reflecting the specific cofactor requirements of the dehydrogenases involved in their formation. Our findings indicate that both the availability of precursors from central carbon metabolism and the accessibility of reduced cofactors contribute to cell redox status modulation of volatile compound formation. PMID:26475113

  8. Restoring NAD(+) Levels with NAD(+) Intermediates, the Second Law of Thermodynamics and Aging Delay.

    PubMed

    Poljsak, Borut; Milisav, Irina

    2018-04-26

    The hypothesis regarding the role of increased nicotinamide adenine dinucleotide (NAD+) levels with reference to the fundamental concepts of ageing and entropy is presented. Considering the second law of thermodynamics, NAD+ seems the appropriate candidate for reversing many aging-associated pathologies. NAD+ is presented as an essential compound that enables organisms to stay highly organized and well-maintained, with a lower entropy state.

  9. Silver-induced reconstruction of an adeninate-based metal–organic framework for encapsulation of luminescent adenine-stabilized silver clusters† †Electronic supplementary information (ESI) available: Experimental details and additional structural, physicochemical and optical characterisation. See DOI: 10.1039/c6tc00260a Click here for additional data file.

    PubMed Central

    Jonckheere, Dries; Coutino-Gonzalez, Eduardo; Baekelant, Wouter; Bueken, Bart; Reinsch, Helge; Stassen, Ivo; Fenwick, Oliver; Richard, Fanny; Samorì, Paolo; Ameloot, Rob; Hofkens, Johan

    2016-01-01

    Bright luminescent silver-adenine species were successfully stabilized in the pores of the MOF-69A (zinc biphenyldicarboxylate) metal–organic framework, starting from the intrinsically blue luminescent bio-MOF-1 (zinc adeninate 4,4′-biphenyldicarboxylate). Bio-MOF-1 is transformed to the MOF-69A framework by selectively leaching structural adenine linkers from the original framework using silver nitrate solutions in aqueous ethanol. Simultaneously, bright blue-green luminescent silver-adenine clusters are formed inside the pores of the recrystallized MOF-69A matrix in high local concentrations. The structural transition and concurrent changes in optical properties were characterized using a range of structural, physicochemical and spectroscopic techniques (steady-state and time-resolved luminescence, quantum yield determination, fluorescence microscopy). The presented results open new avenues for exploring the use of MOFs containing luminescent silver clusters for solid-state lighting and sensor applications. PMID:28496980

  10. Rapid X-ray Photoreduction of Dimetal-Oxygen Cofactors in Ribonucleotide Reductase

    PubMed Central

    Sigfridsson, Kajsa G. V.; Chernev, Petko; Leidel, Nils; Popović-Bijelić, Ana; Gräslund, Astrid; Haumann, Michael

    2013-01-01

    Prototypic dinuclear metal cofactors with varying metallation constitute a class of O2-activating catalysts in numerous enzymes such as ribonucleotide reductase. Reliable structures are required to unravel the reaction mechanisms. However, protein crystallography data may be compromised by x-ray photoreduction (XRP). We studied XPR of Fe(III)Fe(III) and Mn(III)Fe(III) sites in the R2 subunit of Chlamydia trachomatis ribonucleotide reductase using x-ray absorption spectroscopy. Rapid and biphasic x-ray photoreduction kinetics at 20 and 80 K for both cofactor types suggested sequential formation of (III,II) and (II,II) species and similar redox potentials of iron and manganese sites. Comparing with typical x-ray doses in crystallography implies that (II,II) states are reached in <1 s in such studies. First-sphere metal coordination and metal-metal distances differed after chemical reduction at room temperature and after XPR at cryogenic temperatures, as corroborated by model structures from density functional theory calculations. The inter-metal distances in the XPR-induced (II,II) states, however, are similar to R2 crystal structures. Therefore, crystal data of initially oxidized R2-type proteins mostly contain photoreduced (II,II) cofactors, which deviate from the native structures functional in O2 activation, explaining observed variable metal ligation motifs. This situation may be remedied by novel femtosecond free electron-laser protein crystallography techniques. PMID:23400774

  11. Rapid X-ray photoreduction of dimetal-oxygen cofactors in ribonucleotide reductase.

    PubMed

    Sigfridsson, Kajsa G V; Chernev, Petko; Leidel, Nils; Popovic-Bijelic, Ana; Gräslund, Astrid; Haumann, Michael

    2013-04-05

    Prototypic dinuclear metal cofactors with varying metallation constitute a class of O2-activating catalysts in numerous enzymes such as ribonucleotide reductase. Reliable structures are required to unravel the reaction mechanisms. However, protein crystallography data may be compromised by x-ray photoreduction (XRP). We studied XPR of Fe(III)Fe(III) and Mn(III)Fe(III) sites in the R2 subunit of Chlamydia trachomatis ribonucleotide reductase using x-ray absorption spectroscopy. Rapid and biphasic x-ray photoreduction kinetics at 20 and 80 K for both cofactor types suggested sequential formation of (III,II) and (II,II) species and similar redox potentials of iron and manganese sites. Comparing with typical x-ray doses in crystallography implies that (II,II) states are reached in <1 s in such studies. First-sphere metal coordination and metal-metal distances differed after chemical reduction at room temperature and after XPR at cryogenic temperatures, as corroborated by model structures from density functional theory calculations. The inter-metal distances in the XPR-induced (II,II) states, however, are similar to R2 crystal structures. Therefore, crystal data of initially oxidized R2-type proteins mostly contain photoreduced (II,II) cofactors, which deviate from the native structures functional in O2 activation, explaining observed variable metal ligation motifs. This situation may be remedied by novel femtosecond free electron-laser protein crystallography techniques.

  12. The Role of the Plant Hormone Benzyl Adenine to Promote Growth for the Diatom Thalassiosira pseudonana

    NASA Astrophysics Data System (ADS)

    Gutierrez Franco, D.; Vernet, M.; Walters, R. J.; Tan, M.

    2016-02-01

    This study was inspired by the establishment of autoinduction in the model diatom Thalassiosira pseudonana, and the identification of the cytokinin plant hormone benzyl adenine (BA) as a potential autoinducer in this species via comparative genome studies. The effects of a wide range (0.0017518 mg/L-500 mg/L) of concentrations of benzyl adenine on the growth dynamics of T. pseudonana have been explored. The results suggest that a concentration of 5 mg BA/L has the highest positive effect on the growth rate of T. pseudonana batch cultures, compared to the other concentrations tested. Furthermore, concentrations of >100 mg BA/L were lethal. No marked effects on the lag phase length were observed. However, it is possible that some trade-offs between growth rate and lag phase length exist as a result of benzyl adenine. For instance, the BA concentration that exhibited the highest growth rate (5mg BA/L; µ=1.06 d-1) had a negative effect on the lag phase length (6 days), as compared to our control (lag phase length = 5 d; µ=0.81 d-1). On the other hand, at 10 mg BA/L, a slightly smaller growth rate of 1.01 d-1 was observed, with a shorter lag phase length of 4 days, suggesting that benzyl adenine may not have a positive effect on all growth parameters at once. These results provide insight into the physiological and biochemical mechanisms of cell-to-cell communication employed by diatoms, and supports the hypothesis that hormones may play an important role in bloom development.

  13. A new cofactor in prokaryotic enzyme: Tryptophan tryptophylquinone as the redox prosthetic group in methylamine dehydrogenase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    McIntire, W.S.; Wemmer, D.E.; Chistoserdov, A.

    Methylamine dehydrogenase (MADH), an {alpha}{sub 2}{beta}{sub 2} enzyme from numerous methylotrophic soil bacteria, contains a novel quinonoid redox prosthetic group that is covalently bound to its small {beta} subunit through two amino acyl residues. A comparison of the amino acid sequence deduced from the gene sequence of the small subunit for the enzyme from Methylobacterium extorquens AM1 with the published amino acid sequence obtained by Edman degradation method, allowed the identification of the amino acyl constituents of the cofactor as two tryptophyl residues. This information was crucial for interpreting {sup 1}H and {sup 13}C nuclear magnetic resonance, and mass spectralmore » data collected for the semicarbazide- and carboxymethyl-derivatized bis(tripeptidyl)-cofactor of MADH from bacterium W3A1. The cofactor is composed of two cross-linked tryptophyl residues. Although there are many possible isomers, only one is consistent with all the data: The first tryptophyl residue in the peptide sequence exists as an indole-6,7-dione, and is attached at its 4 position to the 2 position of the second, otherwise unmodified, indole side group. Contrary to earlier reports, the cofactor of MADH is not 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), a derivative thereof, of pro-PQQ. This appears to be the only example of two cross-linked, modified amino acyl residues having a functional role in the active site of an enzyme, in the absence of other cofactors or metal ions.« less

  14. [On the influence of local molecular environment on the redox potential of electron transfer cofactors in bacterial photosynthetic reaction centers].

    PubMed

    Krasil'nikov, P M; Noks, P P; Rubin, A B

    2011-01-01

    The addition of cryosolvents (glycerol, dimethylsulfoxide) to a water solution containing bacterial photosynthetic reaction centers changes the redox potential of the bacteriochlorophyll dimer, but does not affect the redox potential of the quinone primary acceptor. It has been shown that the change in redox potential can be produced by changes of the electrostatic interactions between cofactors and the local molecular environment modified by additives entered into the solution. The degree of influence of a solvent on the redox potential of various cofactors is determined by degree of availability of these cofactors for molecules of solvent, which depends on the arrangement of cofactors in the structure of reaction centers.

  15. Hydrophobic patches on SMAD2 and SMAD3 determine selective binding to cofactors.

    PubMed

    Miyazono, Ken-Ichi; Moriwaki, Saho; Ito, Tomoko; Kurisaki, Akira; Asashima, Makoto; Tanokura, Masaru

    2018-03-27

    The transforming growth factor-β (TGF-β) superfamily of cytokines regulates various biological processes, including cell proliferation, immune responses, autophagy, and senescence. Dysregulation of TGF-β signaling causes various diseases, such as cancer and fibrosis. SMAD2 and SMAD3 are core transcription factors involved in TGF-β signaling, and they form heterotrimeric complexes with SMAD4 (SMAD2-SMAD2-SMAD4, SMAD3-SMAD3-SMAD4, and SMAD2-SMAD3-SMAD4) in response to TGF-β signaling. These heterotrimeric complexes interact with cofactors to control the expression of TGF-β-dependent genes. SMAD2 and SMAD3 may promote or repress target genes depending on whether they form complexes with other transcription factors, coactivators, or corepressors; therefore, the selection of specific cofactors is critical for the appropriate activity of these transcription factors. To reveal the structural basis by which SMAD2 and SMAD3 select cofactors, we determined the crystal structures of SMAD3 in complex with the transcription factor FOXH1 and SMAD2 in complex with the transcriptional corepressor SKI. The structures of the complexes show that the MAD homology 2 (MH2) domains of SMAD2 and SMAD3 have multiple hydrophobic patches on their surfaces. The cofactors tether to various subsets of these patches to interact with SMAD2 and SMAD3 in a cooperative or competitive manner to control the output of TGF-β signaling. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  16. Redox-dependent substrate-cofactor interactions in the Michaelis-complex of a flavin-dependent oxidoreductase

    PubMed Central

    Werther, Tobias; Wahlefeld, Stefan; Salewski, Johannes; Kuhlmann, Uwe; Zebger, Ingo; Hildebrandt, Peter; Dobbek, Holger

    2017-01-01

    How an enzyme activates its substrate for turnover is fundamental for catalysis but incompletely understood on a structural level. With redox enzymes one typically analyses structures of enzyme–substrate complexes in the unreactive oxidation state of the cofactor, assuming that the interaction between enzyme and substrate is independent of the cofactors oxidation state. Here, we investigate the Michaelis complex of the flavoenzyme xenobiotic reductase A with the reactive reduced cofactor bound to its substrates by X-ray crystallography and resonance Raman spectroscopy and compare it to the non-reactive oxidized Michaelis complex mimics. We find that substrates bind in different orientations to the oxidized and reduced flavin, in both cases flattening its structure. But only authentic Michaelis complexes display an unexpected rich vibrational band pattern uncovering a strong donor–acceptor complex between reduced flavin and substrate. This interaction likely activates the catalytic ground state of the reduced flavin, accelerating the reaction within a compressed cofactor–substrate complex.

  17. Binding of pixantrone to DNA at CpA dinucleotide sequences and bulge structures.

    PubMed

    Konda, Shyam K; Wang, Haiqiang; Cutts, Suzanne M; Phillips, Don R; Collins, J Grant

    2015-06-07

    The binding of the anti-cancer drug pixantrone to three oligonucleotide sequences, d(TCATATGA)2, d(CCGAGAATTCCGG)2 {double bulge = DB} and the non-self complementary d(TACGATGAGTA) : d(TACCATCGTA) {single bulge = SB}, has been studied by NMR spectroscopy and molecular modelling. The upfield shifts observed for the aromatic resonances of pixantrone upon addition of the drug to each oligonucleotide confirmed the drug bound by intercalation. For the duplex sequence d(TCATATGA)2, NOEs were observed from the pixantrone aromatic H7/8 and aliphatic Ha/Hb protons to the H6/H8 and H1' protons of the C2, A3, T6 and G7 nucleotides, demonstrating that pixantrone preferentially binds at the symmetric CpA sites. However, weaker NOEs observed to various protons from the T4 and A5 residues indicated alternative minor binding sites. NOEs from the H7/H8 and Ha/Hb protons to both major (H6/H8) and minor groove (H1') protons indicated approximately equal proportions of intercalation was from the major and minor groove at the CpA sites. Intermolecular NOEs were observed between the H7/H8 and H4 protons of pixantrone and the A4H1' and G3H1' protons of the oligonucleotide that contains two symmetrically related bulge sites (DB), indicative of binding at the adenine bulge sites. For the oligonucleotide that only contains a single bulge site (SB), NOEs were observed from pixantrone protons to the SB G7H1', A8H1' and G9H1' protons, confirming that the drug bound selectively at the adenine bulge site. A molecular model of pixantrone-bound SB could be constructed with the drug bound from the minor groove at the A8pG9 site that was consistent with the observed NMR data. The results demonstrate that pixantrone preferentially intercalates at adenine bulge sites, compared to duplex DNA, and predominantly from the minor groove.

  18. Long-Range Charge Transport in Adenine-Stacked RNA:DNA Hybrids.

    PubMed

    Li, Yuanhui; Artés, Juan M; Hihath, Joshua

    2016-01-27

    An extremely important biological component, RNA:DNA can also be used to design nanoscale structures such as molecular wires. The conductance of single adenine-stacked RNA:DNA hybrids is rapidly and reproducibly measured using the break junction approach. The conductance decreases slightly over a large range of molecular lengths, suggesting that RNA:DNA can be used as an oligonucleotide wire. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. The History of the Discovery of the Molybdenum Cofactor and Novel Aspects of its Biosynthesis in Bacteria

    PubMed Central

    Leimkühler, Silke; Wuebbens, Margot M.; Rajagopalan, K.V.

    2010-01-01

    Biosynthesis of the molybdenum cofactor in bacteria is described with a detailed analysis of each individual reaction leading to the formation of stable intermediates during the synthesis of molybdopterin from GTP. As a starting point, the discovery of molybdopterin and the elucidation of its structure through the study of stable degradation products are described. Subsequent to molybdopterin synthesis, the molybdenum atom is added to the molybdopterin dithiolene group to form the molybdenum cofactor. This cofactor is either inserted directly into specific molybdoenzymes or is further modified by the addition of nucleotides to the molybdopterin phosphate group or the replacement of ligands at the molybdenum center. PMID:21528011

  20. NifX and NifEN exchange NifB cofactor and the VK-cluster, a newly isolated intermediate of the iron-molybdenum cofactor biosynthetic pathway.

    PubMed

    Hernandez, Jose A; Igarashi, Robert Y; Soboh, Basem; Curatti, Leonardo; Dean, Dennis R; Ludden, Paul W; Rubio, Luis M

    2007-01-01

    The iron-molybdenum cofactor of nitrogenase (FeMo-co) is synthesized in a multistep process catalysed by several Nif proteins and is finally inserted into a pre-synthesized apo-dinitrogenase to generate mature dinitrogenase protein. The NifEN complex serves as scaffold for some steps of this synthesis, while NifX belongs to a family of small proteins that bind either FeMo-co precursors or FeMo-co during cofactor synthesis. In this work, the binding of FeMo-co precursors and their transfer between purified Azotobacter vinelandii NifX and NifEN proteins was studied to shed light on the role of NifX on FeMo-co synthesis. Purified NifX binds NifB cofactor (NifB-co), a precursor to FeMo-co, with high affinity and is able to transfer it to the NifEN complex. In addition, NifEN and NifX exchange another [Fe-S] cluster that serves as a FeMo-co precursor, and we have designated it as the VK-cluster. In contrast to NifB-co, the VK-cluster is electronic paramagnetic resonance (EPR)-active in the reduced and the oxidized states. The NifX/VK-cluster complex is unable to support in vitro FeMo-co synthesis in the absence of NifEN because further processing of the VK-cluster into FeMo-co requires the simultaneous activities of NifEN and NifH. Our in vitro studies suggest that the role of NifX in vivo is to serve as transient reservoir of FeMo-co precursors and thus help control their flux during FeMo-co synthesis.

  1. Lack of hepcidin ameliorates anemia and improves growth in an adenine-induced mouse model of chronic kidney disease

    PubMed Central

    Sureshbabu, Angara; Doty, Steve B.; Zhu, Yuan-Shan; Patino, Edwin; Cunningham-Rundles, Susanna; Choi, Mary E.; Boskey, Adele; Rivella, Stefano

    2016-01-01

    Growth delay is common in children with chronic kidney disease (CKD), often associated with poor quality of life. The role of anemia in uremic growth delay is poorly understood. Here we describe an induction of uremic growth retardation by a 0.2% adenine diet in wild-type (WT) and hepcidin gene (Hamp) knockout (KO) mice, compared with their respective littermates fed a regular diet. Experiments were started at weaning (3 wk). After 8 wk, blood was collected and mice were euthanized. Adenine-fed WT mice developed CKD (blood urea nitrogen 82.8 ± 11.6 mg/dl and creatinine 0.57 ± 0.07 mg/dl) and were 2.1 cm shorter compared with WT controls. WT adenine-fed mice were anemic and had low serum iron, elevated Hamp, and elevated IL6 and TNF-α. WT adenine-fed mice had advanced mineral bone disease (serum phosphorus 16.9 ± 3.1 mg/dl and FGF23 204.0 ± 115.0 ng/ml) with loss of cortical and trabecular bone volume seen on microcomputed tomography. Hamp disruption rescued the anemia phenotype resulting in improved growth rate in mice with CKD, thus providing direct experimental evidence of the relationship between Hamp pathway and growth impairment in CKD. Hamp disruption ameliorated CKD-induced growth hormone-insulin-like growth factor 1 axis derangements and growth plate alterations. Disruption of Hamp did not mitigate the development of uremia, inflammation, and mineral and bone disease in this model. Taken together, these results indicate that an adenine diet can be successfully used to study growth in mice with CKD. Hepcidin appears to be related to pathways of growth retardation in CKD suggesting that investigation of hepcidin-lowering therapies in juvenile CKD is warranted. PMID:27440777

  2. Deproteinization is Necessary for the Accurate Determination of Ammonia Levels by Glutamate Dehydrogenase Assay in Blood Plasma From Subjects With Liver Injury.

    PubMed

    Vodenicarovova, Melita; Skalska, Hana; Holecek, Milan

    2017-11-08

    To determine the effect of presence of high concentrations of nicotinamide adenine dinucleotide (NADH)- and nicotinamide adenine dinucleotide phosphate (NADPH)-consuming enzymes on the accuracy of glutamate dehydrogenase (GLDH) assay for ammonia. We measured ammonia concentrations using GLDH and NADH or NADPH in blood-plasma specimens and specimens deproteinized by sulfosalicylic acid from CCl4-treated or control rats. The nonspecific oxidation of NADH and NADPH was measured in mixtures without GLDH. We observed a gradual decrease (~0.5%) in absorbance in the plasma of controls after the addition of NADH but not after adding NADPH. The decrease in absorbance in plasma of CCl4-treated animals was 13.2% and 5.2% after the addition of NADH and NADPH, respectively. The decrease in absorbance was not detected in deproteinized specimens. The values of ammonia concentration were higher in the plasma specimens compared with the deproteinized ones. Deproteinization is necessary for accurate measurement of ammonia using GLDH assay in the blood plasma of subjects with liver injury. © American Society for Clinical Pathology, 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

  3. A continuous spectrophotometric assay for monitoring adenosine 5'-monophosphate production.

    PubMed

    First, Eric A

    2015-08-15

    A number of biologically important enzymes release adenosine 5'-monophosphate (AMP) as a product, including aminoacyl-tRNA synthetases, cyclic AMP (cAMP) phosphodiesterases, ubiquitin and ubiquitin-like ligases, DNA ligases, coenzyme A (CoA) ligases, polyA deadenylases, and ribonucleases. In contrast to the abundance of assays available for monitoring the conversion of adenosine 5'-triphosphate (ATP) to ADP, there are relatively few assays for monitoring the conversion of ATP (or cAMP) to AMP. In this article, we describe a homogeneous assay that continuously monitors the production of AMP. Specifically, we have coupled the conversion of AMP to inosine 5'-monophosphate (IMP) (by AMP deaminase) to the oxidation of IMP (by IMP dehydrogenase). This results in the reduction of oxidized nicotine adenine dinucleotide (NAD(+)) to reduced nicotine adenine dinucleotide (NADH), allowing AMP formation to be monitored by the change in the absorbance at 340 nm. Changes in AMP concentrations of 5 μM or more can be reliably detected. The ease of use and relatively low expense make the AMP assay suitable for both high-throughput screening and kinetic analyses. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Evaluation of actinic cheilitis using fluorescence lifetime spectroscopy

    NASA Astrophysics Data System (ADS)

    Saito Nogueira, Marcelo; Cosci, Alessandro; Pratavieira, Sebastião.; Takahama, Ademar; Souza Azevedo, Rebeca; Kurachi, Cristina

    2016-03-01

    Actinic cheilitis is a potentially malignant disorder that mostly affects the vermilion border of the lower lip and can lead to squamous cell carcinoma. Because of its heterogeneous clinical aspect, it is difficult to indicate representative biopsy area. Late diagnosis is a limiting factor of therapeutic possibilities available to treat oral cancer. The diagnosis of actinic cheilitis is mainly based on clinical and histopathological analysis and it is a time consuming procedure to get the results. Information about the organization and chemical composition of the tissues can be obtained using fluorescence lifetime spectroscopy techniques without the need for biopsy. The main targeted fluorophores are NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), which have free and bound states, each one with different average lifetimes. The average lifetimes for free and bound NADH and FAD change according to tissue metabolic alterations and allow a quick and non-invasive clinical investigation of injuries and to help clinicians with the early diagnosis of actinic cheilitis. This study aims to evaluate the fluorescence lifetime parameters at the discrimination of three degrees of epithelial dysplasia, the most important predictor of malignant development, described in up to 100% of actinic cheilitis cases.

  5. Mapping absolute tissue endogenous fluorophore concentrations with chemometric wide-field fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Xu, Zhang; Reilley, Michael; Li, Run; Xu, Min

    2017-06-01

    We report chemometric wide-field fluorescence microscopy for imaging the spatial distribution and concentration of endogenous fluorophores in thin tissue sections. Nonnegative factorization aided by spatial diversity is used to learn both the spectral signature and the spatial distribution of endogenous fluorophores from microscopic fluorescence color images obtained under broadband excitation and detection. The absolute concentration map of individual fluorophores is derived by comparing the fluorescence from "pure" fluorophores under the identical imaging condition following the identification of the fluorescence species by its spectral signature. This method is then demonstrated by characterizing the concentration map of endogenous fluorophores (including tryptophan, elastin, nicotinamide adenine dinucleotide, and flavin adenine dinucleotide) for lung tissue specimens. The absolute concentrations of these fluorophores are all found to decrease significantly from normal, perilesional, to cancerous (squamous cell carcinoma) tissue. Discriminating tissue types using the absolute fluorophore concentration is found to be significantly more accurate than that achievable with the relative fluorescence strength. Quantification of fluorophores in terms of the absolute concentration map is also advantageous in eliminating the uncertainties due to system responses or measurement details, yielding more biologically relevant data, and simplifying the assessment of competing imaging approaches.

  6. In vitro characterization of the NAD+ synthetase NadE1 from Herbaspirillum seropedicae.

    PubMed

    Laskoski, Kerly; Santos, Adrian R S; Bonatto, Ana C; Pedrosa, Fábio O; Souza, Emanuel M; Huergo, Luciano F

    2016-05-01

    Nicotinamide adenine dinucleotide synthetase enzyme (NadE) catalyzes the amination of nicotinic acid adenine dinucleotide (NaAD) to form NAD(+). This reaction represents the last step in the majority of the NAD(+) biosynthetic routes described to date. NadE enzymes typically use either glutamine or ammonium as amine nitrogen donor, and the reaction is energetically driven by ATP hydrolysis. Given the key role of NAD(+) in bacterial metabolism, NadE has attracted considerable interest as a potential target for the development of novel antibiotics. The plant-associative nitrogen-fixing bacteria Herbaspirillum seropedicae encodes two putative NadE, namely nadE1 and nadE2. The nadE1 gene is linked to glnB encoding the signal transduction protein GlnB. Here we report the purification and in vitro characterization of H. seropedicae NadE1. Gel filtration chromatography analysis suggests that NadE1 is an octamer. The NadE1 activity was assayed in vitro, and the Michaelis-Menten constants for substrates NaAD, ATP, glutamine and ammonium were determined. Enzyme kinetic and in vitro substrate competition assays indicate that H. seropedicae NadE1 uses glutamine as a preferential nitrogen donor.

  7. The impact of aging, hearing loss, and body weight on mouse hippocampal redox state, measured in brain slices using fluorescence imaging.

    PubMed

    Stebbings, Kevin A; Choi, Hyun W; Ravindra, Aditya; Llano, Daniel Adolfo

    2016-06-01

    The relationships between oxidative stress in the hippocampus and other aging-related changes such as hearing loss, cortical thinning, or changes in body weight are not yet known. We measured the redox ratio in a number of neural structures in brain slices taken from young and aged mice. Hearing thresholds, body weight, and cortical thickness were also measured. We found striking aging-related increases in the redox ratio that were isolated to the stratum pyramidale, while such changes were not observed in thalamus or cortex. These changes were driven primarily by changes in flavin adenine dinucleotide, not nicotinamide adenine dinucleotide hydride. Multiple regression analysis suggested that neither hearing threshold nor cortical thickness independently contributed to this change in hippocampal redox ratio. However, body weight did independently contribute to predicted changes in hippocampal redox ratio. These data suggest that aging-related changes in hippocampal redox ratio are not a general reflection of overall brain oxidative state but are highly localized, while still being related to at least one marker of late aging, weight loss at the end of life. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Primitive Photosynthetic Architectures Based on Self-Organization and Chemical Evolution of Amino Acids and Metal Ions.

    PubMed

    Liu, Kai; Ren, Xiaokang; Sun, Jianxuan; Zou, Qianli; Yan, Xuehai

    2018-06-01

    The emergence of light-energy-utilizing metabolism is likely to be a critical milestone in prebiotic chemistry and the origin of life. However, how the primitive pigment is spontaneously generated still remains unknown. Herein, a primitive pigment model based on adaptive self-organization of amino acids (Cystine, Cys) and metal ions (zinc ion, Zn 2+ ) followed by chemical evolution under hydrothermal conditions is developed. The resulting hybrid microspheres are composed of radially aligned cystine/zinc (Cys/Zn) assembly decorated with carbonate-doped zinc sulfide (C-ZnS) nanocrystals. The part of C-ZnS can work as a light-harvesting antenna to capture ultraviolet and visible light, and use it in various photochemical reactions, including hydrogen (H 2 ) evolution, carbon dioxide (CO 2 ) photoreduction, and reduction of nicotinamide adenine dinucleotide (NAD + ) to nicotinamide adenine dinucleotide hydride (NADH). Additionally, guest molecules (e.g., glutamate dehydrogenase, GDH) can be encapsulated within the hierarchical Cys/Zn framework, which facilitates sustainable photoenzymatic synthesis of glutamate. This study helps deepen insight into the emergent functionality (conversion of light energy) and complexity (hierarchical architecture) from interaction and reaction of prebiotic molecules. The primitive pigment model is also promising to work as an artificial photosynthetic microreactor.

  9. Catabolism of phloroglucinol by the rumen anaerobe coprococcus.

    PubMed

    Patel, T R; Jure, K G; Jones, G A

    1981-12-01

    A rumen isolate, Coprococcus, sp. Pe(1)5, was found to carry phloroglucinol reductase, which catalyzed the initial step in the breakdown of phloroglucinol. The organism uses phloroglucinol as the sole source of carbon and energy when grown in the absence of oxygen. Induced levels of enzyme were detected in cells grown either on phloroglucinol or on other carbon sources in the presence of limiting quantities of phloroglucinol. Although the organism is a strict anaerobe, the enzyme from anaerobically grown cells was insensitive to air. The partially purified enzyme required reduced nicotinamide adenine dinucleotide phosphate as an electron donor and was specific for phloroglucinol. However, partial enzyme activity (14 to 17%) was also detected in the presence of 2-methyl-1,4-naphthoquinone but not in the presence of several other phenolic compounds. The enzyme exhibited a higher affinity for phloroglucinol than for reduced nicotinamide adenine dinucleotide phosphate, with K(m) values of 3.0 x 10 M and 29.0 x 10 M, respectively. The optimum pH for maximal enzyme activity was 7.4, and the molecular weight of the native protein was about 130,000, as determined by the Sephadex gel filtration technique.

  10. Electrochemical Detection of the Molecules of Life

    NASA Technical Reports Server (NTRS)

    Thomson, Seamus; Quinn, Richard; Koehne, Jessica

    2017-01-01

    All forms of life on Earth contain cellular machinery that can transform and regulate chemical energy through metabolic pathways. These processes are oxidation-reduction reactions that are performed by four key classes of molecules: flavins, nicotinamaides, porphyrins, and quinones. By detecting the electrochemical interaction of these redox-active molecules with an electrode, a method of differentiating them by their class could be established and incorporated into future life-detecting missions. This body of work investigates the electrochemistry of ubiquitous molecules found in life and how they may be detected. Molecules can oxidise or reduce the surface of an electrode - giving or receiving electrons - and these interactions are represented by changes in current with respect to an applied voltage. This relationship varies with: electrolyte type and concentration, working electrode material, the redox-active molecule itself, and scan rate. Flavin adenine dinucleotide (FAD), riboflavin, nicotinamide adenine dinucleotide (NADH), and anthraquinone are all molecules found intracellularly in almost all living organisms. An organism-synthesised extracellular redox-active molecule, Plumbagin, was also selected as part of this study. The goal of this work is to detect these molecules in seawater and assess its application in searching for life on Ocean Worlds.

  11. Cofactor-binding sites in proteins of deviating sequence: comparative analysis and clustering in torsion angle, cavity, and fold space.

    PubMed

    Stegemann, Björn; Klebe, Gerhard

    2012-02-01

    Small molecules are recognized in protein-binding pockets through surface-exposed physicochemical properties. To optimize binding, they have to adopt a conformation corresponding to a local energy minimum within the formed protein-ligand complex. However, their conformational flexibility makes them competent to bind not only to homologous proteins of the same family but also to proteins of remote similarity with respect to the shape of the binding pockets and folding pattern. Considering drug action, such observations can give rise to unexpected and undesired cross reactivity. In this study, datasets of six different cofactors (ADP, ATP, NAD(P)(H), FAD, and acetyl CoA, sharing an adenosine diphosphate moiety as common substructure), observed in multiple crystal structures of protein-cofactor complexes exhibiting sequence identity below 25%, have been analyzed for the conformational properties of the bound ligands, the distribution of physicochemical properties in the accommodating protein-binding pockets, and the local folding patterns next to the cofactor-binding site. State-of-the-art clustering techniques have been applied to group the different protein-cofactor complexes in the different spaces. Interestingly, clustering in cavity (Cavbase) and fold space (DALI) reveals virtually the same data structuring. Remarkable relationships can be found among the different spaces. They provide information on how conformations are conserved across the host proteins and which distinct local cavity and fold motifs recognize the different portions of the cofactors. In those cases, where different cofactors are found to be accommodated in a similar fashion to the same fold motifs, only a commonly shared substructure of the cofactors is used for the recognition process. Copyright © 2011 Wiley Periodicals, Inc.

  12. Energy level alignment at the interfaces between typical electrodes and nucleobases: Al/adenine/indium-tin-oxide and Al/thymine/indium-tin-oxide

    NASA Astrophysics Data System (ADS)

    Lee, Younjoo; Lee, Hyunbok; Park, Soohyung; Yi, Yeonjin

    2012-12-01

    We investigated the interfacial electronic structures of Al/adenine/indium-tin-oxide (ITO) and Al/thymine/ITO using in situ ultraviolet and x-ray photoemission spectroscopy and density functional theory calculations. Adenine shows both an interface dipole and level bending, whereas thymine shows only an interface dipole in contact with ITO. In addition, thymine possesses a larger ionization energy than adenine. These are understood with delocalized π states confirmed with theoretical calculations. For the interface between nucleobases and Al, both nucleobases show a prominent reduction of the electron injection barrier from Al to each base in accordance with a downward level shift.

  13. The Carboxy-Terminal Domain of Hsc70 Provides Binding Sites for a Distinct Set of Chaperone Cofactors

    PubMed Central

    Demand, Jens; Lüders, Jens; Höhfeld, Jörg

    1998-01-01

    The modulation of the chaperone activity of the heat shock cognate Hsc70 protein in mammalian cells involves cooperation with chaperone cofactors, such as Hsp40; BAG-1; the Hsc70-interacting protein, Hip; and the Hsc70-Hsp90-organizing protein, Hop. By employing the yeast two-hybrid system and in vitro interaction assays, we have provided insight into the structural basis that underlies Hsc70’s cooperation with different cofactors. The carboxy-terminal domain of Hsc70, previously shown to form a lid over the peptide binding pocket of the chaperone protein, mediates the interaction of Hsc70 with Hsp40 and Hop. Remarkably, the two cofactors bind to the carboxy terminus of Hsc70 in a noncompetitive manner, revealing the existence of distinct binding sites for Hsp40 and Hop within this domain. In contrast, Hip interacts exclusively with the amino-terminal ATPase domain of Hsc70. Hence, Hsc70 possesses separate nonoverlapping binding sites for Hsp40, Hip, and Hop. This appears to enable the chaperone protein to cooperate simultaneously with multiple cofactors. On the other hand, BAG-1 and Hip have recently been shown to compete in binding to the ATPase domain. Our data thus establish the existence of a network of cooperating and competing cofactors regulating the chaperone activity of Hsc70 in the mammalian cell. PMID:9528774

  14. Adenine-functionalized Spongy Graphene for Green and High-Performance Supercapacitors

    PubMed Central

    El-Gendy, Dalia M.; Ghany, Nabil A. Abdel; El Sherbini, E. E. Foad; Allam, Nageh K.

    2017-01-01

    A simple method is demonstrated to prepare spongy adenine-functionalized graphene (SFG) as interconnected, porous 3-dimensional (3D) network crinkly sheets. Such 3D network structure provides better contact at the electrode/electrolyte interface and facilitates the charge transfer kinetics. The fabricated SFG was characterized by X-ray diffraction (XRD), FTIR, scanning electron microscopy (FESEM), Raman spectroscopy, thermogravimetric analysis (TGA), UV−vis absorption spectroscopy, and transmission electron microscopy (TEM). The synthesized materials have been evaluated as supercapacitor materials in 0.5 M H2SO4 using cyclic voltammetry (CV) at different potential scan rates, and galvanostatic charge/discharge tests at different current densities. The SFG electrodes showed a maximum specific capacitance of 333 F/g at scan rate of 1 mV/s and exhibited excellent cycling retention of 102% after 1000 cycles at 200 mV/s. The energy density was 64.42 Wh/kg with a power density of 599.8 W/kg at 1.0 A/g. Those figures of merit are much higher than those reported for graphene-based materials tested under similar conditions. The observed high performance can be related to the synergistic effects of the spongy structure and the adenine functionalization. PMID:28216668

  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. Effect of aqueous extract and anthocyanins of calyces of Hibiscus sabdariffa (Malvaceae) in rats with adenine-induced chronic kidney disease.

    PubMed

    Ali, Badreldin H; Cahliková, Lucie; Opletal, Lubomir; Karaca, Turan; Manoj, Priyadarsini; Ramkumar, Aishwarya; Al Suleimani, Yousuf M; Al Za'abi, Mohammed; Nemmar, Abderrahim; Chocholousova-Havlikova, Lucie; Locarek, Miroslav; Siatka, Tomas; Blunden, Gerald

    2017-09-01

    The aim of this work was to assess the possible beneficial effects of aqueous extracts of Hibiscus sabdariffa L. calyces and anthocyanins isolated therefrom in an adenine-induced chronic kidney disease (CKD) model. Rats were orally given, for 28 consecutive days, either adenine alone or together with either aqueous extract of H. sabdariffa calyces (5 and 10%) or anthocyanins (50, 100 and 200 mg/kg of anthocyanin concentrate). For comparative purposes, two groups of rats were given lisinopril (10 mg/kg). When either H. sabdariffa aqueous extract or the anthocyanins isolated from it was administered along with adenine, the adverse effects of adenine-induced CKD were significantly lessened, mostly in a dose-dependent manner. The positive effects were similar to those obtained by administration of lisinopril. The results obtained show that both H. sabdariffa and its anthocyanins could be considered as possible promising safe dietary agents that could be used to attenuate the progression of human CKD. This could have added significance as H. sabdariffa tea is widely consumed in many parts of Africa and Asia and is thus readily available. © 2017 Royal Pharmaceutical Society.

  17. The superfamily keeps growing: Identification in trypanosomatids of RibJ, the first riboflavin transporter family in protists.

    PubMed

    Balcazar, Darío E; Vanrell, María Cristina; Romano, Patricia S; Pereira, Claudio A; Goldbaum, Fernando A; Bonomi, Hernán R; Carrillo, Carolina

    2017-04-01

    Trypanosomatid parasites represent a major health issue affecting hundreds of million people worldwide, with clinical treatments that are partially effective and/or very toxic. They are responsible for serious human and plant diseases including Trypanosoma cruzi (Chagas disease), Trypanosoma brucei (Sleeping sickness), Leishmania spp. (Leishmaniasis), and Phytomonas spp. (phytoparasites). Both, animals and trypanosomatids lack the biosynthetic riboflavin (vitamin B2) pathway, the vital precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) cofactors. While metazoans obtain riboflavin from the diet through RFVT/SLC52 transporters, the riboflavin transport mechanisms in trypanosomatids still remain unknown. Here, we show that riboflavin is imported with high affinity in Trypanosoma cruzi, Trypanosoma brucei, Leishmania (Leishmania) mexicana, Crithidia fasciculata and Phytomonas Jma using radiolabeled riboflavin transport assays. The vitamin is incorporated through a saturable carrier-mediated process. Effective competitive uptake occurs with riboflavin analogs roseoflavin, lumiflavin and lumichrome, and co-factor derivatives FMN and FAD. Moreover, important biological processes evaluated in T. cruzi (i.e. proliferation, metacyclogenesis and amastigote replication) are dependent on riboflavin availability. In addition, the riboflavin competitive analogs were found to interfere with parasite physiology on riboflavin-dependent processes. By means of bioinformatics analyses we identified a novel family of riboflavin transporters (RibJ) in trypanosomatids. Two RibJ members, TcRibJ and TbRibJ from T. cruzi and T. brucei respectively, were functionally characterized using homologous and/or heterologous expression systems. The RibJ family represents the first riboflavin transporters found in protists and the third eukaryotic family known to date. The essentiality of riboflavin for trypanosomatids, and the structural/biochemical differences that RFVT

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Podzelinska, K.; Latimer, R; Bhattacharya, A

    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, themore » 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.« less

  19. The superfamily keeps growing: Identification in trypanosomatids of RibJ, the first riboflavin transporter family in protists

    PubMed Central

    Balcazar, Darío E.; Vanrell, María Cristina; Romano, Patricia S.; Pereira, Claudio A.; Goldbaum, Fernando A.; Bonomi, Hernán R.; Carrillo, Carolina

    2017-01-01

    Background Trypanosomatid parasites represent a major health issue affecting hundreds of million people worldwide, with clinical treatments that are partially effective and/or very toxic. They are responsible for serious human and plant diseases including Trypanosoma cruzi (Chagas disease), Trypanosoma brucei (Sleeping sickness), Leishmania spp. (Leishmaniasis), and Phytomonas spp. (phytoparasites). Both, animals and trypanosomatids lack the biosynthetic riboflavin (vitamin B2) pathway, the vital precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) cofactors. While metazoans obtain riboflavin from the diet through RFVT/SLC52 transporters, the riboflavin transport mechanisms in trypanosomatids still remain unknown. Methodology/Principal findings Here, we show that riboflavin is imported with high affinity in Trypanosoma cruzi, Trypanosoma brucei, Leishmania (Leishmania) mexicana, Crithidia fasciculata and Phytomonas Jma using radiolabeled riboflavin transport assays. The vitamin is incorporated through a saturable carrier-mediated process. Effective competitive uptake occurs with riboflavin analogs roseoflavin, lumiflavin and lumichrome, and co-factor derivatives FMN and FAD. Moreover, important biological processes evaluated in T. cruzi (i.e. proliferation, metacyclogenesis and amastigote replication) are dependent on riboflavin availability. In addition, the riboflavin competitive analogs were found to interfere with parasite physiology on riboflavin-dependent processes. By means of bioinformatics analyses we identified a novel family of riboflavin transporters (RibJ) in trypanosomatids. Two RibJ members, TcRibJ and TbRibJ from T. cruzi and T. brucei respectively, were functionally characterized using homologous and/or heterologous expression systems. Conclusions/Significance The RibJ family represents the first riboflavin transporters found in protists and the third eukaryotic family known to date. The essentiality of riboflavin for

  20. Membrane Topology Mapping of the Na+-Pumping NADH: Quinone Oxidoreductase from Vibrio cholerae by PhoA- Green Fluorescent Protein Fusion Analysis▿

    PubMed Central

    Duffy, Ellen B.; Barquera, Blanca

    2006-01-01

    The membrane topologies of the six subunits of Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae were determined by a combination of topology prediction algorithms and the construction of C-terminal fusions. Fusion expression vectors contained either bacterial alkaline phosphatase (phoA) or green fluorescent protein (gfp) genes as reporters of periplasmic and cytoplasmic localization, respectively. A majority of the topology prediction algorithms did not predict any transmembrane helices for NqrA. A lack of PhoA activity when fused to the C terminus of NqrA and the observed fluorescence of the green fluorescent protein C-terminal fusion confirm that this subunit is localized to the cytoplasmic side of the membrane. Analysis of four PhoA fusions for NqrB indicates that this subunit has nine transmembrane helices and that residue T236, the binding site for flavin mononucleotide (FMN), resides in the cytoplasm. Three fusions confirm that the topology of NqrC consists of two transmembrane helices with the FMN binding site at residue T225 on the cytoplasmic side. Fusion analysis of NqrD and NqrE showed almost mirror image topologies, each consisting of six transmembrane helices; the results for NqrD and NqrE are consistent with the topologies of Escherichia coli homologs YdgQ and YdgL, respectively. The NADH, flavin adenine dinucleotide, and Fe-S center binding sites of NqrF were localized to the cytoplasm. The determination of the topologies of the subunits of Na+-NQR provides valuable insights into the location of cofactors and identifies targets for mutagenesis to characterize this enzyme in more detail. The finding that all the redox cofactors are localized to the cytoplasmic side of the membrane is discussed. PMID:17041063

  1. Tracking gene expression and oxidative damage of O2-stressed Clostridioides difficile by a multi-omics approach.

    PubMed

    Neumann-Schaal, Meina; Metzendorf, Nicole G; Troitzsch, Daniel; Nuss, Aaron Mischa; Hofmann, Julia Danielle; Beckstette, Michael; Dersch, Petra; Otto, Andreas; Sievers, Susanne

    2018-05-31

    Clostridioides difficile is the major pathogen causing diarrhea following antibiotic treatment. It is considered to be a strictly anaerobic bacterium, however, previous studies have shown a certain and strain-dependent oxygen tolerance. In this study, the model strain C. difficile 630Δerm was shifted to micro-aerobiosis and was found to stay growing to the same extent as anaerobically growing cells with only few changes in the metabolite pattern. However, an extensive change in gene expression was determined by RNA-Seq. The most striking adaptation strategies involve a change in the reductive fermentation pathways of the amino acids proline, glycine and leucine. But also a far-reaching restructuring in the carbohydrate metabolism was detected with changes in the phosphotransferase system (PTS) facilitated uptake of sugars and a repression of enzymes of glycolysis and butyrate fermentation. Furthermore, a temporary induction in the synthesis of cofactor riboflavin was detected possibly due to an increased demand for flavin mononucleotid (FMN) and flavin adenine dinucleotide (FAD) in redox reactions. However, biosynthesis of the cofactors thiamin pyrophosphate and cobalamin were repressed deducing oxidation-prone enzymes and intermediates in these pathways. Micro-aerobically shocked cells were characterized by an increased demand for cysteine and a thiol redox proteomics approach revealed a dramatic increase in the oxidative state of cysteine in more than 800 peptides after 15 min of micro-aerobic shock. This provides not only a catalogue of oxidation-prone cysteine residues in the C. difficile proteome but also puts the amino acid cysteine into a key position in the oxidative stress response. Our study suggests that tolerance of C. difficile towards O 2 is based on a complex and far-reaching adjustment of global gene expression which leads to only a slight change in phenotype. Copyright © 2018. Published by Elsevier Ltd.

  2. STUDIES ON MAMMALIAN AND HUMAN PYRUVATE AND ALPHA-KETOGLUTARATE DEHYDROGENATION COMPLEXES.

    DTIC Science & Technology

    Enzyme systems that catalyze a coenzyme A- and nicotinamide adenine dinucleotide-linked oxidative decarboxylation of pyruvate and alpha - ketoglutarate ...The pig heart pyruvate dehydrogenase complex was strongly inhibited by EDTA at low concentration, but the pig heart alpha - ketoglutarate ...On the oxidative decarboxylation of alpha -keto acids in pig heart complexes, Ca(2+) was strongly stimulatory to the same or more extent than Mg(2

  3. Death by Protein Damage in Irradiated Cells

    DTIC Science & Technology

    2011-01-01

    tardigrades, J. Exp. Biol. 212 (2009) 4033– 4039. [63] A. Oren, N. Gunde-Cimerman, Mycosporines and mycosporine - like amino acids : UV protectants or...catalytically remove superoxide via a disproportionation mechanism [29,46]; and amino acids and peptides, which scav- enge hydroxyl radicals very efficiently...most radiation resistant mutants of B. pumilus displayed mul- tiple amino acid auxotrophies and a requirement for nicotinamide adenine dinucleotide

  4. Anthocyanin copigmentation and color of wine: The effect of naturally obtained hydroxycinnamic acids as cofactors.

    PubMed

    Bimpilas, Andreas; Panagopoulou, Marilena; Tsimogiannis, Dimitrios; Oreopoulou, Vassiliki

    2016-04-15

    Copigmentation of anthocyanins accounts for over 30% of fresh red wine color, while during storage, the color of polymeric pigments formed between anthocyanins and proanthocyanidins predominates. Rosmarinic acid and natural extracts rich in hydroxycinnamic acids, obtained from aromatic plants (Origanum vulgare and Satureja thymbra), were examined as cofactors to fresh Merlot wine and the effect on anthocyanin copigmentation and wine color was studied during storage for 6months. An increase of the copigmented anthocyanins that enhanced color intensity by 15-50% was observed, confirming the ability of complex hydroxycinnamates to form copigments. The samples with added cofactors retained higher percentages of copigmented anthocyanins and higher color intensity, compared to the control wine, up to 3 months. However, the change in the equilibrium between monomeric and copigmented anthocyanins that was induced by added cofactors, did not affect the rate of polymerization reactions during storage. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Understanding the Sequence Preference of Recurrent RNA Building Blocks using Quantum Chemistry: The Intrastrand RNA Dinucleotide Platform.

    PubMed

    Mládek, Arnošt; Sponer, Judit E; Kulhánek, Petr; Lu, Xiang-Jun; Olson, Wilma K; Sponer, Jiřĺ

    2012-01-10

    Folded RNA molecules are shaped by an astonishing variety of highly conserved noncanonical molecular interactions and backbone topologies. The dinucleotide platform is a widespread recurrent RNA modular building submotif formed by the side-by-side pairing of bases from two consecutive nucleotides within a single strand, with highly specific sequence preferences. This unique arrangement of bases is cemented by an intricate network of noncanonical hydrogen bonds and facilitated by a distinctive backbone topology. The present study investigates the gas-phase intrinsic stabilities of the three most common RNA dinucleotide platforms - 5'-GpU-3', ApA, and UpC - via state-of-the-art quantum-chemical (QM) techniques. The mean stability of base-base interactions decreases with sequence in the order GpU > ApA > UpC. Bader's atoms-in-molecules analysis reveals that the N2(G)…O4(U) hydrogen bond of the GpU platform is stronger than the corresponding hydrogen bonds in the other two platforms. The mixed-pucker sugar-phosphate backbone conformation found in most GpU platforms, in which the 5'-ribose sugar (G) is in the C2'-endo form and the 3'-sugar (U) in the C3'-endo form, is intrinsically more stable than the standard A-RNA backbone arrangement, partially as a result of a favorable O2'…O2P intra-platform interaction. Our results thus validate the hypothesis of Lu et al. (Lu Xiang-Jun, et al. Nucleic Acids Res. 2010, 38, 4868-4876), that the superior stability of GpU platforms is partially mediated by the strong O2'…O2P hydrogen bond. In contrast, ApA and especially UpC platform-compatible backbone conformations are rather diverse and do not display any characteristic structural features. The average stabilities of ApA and UpC derived backbone conformers are also lower than those of GpU platforms. Thus, the observed structural and evolutionary patterns of the dinucleotide platforms can be accounted for, to a large extent, by their intrinsic properties as described by

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

    PubMed Central

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

    2015-01-01

    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 a broad spectrum of biological activities, among which energetic metabolism and chromatin remodeling. Subcellular localisation of FAD synthase (EC 2.7.7.2, FADS), the second enzyme in the FAD forming pathway, is addressed here in HepG2 cells by confocal microscopy, in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalyzed 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 synthesizing 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 lysine-specific demethylase 1 (LSD1) or a mitochondrial dimethylglycine dehydrogenase (Me2GlyDH, EC 1.5.8.4). Both enzymes carry out similar reactions of oxidative demethylation, in which tetrahydrofolate is converted into 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. PMID:25954742

  7. Gum acacia mitigates genetic damage in adenine-induced chronic renal failure in rats.

    PubMed

    Ali, B H; Al Balushi, K; Al-Husseini, I; Mandel, P; Nemmar, A; Schupp, N; Ribeiro, D A

    2015-12-01

    Subjects with chronic renal failure (CRF) exhibit oxidative genome damage, which may predispose to carcinogenesis, and Gum acacia (GumA) ameliorates this condition in humans and animals. We evaluated here renal DNA damage and urinary excretion of four nucleic acid oxidation adducts namely 8-oxoguanine (8-oxoGua), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxoguanosine (8-oxoGuo) and 8-hydroxy-2-deoxyguanisone (8-OHdg) in rats with adenine (ADE)-induced CRF with and without GumA treatment. Twenty-four rats were divided into four equal groups and treated for 4 weeks. The first group was given normal food and water (control). The second group was given normal food and GumA (15% w/v) in drinking water. The third group was fed powder diet containing adenine (ADE) (0·75% w/w in feed). The fourth group was fed like in the third group, plus GumA in drinking water (15%, w/v). ADE feeding induced CRF (as measured by several physiological, biochemical and histological indices) and also caused a significant genetic damage and significant decreases in urinary 8-oxo Gua and 8-oxoGuo, but not in the other nucleic acids. However, concomitant GumA treatment reduced the level of genetic damage in kidney cells as detected by Comet assay and significantly reversed the effect of adenine on urinary 8-oxoGuo. Treatment with GumA is able to mitigate genetic damage in renal tissues of rats with ADE-induced CRF. © 2015 Stichting European Society for Clinical Investigation Journal Foundation.

  8. Evidence of the Importance of Nox4 in Production of Hypertension in Dahl Salt-Sensitive Rats.

    PubMed

    Cowley, Allen W; Yang, Chun; Zheleznova, Nadezhda N; Staruschenko, Alexander; Kurth, Theresa; Rein, Lisa; Kumar, Vikash; Sadovnikov, Katherine; Dayton, Alex; Hoffman, Matthew; Ryan, Robert P; Skelton, Meredith M; Salehpour, Fahimeh; Ranji, Mahsa; Geurts, Aron

    2016-02-01

    This study reports the consequences of knocking out NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 4 (Nox4) on the development of hypertension and kidney injury in the Dahl salt-sensitive (SS) rat. Zinc finger nuclease injection of single-cell SS embryos was used to create an 8 base-pair frame-shift deletion of Nox4, resulting in a loss of the ≈68 kDa band in Western blot analysis of renal cortical tissue of the knock out of Nox4 in the SS rat (SS(Nox4-/-)) rats. SS(Nox4-/-) rats exhibited a significant reduction of salt-induced hypertension compared with SS rats after 21 days of 4.0% NaCl diet (134±5 versus 151±3 mm Hg in SS) and a significant reduction of albuminuria, tubular casts, and glomerular injury. Optical fluorescence 3-dimensional cryoimaging revealed significantly higher redox ratios (NADH/FAD [reduced nicotinamide adenine dinucleotide/flavin adenine dinucleotide]) in the kidneys of SS(Nox4-/-) rats even when fed the 0.4% NaCl diet, indicating greater levels of mitochondrial electron transport chain metabolic activity and reduced oxidative stress compared with SS rats. Before the development of hypertension, RNA expression levels of Nox subunits Nox2, p67(phox), and p22(phox) were found to be significantly lower (P<0.05) in SS(Nox4-/-) compared with SS rats in the renal cortex. Thus, the mutation of Nox4 seems to modify transcription of several genes in ways that contribute to the protective effects observed in the SS(Nox4-/-) rats. We conclude that the reduced renal injury and attenuated blood pressure response to high salt in the SS(Nox4-/-) rat could be the result of multiple pathways, including gene transcription, mitochondrial energetics, oxidative stress, and protein matrix production impacted by the knock out of Nox4. © 2015 American Heart Association, Inc.

  9. Probing adenine rings and backbone linkages using base specific isotope-edited Raman spectroscopy: application to group II intron ribozyme domain V.

    PubMed

    Chen, Yuanyuan; Eldho, Nadukkudy V; Dayie, T Kwaku; Carey, Paul R

    2010-04-27

    Raman difference spectroscopy is used to probe the properties of a 36-nt RNA molecule, "D5", which lies at the heart of the catalytic apparatus in group II introns. For D5 that has all of its adenine residues labeled with (13)C and (15)N and utilizing Raman difference spectroscopy, we identify the conformationally sensitive -C-O-P-O-C- stretching modes of the unlabeled bonds adjacent to adenine bases, as well as the adenine ring modes themselves. The phosphodiester modes can be assigned to individual adenine residues based on earlier NMR data. The effect of Mg(2+) binding was explored by analyzing the Raman difference spectra for [D5 + Mg(2+)] minus [D5 no Mg(2+)], for D5 unlabeled, or D5 labeled with (13)C/(15)N-enriched adenine. In both sets of data we assign differential features to G ring modes perturbed by Mg(2+) binding at the N7 position. In the A-labeled spectra we attribute a Raman differential near 1450 cm(-1) and changes of intensity at 1296 cm(-1) to Mg binding at the N7 position of adenine bases. The A and G bases involved in Mg(2+) binding again can be identified using earlier NMR results. For the unlabeled D5, a change in the C-O-P-O-C stretch profile at 811 cm(-1) upon magnesium binding is due to a "tightening up" (in the sense of a more rigid molecule with less dynamic interchange among competing ribose conformers) of the D5 structure. For adenine-labeled D5, small changes in the adenine backbone bond signatures in the 810-830 cm(-1) region suggest that small conformational changes occur in the tetraloop and bulge regions upon binding of Mg(2+). The PO(2)(-) stretching vibration, near 1100 cm(-1), from the nonbridging phosphate groups, probes the effect of Mg(2+)-hydrate inner-sphere interactions that cause an upshift. In turn, the upshift is modulated by the presence of monovalent cations since in the presence of Na(+) and Li(+) the upshift is 23 +/- 2 cm(-1) while in the presence of K(+) and Cs(+) it is 13 +/- 3 cm(-1), a finding that correlates

  10. Stability and reactivity of liposome-encapsulated formate dehydrogenase and cofactor system in carbon dioxide gas-liquid flow.

    PubMed

    Yoshimoto, Makoto; Yamashita, Takayuki; Yamashiro, Takuya

    2010-01-01

    Formate dehydrogenase from Candida boidinii (CbFDH) is potentially applicable in reduction of CO(2) through oxidation of cofactor NADH into NAD(+). For this, the CbFDH activity needs to be maintained under practical reaction conditions, such as CO(2) gas-liquid flow. In this work, CbFDH and cofactor were encapsulated in liposomes and the liposomal enzymes were characterized in an external loop airlift bubble column. The airlift was operated at 45 degrees C with N(2) or CO(2) as gas phase at the superficial gas velocity U(G) of 2.0 or 3.0 cm/s. The activities of liposomal CbFDH/cofactor systems were highly stable in the airlift regardless of the type of gas phase because liposome membranes prevented interactions of the encapsulated enzyme and cofactor molecules with the gas-liquid interface of bubbles. On the other hand, free CbFDH was deactivated in the airlift especially at high U(G) with CO(2) bubbles. The liposomal CbFDH/NADH could catalyze reduction of CO(2) in the airlift giving the fractional oxidation of the liposomal NADH of 23% at the reaction time of 360 min. The cofactor was kept inside liposomes during the reaction operation with less than 10% of leakage. All of the results obtained demonstrate that the liposomal CbFDH/NADH functions as a stable catalyst for reduction of CO(2) in the airlift. (c) 2010 American Institute of Chemical Engineers

  11. Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor.

    PubMed

    Spatzal, Thomas; Perez, Kathryn A; Howard, James B; Rees, Douglas C

    2015-12-16

    Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32-1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis.

  12. A live zebrafish-based screening system for human nuclear receptor ligand and cofactor discovery.

    PubMed

    Tiefenbach, Jens; Moll, Pamela R; Nelson, Meryl R; Hu, Chun; Baev, Lilia; Kislinger, Thomas; Krause, Henry M

    2010-03-22

    Nuclear receptors (NRs) belong to a superfamily of transcription factors that regulate numerous homeostatic, metabolic and reproductive processes. Taken together with their modulation by small lipophilic molecules, they also represent an important and successful class of drug targets. Although many NRs have been targeted successfully, the majority have not, and one third are still orphans. Here we report the development of an in vivo GFP-based reporter system suitable for monitoring NR activities in all cells and tissues using live zebrafish (Danio rerio). The human NR fusion proteins used also contain a new affinity tag cassette allowing the purification of receptors with bound molecules from responsive tissues. We show that these constructs 1) respond as expected to endogenous zebrafish hormones and cofactors, 2) facilitate efficient receptor and cofactor purification, 3) respond robustly to NR hormones and drugs and 4) yield readily quantifiable signals. Transgenic lines representing the majority of human NRs have been established and are available for the investigation of tissue- and isoform-specific ligands and cofactors.

  13. Protein Cofactors Are Essential for High-Affinity DNA Binding by the Nuclear Factor κB RelA Subunit.

    PubMed

    Mulero, Maria Carmen; Shahabi, Shandy; Ko, Myung Soo; Schiffer, Jamie M; Huang, De-Bin; Wang, Vivien Ya-Fan; Amaro, Rommie E; Huxford, Tom; Ghosh, Gourisankar

    2018-05-22

    Transcription activator proteins typically contain two functional domains: a DNA binding domain (DBD) that binds to DNA with sequence specificity and an activation domain (AD) whose established function is to recruit RNA polymerase. In this report, we show that purified recombinant nuclear factor κB (NF-κB) RelA dimers bind specific κB DNA sites with an affinity significantly lower than that of the same dimers from nuclear extracts of activated cells, suggesting that additional nuclear cofactors might facilitate DNA binding by the RelA dimers. Additionally, recombinant RelA binds DNA with relatively low affinity at a physiological salt concentration in vitro. The addition of p53 or RPS3 (ribosomal protein S3) increases RelA:DNA binding affinity 2- to >50-fold depending on the protein and ionic conditions. These cofactor proteins do not form stable ternary complexes, suggesting that they stabilize the RelA:DNA complex through dynamic interactions. Surprisingly, the RelA-DBD alone fails to bind DNA under the same solution conditions even in the presence of cofactors, suggesting an important role of the RelA-AD in DNA binding. Reduced RelA:DNA binding at a physiological ionic strength suggests that multiple cofactors might be acting simultaneously to mitigate the electrolyte effect and stabilize the RelA:DNA complex in vivo. Overall, our observations suggest that the RelA-AD and multiple cofactor proteins function cooperatively to prime the RelA-DBD and stabilize the RelA:DNA complex in cells. Our study provides a mechanism for nuclear cofactor proteins in NF-κB-dependent gene regulation.

  14. Streptozotocin Diabetes CORRELATION WITH EXTENT OF DEPRESSION OF PANCREATIC ISLET NICOTINAMIDE ADENINE DINUCLEOTIDE

    PubMed Central

    Anderson, Tom; Schein, Philip S.; McMenamin, Mary G.; Cooney, David A.

    1974-01-01

    The diabetogenic activity of streptozotocin has been correlated with a reduction in pyridine nucleotide synthesis in the mouse pancreatic islet. To determine the specificity of this reduction for diabetogenicity, a comparative study of streptozotocin, its cytotoxic moiety, 1-methyl-1-nitrosourea, and alloxan was performed. Streptozotocin administered intraperitoneally (i.p.) producd a dose-related reduction in islet NAD which was proportional to the degree of diabetogenicity. A diabetogenic dose, 200 mg/kg, attained a peak plasma N-nitroso intact streptozotocin concentration of 0.224 μmol/ml and reduced the mean islet NAD from a control of 0.78 to 0.15 pmol. At borderline, 150 mg/kg, and nondiabetogenic, 100 mg/kg, doses, plasma concentrations reached 0.161 and 0.136 μmol/ml, and NAD was 0.36 and 0.86 pmol/islet, respectively. 1-Methyl-1-nitrosourea, 100 mg/kg, attained a maximum N-nitroso intact 1-methyl-1-nitrosourea concentration of 0.162 μmol/ml and reduced the mean NAD to 0.58 pmol/islet, and was nondiabetogenic; 200 mg/kg attained a peak plasma concentration of 0.344 μmol/ml and depressed NAD to 0.38 pmol/islet, and was inconsistently diabetogenic. Islet NAD of 0.4 pmol/islet or greater is required for integrity of the beta cell. A diabetogenic dose of alloxan, 500 mg/kg, did not depress NAD, 0.85 pmol/islet, therefore confirming that its mechanism of diabetogenicity differs from that of streptozotocin. In vivo uptake of [methyl-14C]streptozotocin by islets was 3.8 times that of [methyl-14C]-1-methyl-1-nitrosourea, whereas uptake by the exocrine pancreas favored 1-methyl-1-nitrosourea over streptozotocin 2.4:1. The decreased islet uptake of 1-methyl-1-nitrosourea correlates with the 3.5 times increased molar dosage required to produce islet NAD depression comparable to that of streptozotocin, 150 mg/kg. These studies indicate that the glucose carrier of streptozotocin facilitates uptake of its cytotoxic group, 1-methyl-1-nitrosourea, into islets. PMID:4369217

  15. DNA adenine methylation of sams1 gene in symbiont-bearing Amoeba proteus.

    PubMed

    Jeon, Taeck J

    2008-10-01

    The expression of amoeba sams genes is switched from sams1 to sams2 when amoebae are infected with Legionella jeonii. To elucidate the mechanism for the inactivation of host sams1 gene by endosymbiotic bacteria, methylation states of the sams1 gene of D and xD amoebae was compared in this study. The sams1 gene of amoebae was methylated at an internal adenine residue of GATC site in symbiont-bearing xD amoebae but not in symbiont-free D amoebae, suggesting that the modification might have caused the inactivation of sams1 in xD amoebae. The sams1 gene of xD amoebae was inactivated at the transcriptional level. Analysis of DNA showed that adenine residues in L. jeonii sams were also methylated, implying that L. jeonii bacteria belong to a Dam methylase-positive strain. In addition, both SAM and Met appeared to act as negative regulators for the expression of sams1 whereas the expression of sams2 was not affected in amoebae.

  16. DNA adenine methylation modulates pathogenicity of Klebsiella pneumoniae genotype K1.

    PubMed

    Fang, Chi-Tai; Yi, Wen-Ching; Shun, Chia-Tung; Tsai, Shih-Feng

    2017-08-01

    Klebsiella pneumoniae genotype K1 is a highly virulent pathogen that causes liver abscess and metastatic endophthalmitis/meningitis. Whether its pathogenicity is controlled by DNA adenine methylase (Dam), an epigenetic regulator of bacterial virulence gene expression, is yet unknown. We aimed to study the role of DNA adenine methylation in the pathogenicity of K. pneumoniae genotype K1. We identified the dam gene in the prototype tissue-invasive strain (NTUH-K2044) of K. pneumoniae genotype K1, using the strain's complete genome sequence in GenBank. We constructed a dam - mutant and compared it with the wild type, in terms of in vitro serum resistance and in vivo BALB/cByl mice inoculation. Loss of Dam activity in the mutant was verified by MboI restriction digestion of the genomic DNA and a 1000-fold increase in spontaneous mutation rate. The dam mutant lost at least 68% of serum resistance when compared with the wild type (survival ratio at 1 hour: 2.6 ± 0.4 vs. 8.2 ± 1.9; at 2 hours: 3.9 ± 1.6 vs. 17.4 ± 3.6; p values < 0.05). Likewise, virulence to mice decreased by 40-fold in an intraperitoneal injection model [lethal dose, 50% (LD 50 ): 2 × 10 3 colony-forming units (CFUs) vs. 5 × 10 1 CFUs] and by sixfold in a gastric ingestion model (LD 50 : 3 × 10 4 CFUs vs. 5 × 10 3 CFUs). Attenuation of the dam mutant was not attributable to its growth rate, which was similar to that of the wild type. Our results support the view that DNA adenine methylation plays an important role in modulating the pathogenicity of K. pneumoniae genotype K1. The incomplete attenuation indicates the existence of other regulatory factors. Copyright © 2015. Published by Elsevier B.V.

  17. Efficacy of Adenine in the Treatment of Leukopenia and Neutropenia Associated with an Overdose of Antipsychotics or Discontinuation of Lithium Carbonate Administration: Three Case Studies.

    PubMed

    Tomita, Takashi; Goto, Hidekazu; Sumiya, Kenji; Yoshida, Tadashi; Tanaka, Katsuya; Kohda, Yukinao

    2016-11-30

    Because adenine is effective for managing cases of radiation-induced and drug-induced leukopenia, it may be effective in cases of antipsychotic-induced leukopenia and neutropenia. Here, we report our experience with patients with leukopenia and neutropenia caused by an antipsychotic overdose or discontinuation of lithium carbonate, in whom adenine administration ameliorated the white blood cell and neutrophil counts. The progress of patients suggests that adenine is effective in cases of leukopenia and neutropenia associated with lithium carbonate discontinuation and an antipsychotic overdose.

  18. Effects of low-molecular-weight-chitosan on the adenine-induced chronic renal failure rats in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Zhi, Xuan; Han, Baoqin; Sui, Xianxian; Hu, Rui; Liu, Wanshun

    2015-02-01

    The effects of low-molecular-weight-chitosan (LMWC) on chronic renal failure (CRF) rats induced by adenine were investigated in vivo and in vitro. Chitosan were hydrolyzed using chitosanase at pH 6-7 and 37° for 24 h to obtain LMWC. In vitro, the effect of LMWC on the proliferation of renal tubular epithelial cells (RTEC) showed that it had no cytotoxic effect and could promote cell growth. For the in vivo experiment, chronic renal failure rats induced by adenine were randomly divided into control group, Niaoduqing group, and high-, medium- and low-dose LMWC groups. For each group, we detected serum creatinine (SCR), blood urea nitrogen (BUN), and total superoxide dismutase (T-SOD), glutathione oxidase (GSH-Px) activities of renal tissue, and obtained the ratio of kidney weight/body weight, pathological changes of kidney. The levels of serum SCR, BUN were higher in the adenine-induced rats than those in the control group, indicating that the rat chronic renal failure model worked successfully. The results after treatment showed that LMWC could reduce the SCR and BUN levels and enhance the activities/levels of T-SOD and GSH-PX in kidney compared to control group. Histopathological examination revealed that adenine-induced renal alterations were restored by LMWC at three tested dosages, especially at the low dosage of 100 mg kg-1 d-1.

  19. Adenine radicals generated in alternating AT duplexes by direct absorption of low-energy UV radiation.

    PubMed

    Banyasz, Akos; Ketola, Tiia; Martínez-Fernández, Lara; Improta, Roberto; Markovitsi, Dimitra

    2018-04-17

    There is increasing evidence that the direct absorption of photons with energies that are lower than the ionization potential of nucleobases may result in oxidative damage to DNA. The present work, which combines nanosecond transient absorption spectroscopy and quantum mechanical calculations, studies this process in alternating adenine-thymine duplexes (AT)n. We show that the one-photon ionization quantum yield of (AT)10 at 266 nm (4.66 eV) is (1.5 ± 0.3) × 10-3. According to our PCM/TD-DFT calculations carried out on model duplexes composed of two base pairs, (AT)1 and (TA)1, simultaneous base pairing and stacking does not induce important changes in the absorption spectra of the adenine radical cation and deprotonated radical. The adenine radicals, thus identified in the time-resolved spectra, disappear with a lifetime of 2.5 ms, giving rise to a reaction product that absorbs at 350 nm. In parallel, the fingerprint of reaction intermediates other than radicals, formed directly from singlet excited states and assigned to AT/TA dimers, is detected at shorter wavelengths. PCM/TD-DFT calculations are carried out to map the pathways leading to such species and to characterize their absorption spectra; we find that, in addition to the path leading to the well-known TA* photoproduct, an AT photo-dimerization path may be operative in duplexes.

  20. Caenorhabditis elegans UBX cofactors for CDC-48/p97 control spermatogenesis.

    PubMed

    Sasagawa, Yohei; Yamanaka, Kunitoshi; Saito-Sasagawa, Yuko; Ogura, Teru

    2010-12-01

    UBX (ubiquitin regulatory X) domain-containing proteins act as cofactors for CDC-48/p97. CDC-48/p97 is essential for various cellular processes including retro-translocation in endoplasmic reticulum-associated degradation, homotypic membrane fusion, nuclear envelope assembly, degradation of ubiquitylated proteins, and cell cycle progression. CDC-48/p97-dependent processes are determined by differential binding of cofactors including UBX proteins, but the cellular functions of UBX proteins have not yet been elucidated, especially in multicellular organisms. Therefore, we investigated the functions of UBX family members using Caenorhabditis elegans, which expresses six UBX proteins, UBXN-1 to UBXN-6. All six UBXN proteins directly interacted with CDC-48.1 and CDC-48.2, and simultaneous knockdown of the expression of three genes, ubxn-1, ubxn-2 and ubxn-3, induced embryonic lethal and sterile phenotypes, but knockdown of either one or two did not. The sterile worms had a feminized germ-line phenotype, producing oocytes but no sperm. UBXN-1, UBXN-2 and UBXN-3 colocalized with CDC-48 in spermatocytes but not mature sperm. TRA-1A, which is a key factor in the sex determination pathway and inhibits spermatogenesis, accumulated in worms in which UBXN-1, UBXN-2 and UBXN-3 had been simultaneously knocked down. Taken together, these results suggest that UBXN-1, UBXN-2 and UBXN-3 are redundant cofactors for CDC-48/p97 and control spermatogenesis via the degradation of TRA-1A. © 2010 The Authors. Journal compilation © 2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

  1. Yeast ERV2p is the first microsomal FAD-linked sulfhydryl oxidase of the Erv1p/Alrp protein family.

    PubMed

    Gerber, J; Mühlenhoff, U; Hofhaus, G; Lill, R; Lisowsky, T

    2001-06-29

    Saccharomyces cerevisiae Erv2p was identified previously as a distant homologue of Erv1p, an essential mitochondrial protein exhibiting sulfhydryl oxidase activity. Expression of the ERV2 (essential for respiration and vegetative growth 2) gene from a high-copy plasmid cannot substitute for the lack of ERV1, suggesting that the two proteins perform nonredundant functions. Here, we show that the deletion of the ERV2 gene or the depletion of Erv2p by regulated gene expression is not associated with any detectable growth defects. Erv2p is located in the microsomal fraction, distinguishing it from the mitochondrial Erv1p. Despite their distinct subcellular localization, the two proteins exhibit functional similarities. Both form dimers in vivo and in vitro, contain a conserved YPCXXC motif in their carboxyl-terminal part, bind flavin adenine dinucleotide (FAD) as a cofactor, and catalyze the formation of disulfide bonds in protein substrates. The catalytic activity, the ability to form dimers, and the binding of FAD are associated with the carboxyl-terminal domain of the protein. Our findings identify Erv2p as the first microsomal member of the Erv1p/Alrp protein family of FAD-linked sulfhydryl oxidases. We propose that Erv2p functions in the generation of microsomal disulfide bonds acting in parallel with Ero1p, the essential, FAD-dependent oxidase of protein disulfide isomerase.

  2. Structural studies of Neurospora crassa LPMO9D and redox partner CDHIIA using neutron crystallography and small-angle scattering.

    PubMed

    Bodenheimer, Annette M; O'Dell, William B; Stanley, Christopher B; Meilleur, Flora

    2017-08-07

    Sensitivity to hydrogen/deuterium and lack of observable radiation damage makes cold neutrons an ideal probe the structural studies of proteins with highly photosensitive groups such as the copper center of lytic polysaccharide monooxygenases (LPMOs) and flavin adenine dinucleotide (FAD) and heme redox cofactors of cellobiose dehydrogenases (CDHs). Here, neutron crystallography and small-angle neutron scattering are used to investigate Neurospora crassa LPMO9D (NcLPMO9D) and CDHIIA (NcCDHIIA), respectively. The presence of LPMO greatly enhances the efficiency of commercial glycoside hydrolase cocktails in the depolymerization of cellulose. LPMOs can receive electrons from CDHs to activate molecular dioxygen for the oxidation of cellulose resulting in chain cleavage and disruption of local crystallinity. Using neutron protein crystallography, the hydrogen/deuterium atoms of NcLPMO9D could be located throughout the structure. At the copper active site, the protonation states of the side chains of His1, His84, His157 and Tyr168, and the orientation of water molecules could be determined. Small-angle neutron scattering measurements provided low resolution models of NcCDHIIA with both the dehydrogenase and cytochrome domains in oxidized states that exhibited elongated conformations. This work demonstrates the suitability of neutron diffraction and scattering for characterizing enzymes critical to oxidative cellulose deconstruction. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Biosynthesis of Cutin

    PubMed Central

    Kolattukudy, P.E.; Croteau, Rodney; Walton, T.J.

    1975-01-01

    Long chain dicarboxylic acids are constituents of the protective biopolymers cutin and suberin of plants. Cell-free extracts from the excised epidermis of Vicia faba leaves catalyzed conversion of 16-hydroxy[G-3H]hexadecanoic acid to the corresponding dicarboxylic acid with nicotinamide-adenine dinucleotide phosphate as the preferred cofactor. This enzymatic activity, located largely in the 100,000g supernatant fraction, had a pH optimum near 8. This dehydrogenase showed an apparent Km of 1.25 × 10−5m and 3.6 × 10−4m for 16-hydroxyhexadecanoic acid and NADP, respectively. Modification of the substrate, either by esterification of the carboxyl group or by introduction of another hydroxyl group at C-10, resulted in a substantial (two-thirds) decrease in the rate of reaction, and hexadecanol was not a good substrate. The enzyme was inhibited by thiol reagents such as N-ethylmaleimide and p-chloromercuribenzoate. The aldehyde intermediate was trapped by the inclusion of dinitrophenyl hydrazine in the reaction mixture, and the 16-oxo compound was regenerated and identified. Furthermore, synthetic 16-oxo-[G-3H] hexadecanoic acid was readily converted to the dicarboxylic acid by the cell-free preparation. These results demonstrate that epidermis of Vicia faba contains an ω-hydroxyacid dehydrogenase and an ω-oxoacid dehydrogenase. PMID:16659184

  4. Role of Sirt1 during the ageing process: relevance to protection of synapses in the brain.

    PubMed

    Godoy, Juan A; Zolezzi, Juan M; Braidy, Nady; Inestrosa, Nibaldo C

    2014-12-01

    Ageing is a stochastic process associated with a progressive decline in physiological functions which predispose to the pathogenesis of several neurodegenerative diseases. The intrinsic complexity of ageing remains a significant challenge to understand the cause of this natural phenomenon. At the molecular level, ageing is thought to be characterized by the accumulation of chronic oxidative damage to lipids, proteins and nucleic acids caused by free radicals. Increased oxidative stress and misfolded protein formations, combined with impaired compensatory mechanisms, may promote neurodegenerative disorders with age. Nutritional modulation through calorie restriction has been shown to be effective as an anti-ageing factor, promoting longevity and protecting against neurodegenerative pathology in yeast, nematodes and murine models. Calorie restriction increases the intracellular levels of the essential pyridine nucleotide, nicotinamide adenine dinucleotide (NAD(+)), a co-substrate for the sirtuin 1 (Sirt1, silent mating-type information regulator 2 homolog 1) activity and a cofactor for oxidative phosphorylation and ATP synthesis. Promotion of intracellular NAD(+) anabolism is speculated to induce neuroprotective effects against amyloid-β-peptide (Aβ) toxicity in some models for Alzheimer's disease (AD). The NAD(+)-dependent histone deacetylase, Sirt1, has been implicated in the ageing process. Sirt1 serves as a deacetylase for numerous proteins involved in several cellular pathways, including stress response and apoptosis, and plays a protective role in neurodegenerative disorders, such as AD.

  5. Structural analysis of fungus-derived FAD glucose dehydrogenase

    PubMed Central

    Yoshida, Hiromi; Sakai, Genki; Mori, Kazushige; Kojima, Katsuhiro; Kamitori, Shigehiro; Sode, Koji

    2015-01-01

    We report the first three-dimensional structure of fungus-derived glucose dehydrogenase using flavin adenine dinucleotide (FAD) as the cofactor. This is currently the most advanced and popular enzyme used in glucose sensor strips manufactured for glycemic control by diabetic patients. We prepared recombinant nonglycosylated FAD-dependent glucose dehydrogenase (FADGDH) derived from Aspergillus flavus (AfGDH) and obtained the X-ray structures of the binary complex of enzyme and reduced FAD at a resolution of 1.78 Å and the ternary complex with reduced FAD and D-glucono-1,5-lactone (LGC) at a resolution of 1.57 Å. The overall structure is similar to that of fungal glucose oxidases (GOxs) reported till date. The ternary complex with reduced FAD and LGC revealed the residues recognizing the substrate. His505 and His548 were subjected for site-directed mutagenesis studies, and these two residues were revealed to form the catalytic pair, as those conserved in GOxs. The absence of residues that recognize the sixth hydroxyl group of the glucose of AfGDH, and the presence of significant cavity around the active site may account for this enzyme activity toward xylose. The structural information will contribute to the further engineering of FADGDH for use in more reliable and economical biosensing technology for diabetes management. PMID:26311535

  6. Crystal structures and atomic model of NADPH oxidase.

    PubMed

    Magnani, Francesca; Nenci, Simone; Millana Fananas, Elisa; Ceccon, Marta; Romero, Elvira; Fraaije, Marco W; Mattevi, Andrea

    2017-06-27

    NADPH oxidases (NOXs) are the only enzymes exclusively dedicated to reactive oxygen species (ROS) generation. Dysregulation of these polytopic membrane proteins impacts the redox signaling cascades that control cell proliferation and death. We describe the atomic crystal structures of the catalytic flavin adenine dinucleotide (FAD)- and heme-binding domains of Cylindrospermum stagnale NOX5. The two domains form the core subunit that is common to all seven members of the NOX family. The domain structures were then docked in silico to provide a generic model for the NOX family. A linear arrangement of cofactors (NADPH, FAD, and two membrane-embedded heme moieties) injects electrons from the intracellular side across the membrane to a specific oxygen-binding cavity on the extracytoplasmic side. The overall spatial organization of critical interactions is revealed between the intracellular loops on the transmembrane domain and the NADPH-oxidizing dehydrogenase domain. In particular, the C terminus functions as a toggle switch, which affects access of the NADPH substrate to the enzyme. The essence of this mechanistic model is that the regulatory cues conformationally gate NADPH-binding, implicitly providing a handle for activating/deactivating the very first step in the redox chain. Such insight provides a framework to the discovery of much needed drugs that selectively target the distinct members of the NOX family and interfere with ROS signaling.

  7. Regulation of the Nampt-mediated NAD salvage pathway and its therapeutic implications in pancreatic cancer.

    PubMed

    Ju, Huai-Qiang; Zhuang, Zhuo-Nan; Li, Hao; Tian, Tian; Lu, Yun-Xin; Fan, Xiao-Qiang; Zhou, Hai-Jun; Mo, Hai-Yu; Sheng, Hui; Chiao, Paul J; Xu, Rui-Hua

    2016-08-28

    Nicotinamide adenine dinucleotide (NAD) is a crucial cofactor for the redox reactions in the metabolic pathways of cancer cells that have elevated aerobic glycolysis (Warburg effect). Cancer cells are reported to rely on NAD recycling and inhibition of the NAD salvage pathway causes metabolic collapse and cell death. However, the underlying regulatory mechanisms and clinical implications for the NAD salvage pathway in pancreatic ductal adenocarcinoma (PDAC) remain unclear. This study showed that the expression of Nampt, the rate-limiting enzyme of the NAD salvage pathway, was significantly increased in PDAC cells and PDAC tissues. Additionally, inhibition of Nampt impaired tumor growth in vitro and tumorigenesis in vivo, which was accompanied by a decreased cellular NAD level and glycolytic activity. Mechanistically, the Nampt expression was independent of Kras and p16 status, but it was directly regulated by miR-206, which was inversely correlated with the expression of Nampt in PDAC tissues. Importantly, pharmacological inhibition of Nampt by its inhibitor, FK866, significantly enhanced the antitumor activity of gemcitabine in PDAC cells and in orthotopic xenograft mouse models. In conclusion, the present study revealed a novel regulatory mechanism for Nampt in PDAC and suggested that Nampt inhibition may override gemcitabine resistance by decreasing the NAD level and suppressing glycolytic activity, warranting further clinical investigation for pancreatic cancer treatment. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  8. Nicotinamide mononucleotide inhibits post-ischemic NAD(+) degradation and dramatically ameliorates brain damage following global cerebral ischemia.

    PubMed

    Park, Ji H; Long, Aaron; Owens, Katrina; Kristian, Tibor

    2016-11-01

    Nicotinamide adenine dinucleotide (NAD(+)) is an essential cofactor for multiple cellular metabolic reactions and has a central role in energy production. Brain ischemia depletes NAD(+) pools leading to bioenergetics failure and cell death. Nicotinamide mononucleotide (NMN) is utilized by the NAD(+) salvage pathway enzyme, nicotinamide adenylyltransferase (Nmnat) to generate NAD(+). Therefore, we examined whether NMN could protect against ischemic brain damage. Mice were subjected to transient forebrain ischemia and treated with NMN or vehicle at the start of reperfusion or 30min after the ischemic insult. At 2, 4, and 24h of recovery, the proteins poly-ADP-ribosylation (PAR), hippocampal NAD(+) levels, and expression levels of NAD(+) salvage pathway enzymes were determined. Furthermore, animal's neurologic outcome and hippocampal CA1 neuronal death was assessed after six days of reperfusion. NMN (62.5mg/kg) dramatically ameliorated the hippocampal CA1 injury and significantly improved the neurological outcome. Additionally, the post-ischemic NMN treatment prevented the increase in PAR formation and NAD(+) catabolism. Since the NMN administration did not affect animal's temperature, blood gases or regional cerebral blood flow during recovery, the protective effect was not a result of altered reperfusion conditions. These data suggest that administration of NMN at a proper dosage has a strong protective effect against ischemic brain injury. Published by Elsevier Inc.

  9. Simultaneous measurement of NAD metabolome in aged mice tissue using liquid chromatography tandem-mass spectrometry.

    PubMed

    Yaku, Keisuke; Okabe, Keisuke; Nakagawa, Takashi

    2018-06-01

    Nicotinamide adenine dinucleotide (NAD) is a major co-factor that mediates multiple biological processes including redox reaction and gene expression. Recently, NAD metabolism has received considerable attention because administration of NAD precursors exhibited beneficial effects against aging-related metabolic disorders in animals. Although numerous studies have reported that NAD levels decline with aging in multiple animal tissues, the pathway and kinetics of NAD metabolism in aged organs are not completely understood. To determine the NAD metabolism upon aging, we developed targeted metabolomics based on an LC/MS/MS system. Our method is simple and applicable to crude biological samples, including culture cells and animal tissues. Unlike a conventional enzymatic cycling assay, our approach can determine NAD and NADH (reduced form of NAD) by performing a single sample preparation. Further, we validated our method using biological samples and investigated the alteration of the NAD metabolome during aging. Consistent with previous reports, the NAD levels in the liver and skeletal muscle decreased with aging. Further, we detected a significant increase in nicotinamide mononucleotide and nicotinamide riboside in the kidney upon aging. The LC/MS/MS-based NAD metabolomics that we have developed is extensively applicable to biomedical studies, and the results will present innovative ideas for the aging studies, especially for that of NAD metabolism. Copyright © 2018 John Wiley & Sons, Ltd.

  10. NAD and the aging process: Role in life, death and everything in between.

    PubMed

    Chini, Claudia C S; Tarragó, Mariana G; Chini, Eduardo N

    2017-11-05

    Life as we know it cannot exist without the nucleotide nicotinamide adenine dinucleotide (NAD). From the simplest organism, such as bacteria, to the most complex multicellular organisms, NAD is a key cellular component. NAD is extremely abundant in most living cells and has traditionally been described to be a cofactor in electron transfer during oxidation-reduction reactions. In addition to participating in these reactions, NAD has also been shown to play a key role in cell signaling, regulating several pathways from intracellular calcium transients to the epigenetic status of chromatin. Thus, NAD is a molecule that provides an important link between signaling and metabolism, and serves as a key molecule in cellular metabolic sensoring pathways. Importantly, it has now been clearly demonstrated that cellular NAD levels decline during chronological aging. This decline appears to play a crucial role in the development of metabolic dysfunction and age-related diseases. In this review we will discuss the molecular mechanisms responsible for the decrease in NAD levels during aging. Since other reviews on this subject have been recently published, we will concentrate on presenting a critical appraisal of the current status of the literature and will highlight some controversial topics in the field. In particular, we will discuss the potential role of the NADase CD38 as a driver of age-related NAD decline. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  11. Miniaturised enzymatic conductometric biosensor with Nafion membrane for the direct determination of formaldehyde in water samples.

    PubMed

    Nguyen-Boisse, Thanh-Thuy; Saulnier, Joëlle; Jaffrezic-Renault, Nicole; Lagarde, Florence

    2014-02-01

    A new conductometric enzyme-based biosensor was developed for the determination of formaldehyde (FA) in aqueous solutions. The biosensor was prepared by cross-linking formaldehyde dehydrogenase from Pseudomonas putida with bovine serum albumin in saturated glutaraldehyde vapours (GA) at the surface of interdigitated gold microelectrodes. Nicotinamide adenine dinucleotide cofactor (NAD(+)) was added in solution at each measurement to maintain enzyme activity. Addition of a Nafion layer over the enzyme modified electrode resulted in a significant increase of biosensor signal due to enhanced accumulation of protons generated by enzymatic reaction at the electrode surface. Different parameters affecting enzyme activity or playing a role in ionic transfer through the Nafion membrane were optimised. In optimal conditions (0.045 mg enzyme, 30 min exposure to GA, 0.3 μL of a 1% (v/v) Nafion solution deposit, measurement in 5 mM phosphate buffer pH 7 containing 20 μM NAD(+)), the biosensor signal was linear up to 10 mM FA, and the detection limit was 18 μM. Relative standard deviations calculated from five consecutive replicates of FA solutions were lower than 5% in the 1-10 mM range. The biosensor was successfully applied to the determination of FA in spiked water samples (tap water and Rhone river water), with recoveries in the 95-110% range.

  12. Structural studies of Neurospora crassa LPMO9D and redox partner CDHIIA using neutron crystallography and small-angle scattering

    DOE PAGES

    Bodenheimer, Annette M.; O'Dell, William B.; Stanley, Christopher B.; ...

    2017-03-04

    Sensitivity to hydrogen/deuterium and lack of observable radiation damage makes cold neutrons an ideal probe the structural studies of proteins with highly photosensitive groups such as the copper center of lytic polysaccharide monooxygenases (LPMOs) and flavin adenine dinucleotide (FAD) and heme redox cofactors of cellobiose dehydrogenases (CDHs). In this paper, neutron crystallography and small-angle neutron scattering are used to investigate Neurospora crassa LPMO9D (NcLPMO9D) and CDHIIA (NcCDHIIA), respectively. The presence of LPMO greatly enhances the efficiency of commercial glycoside hydrolase cocktails in the depolymerization of cellulose. LPMOs can receive electrons from CDHs to activate molecular dioxygen for the oxidation ofmore » cellulose resulting in chain cleavage and disruption of local crystallinity. Using neutron protein crystallography, the hydrogen/deuterium atoms of NcLPMO9D could be located throughout the structure. At the copper active site, the protonation states of the side chains of His1, His84, His157 and Tyr168, and the orientation of water molecules could be determined. Small-angle neutron scattering measurements provided low resolution models of NcCDHIIA with both the dehydrogenase and cytochrome domains in oxidized states that exhibited elongated conformations. Finally, this work demonstrates the suitability of neutron diffraction and scattering for characterizing enzymes critical to oxidative cellulose deconstruction.« less

  13. Structural studies of Neurospora crassa LPMO9D and redox partner CDHIIA using neutron crystallography and small-angle scattering

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bodenheimer, Annette M.; O'Dell, William B.; Stanley, Christopher B.

    Sensitivity to hydrogen/deuterium and lack of observable radiation damage makes cold neutrons an ideal probe the structural studies of proteins with highly photosensitive groups such as the copper center of lytic polysaccharide monooxygenases (LPMOs) and flavin adenine dinucleotide (FAD) and heme redox cofactors of cellobiose dehydrogenases (CDHs). In this paper, neutron crystallography and small-angle neutron scattering are used to investigate Neurospora crassa LPMO9D (NcLPMO9D) and CDHIIA (NcCDHIIA), respectively. The presence of LPMO greatly enhances the efficiency of commercial glycoside hydrolase cocktails in the depolymerization of cellulose. LPMOs can receive electrons from CDHs to activate molecular dioxygen for the oxidation ofmore » cellulose resulting in chain cleavage and disruption of local crystallinity. Using neutron protein crystallography, the hydrogen/deuterium atoms of NcLPMO9D could be located throughout the structure. At the copper active site, the protonation states of the side chains of His1, His84, His157 and Tyr168, and the orientation of water molecules could be determined. Small-angle neutron scattering measurements provided low resolution models of NcCDHIIA with both the dehydrogenase and cytochrome domains in oxidized states that exhibited elongated conformations. Finally, this work demonstrates the suitability of neutron diffraction and scattering for characterizing enzymes critical to oxidative cellulose deconstruction.« less

  14. Rational modification of Corynebacterium glutamicum dihydrodipicolinate reductase to switch the nucleotide-cofactor specificity for increasing l-lysine production.

    PubMed

    Xu, Jian-Zhong; Yang, Han-Kun; Liu, Li-Ming; Wang, Ying-Yu; Zhang, Wei-Guo

    2018-03-25

    l-lysine is an important amino acid in animals and humans and NADPH is a vital cofactor for maximizing the efficiency of l-lysine fermentation. Dihydrodipicolinate reductase (DHDPR), an NAD(P)H-dependent enzyme, shows a variance in nucleotide-cofactor affinity in bacteria. In this study, we rationally engineered Corynebacterium glutamicum DHDPR (CgDHDPR) to switch its nucleotide-cofactor specificity resulting in an increase in final titer (from 82.6 to 117.3 g L -1 ), carbon yield (from 0.35 to 0.44 g [g glucose] -1 ) and productivity (from 2.07 to 2.93 g L -1  hr -1 ) of l-lysine in JL-6 ΔdapB::Ec-dapB C115G,G116C in fed-batch fermentation. To do this, we comparatively analyzed the characteristics of CgDHDPR and Escherichia coli DHDPR (EcDHDPR), indicating that hetero-expression of NADH-dependent EcDHDPR increased l-lysine production. Subsequently, we rationally modified the conserved structure of cofactor-binding motif, and results indicated that introducing the mutation K11A or R13A in CgDHDPR and introducing the mutation R16A or R39A in EcDHDPR modifies the nucleotide-cofactor affinity of DHDPR. Lastly, the effects of these mutated DHDPRs on l-lysine production were investigated. The highest increase (26.2%) in l-lysine production was observed for JL-6 ΔdapB::Ec-dapB C115G,G116C , followed by JL-6 Cg-dapB C37G,G38C (21.4%) and JL-6 ΔdapB::Ec-dapB C46G,G47C (15.2%). This is the first report of a rational modification of DHDPR that enhances the l-lysine production and yield through the modulation of nucleotide-cofactor specificity. © 2018 Wiley Periodicals, Inc.

  15. Light-driven enzymatic catalysis of DNA repair: a review of recent biophysical studies on photolyase.

    PubMed

    Weber, Stefan

    2005-02-25

    More than 50 years ago, initial experiments on enzymatic photorepair of ultraviolet (UV)-damaged DNA were reported [Proc. Natl. Acad. Sci. U. S. A. 35 (1949) 73]. Soon after this discovery, it was recognized that one enzyme, photolyase, is able to repair UV-induced DNA lesions by effectively reversing their formation using blue light. The enzymatic process named DNA photoreactivation depends on a non-covalently bound cofactor, flavin adenine dinucleotide (FAD). Flavins are ubiquitous redox-active catalysts in one- and two-electron transfer reactions of numerous biological processes. However, in the case of photolyase, not only the ground-state redox properties of the FAD cofactor are exploited but also, and perhaps more importantly, its excited-state properties. In the catalytically active, fully reduced redox form, the FAD absorbs in the blue and near-UV ranges of visible light. Although there is no direct experimental evidence, it appears generally accepted that starting from the excited singlet state, the chromophore initiates a reductive cleavage of the two major DNA photodamages, cyclobutane pyrimidine dimers and (6-4) photoproducts, by short-distance electron transfer to the DNA lesion. Back electron transfer from the repaired DNA segment is believed to eventually restore the initial redox states of the cofactor and the DNA nucleobases, resulting in an overall reaction with net-zero exchanged electrons. Thus, the entire process represents a true catalytic cycle. Many biochemical and biophysical studies have been carried out to unravel the fundamentals of this unique mode of action. The work has culminated in the elucidation of the three-dimensional structure of the enzyme in 1995 that revealed remarkable details, such as the FAD-cofactor arrangement in an unusual U-shaped configuration. With the crystal structure of the enzyme at hand, research on photolyases did not come to an end but, for good reason, intensified: the geometrical structure of the enzyme

  16. Label-free in vivo flow cytometry in zebrafish using two-photon autofluorescence imaging.

    PubMed

    Zeng, Yan; Xu, Jin; Li, Dong; Li, Li; Wen, Zilong; Qu, Jianan Y

    2012-07-01

    We demonstrate a label-free in vivo flow cytometry in zebrafish blood vessels based on two-photon excited autofluorescence imaging. The major discovery in this work is the strong autofluorescence emission from the plasma in zebrafish blood. The plasma autofluorescence provides excellent contrast for visualizing blood vessels and counting blood cells. In addition, the cellular nicotinamide adenine dinucleotide autofluorescence enables in vivo imaging and counting of white blood cells (neutrophils).

  17. ROS open roads to roundworm infection.

    PubMed

    Feng, Baomin; Shan, Libo

    2014-04-08

    The rapid production of reactive oxygen species (ROS) upon pathogen attack is generally considered a defense mechanism for microbial killing and an initiation of host defense responses in plants and animals. In this issue, Siddique et al. show that nicotinamide adenine dinucleotide phosphate oxidase-derived ROS function as a pathogenicity factor to promote the roundworm nematode infection in Arabidopsis thaliana, revealing the complex action of ROS in host-pathogen interactions.

  18. The role of FeS clusters for molybdenum cofactor biosynthesis and molybdoenzymes in bacteria

    PubMed Central

    Yokoyama, Kenichi; Leimkühler, Silke

    2016-01-01

    Molybdenum is the only second row transition metal essential for biological systems, which is biologically available as molybdate ion. In eukarya, bacteria and archaea, molybdenum is bound to either to a tricyclic pyranopterin, thereby forming the molybdenum cofactor (Moco), or in some bacteria to the FeS cluster based iron-molybdenum cofactor (FeMoco), which forms the active site of nitrogenase. To date more than 50 Moco-containing enzymes have been purified and biochemically or structurally characterized. The physiological role of molybdenum in these enzymes is fundamental to organisms, since the reactions include the catalysis of key steps in carbon, nitrogen and sulfur metabolism. The catalyzed reactions are in most cases oxo-transfer reactions or the hydroxylation of carbon centers. The biosynthesis of Moco has been intensively studied, in addition to its insertion into molybdoenzymes. In particular, a link between the biosynthesis and maturation of molybdoenzymes and the biosynthesis and distribution of FeS clusters has been identified in the last years: 1) The synthesis of the first intermediate in Moco biosynthesis requires an FeS-cluster containing protein, 2) The sulfurtransferase for the dithiolene group in Moco is common also for the synthesis of FeS clusters, thiamin and thiolated tRNAs, 3) the modification of the active site with a sulfur atom additionally involves a sulfurtransferase, 4) most molybdoenzymes in bacteria require FeS clusters as additional redox active cofactors. In this review we will focus on the biosynthesis of the molybdenum cofactor in bacteria, its modification and insertion into molybdoenzymes, with an emphasis to its link to FeS cluster biosynthesis and sulfur transfer. PMID:25268953

  19. Metabolic adaptation to long term changes in gravity environment

    NASA Astrophysics Data System (ADS)

    Slenzka, K.; Appel, R.; Rahmann, H.

    Biochemical analyses of the brain of Cichlid fish larvae, exposed during their very early development for 7 days to an increased acceleration of 3g (hyper-gravity), revealed a decrease in brain nucleoside diphosphate kinase (NDPK) as well as creatine kinase (BB-CK) activity. Using high performance liquid chromatography (HPLC) the concentrations of adenine nucleotides (AMP, ADP, ATP), phosphocreatine (CP), as well as of nicotineamide adenine dinucleotides (NAD, NADP) were analyzed in the brain of hyper-g exposed larvae vs. 1g controls. A slight reduction in the total adenine nucleotides (TAN) as well as the adenylate energy charge (AEC) was found. In parallel a significant increase in the NAD concentration and a corresponding decrease in NADP concentration occurred in larva's hyper-g brains vs. 1g controls. These results give further evidence for an influence of gravity on cellular level and furthermore contribute to a clarification of the cellular signal-response chain for gravity perception.

  20. Targeted redox and energy cofactor metabolomics in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

    DOE PAGES

    Sander, Kyle; Asano, Keiji G.; Bhandari, Deepak; ...

    2017-11-30

    Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are prominent candidate biocatalysts that, together, can enable the direct biotic conversion of lignocellulosic biomass to ethanol. The imbalance and suboptimal turnover rates of redox cofactors are currently hindering engineering efforts to achieve higher bioproductivity in both organisms. Measuring relevant intracellular cofactor concentrations will help understand redox state of these cofactors and help identify a strategy to overcome these limitations; however, metabolomic determinations of these labile metabolites have historically proved challenging.Results: Through our validations, we verified the handling and storage stability of these metabolites, and verified extraction matrices and extraction solvent were not suppressing massmore » spectrometry signals. We recovered adenylate energy charge ratios (a main quality indicator) above 0.82 for all extractions. NADH/NAD+ values of 0.26 and 0.04 for an adhE-deficient strain of C. thermocellum and its parent, respectively, reflect the expected shift to a more reduced redox potential when a species lacks the ability to re-oxidize NADH by synthesizing ethanol. This method failed to yield reliable results with C. bescii and poor-growing strains of T. saccharolyticum. Lastly, our validated protocols demonstrate and validate the extraction and analysis of selected redox and energy-related metabolites from two candidate consolidated bioprocessing biocatalysts, C. thermocellum and T. saccharolyticum. This development and validation highlights the important, but often neglected, need to optimize and validate metabolomic protocols when adapting them to new cell or tissue types.« less

  1. Targeted redox and energy cofactor metabolomics in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sander, Kyle; Asano, Keiji G.; Bhandari, Deepak

    Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are prominent candidate biocatalysts that, together, can enable the direct biotic conversion of lignocellulosic biomass to ethanol. The imbalance and suboptimal turnover rates of redox cofactors are currently hindering engineering efforts to achieve higher bioproductivity in both organisms. Measuring relevant intracellular cofactor concentrations will help understand redox state of these cofactors and help identify a strategy to overcome these limitations; however, metabolomic determinations of these labile metabolites have historically proved challenging.Results: Through our validations, we verified the handling and storage stability of these metabolites, and verified extraction matrices and extraction solvent were not suppressing massmore » spectrometry signals. We recovered adenylate energy charge ratios (a main quality indicator) above 0.82 for all extractions. NADH/NAD+ values of 0.26 and 0.04 for an adhE-deficient strain of C. thermocellum and its parent, respectively, reflect the expected shift to a more reduced redox potential when a species lacks the ability to re-oxidize NADH by synthesizing ethanol. This method failed to yield reliable results with C. bescii and poor-growing strains of T. saccharolyticum. Lastly, our validated protocols demonstrate and validate the extraction and analysis of selected redox and energy-related metabolites from two candidate consolidated bioprocessing biocatalysts, C. thermocellum and T. saccharolyticum. This development and validation highlights the important, but often neglected, need to optimize and validate metabolomic protocols when adapting them to new cell or tissue types.« less

  2. Molybdenum cofactor (chlorate-resistant) mutants of Klebsiella pneumoniae M5al can use hypoxanthine as the sole nitrogen source.

    PubMed Central

    Garzón, A; Li, J; Flores, A; Casadesus, J; Stewart, V

    1992-01-01

    Selection for chlorate resistance yields mol (formerly chl) mutants with defects in molybdenum cofactor synthesis. Complementation and genetic mapping analyses indicated that the Klebsiella pneumoniae mol genes are functionally homologous to those of Escherichia coli and occupy analogous genetic map positions. Hypoxanthine utilization in other organisms requires molybdenum cofactor as a component of xanthine dehydrogenase, and thus most chlorate-resistant mutants cannot use hypoxanthine as a sole source of nitrogen. Surprisingly, the K. pneumoniae mol mutants and the mol+ parent grew equally well with hypoxanthine as the sole nitrogen source, suggesting that K. pneumoniae has a molybdenum cofactor-independent pathway for hypoxanthine utilization. PMID:1400180

  3. The 2.5 Å Crystal Structure of the SIRT1 Catalytic Domain Bound to Nicotinamide Adenine Dinucleotide (NAD + ) and an Indole (EX527 Analogue) Reveals a Novel Mechanism of Histone Deacetylase Inhibition

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhao, Xun; Allison, Dagart; Condon, Bradley

    2013-02-14

    The sirtuin SIRT1 is a NAD+-dependent histone deacetylase, a Sir2 family member, and one of seven human sirtuins. Sirtuins are conserved from archaea to mammals and regulate transcription, genome stability, longevity, and metabolism. SIRT1 regulates transcription via deacetylation of transcription factors such as PPARγ, NFκB, and the tumor suppressor protein p53. EX527 (27) is a nanomolar SIRT1 inhibitor and a micromolar SIRT2 inhibitor. To elucidate the mechanism of SIRT inhibition by 27, we determined the 2.5 Å crystal structure of the SIRT1 catalytic domain (residues 241–516) bound to NAD+ and the 27 analogue compound 35. 35 binds deep in themore » catalytic cleft, displacing the NAD+ nicotinamide and forcing the cofactor into an extended conformation. The extended NAD+ conformation sterically prevents substrate binding. The SIRT1/NAD+/35 crystal structure defines a novel mechanism of histone deacetylase inhibition and provides a basis for understanding, and rationally improving, inhibition of this therapeutically important target by drug-like molecules.« less

  4. Involvement of the Cys-Tyr cofactor on iron binding in the active site of human cysteine dioxygenase.

    PubMed

    Arjune, Sita; Schwarz, Guenter; Belaidi, Abdel A

    2015-01-01

    Sulfur metabolism has gained increasing medical interest over the last years. In particular, cysteine dioxygenase (CDO) has been recognized as a potential marker in oncology due to its altered gene expression in various cancer types. Human CDO is a non-heme iron-dependent enzyme, which catalyzes the irreversible oxidation of cysteine to cysteine sulfinic acid, which is further metabolized to taurine or pyruvate and sulfate. Several studies have reported a unique post-translational modification of human CDO consisting of a cross-link between cysteine 93 and tyrosine 157 (Cys-Tyr), which increases catalytic efficiency in a substrate-dependent manner. However, the reaction mechanism by which the Cys-Tyr cofactor increases catalytic efficiency remains unclear. In this study, steady-state kinetics were determined for wild type CDO and two different variants being either impaired or saturated with the Cys-Tyr cofactor. Cofactor formation in CDO resulted in an approximately fivefold increase in k cat and tenfold increase in k cat/K m over the cofactor-free CDO variant. Furthermore, iron titration experiments revealed an 18-fold decrease in K d of iron upon cross-link formation. This finding suggests a structural role of the Cys-Tyr cofactor in coordinating the ferrous iron in the active site of CDO in accordance with the previously postulated reaction mechanism of human CDO. Finally, we identified product-based inhibition and α-ketoglutarate and glutarate as CDO inhibitors using a simplified well plate-based activity assay. This assay can be used for high-throughput identification of additional inhibitors, which may contribute to understand the functional importance of CDO in sulfur amino acid metabolism and related diseases.

  5. The effect of swimming exercise on adenine-induced kidney disease in rats, and the influence of curcumin or lisinopril thereon

    PubMed Central

    Karaca, Turan; Al Suleimani, Yousuf; Al Za'abi, Mohammed; Al Kalbani, Jamila; Ashique, Mohammed; Nemmar, Abderrahim

    2017-01-01

    Patients with chronic kidney disease (CKD) have been reported to benefit from different types of exercises. It has also been shown that the ACE inhibitor lisinopril, and the natural product curcumin are also beneficial in different models of CKD in rats. We assessed the influence of moderate swimming exercise (SE) on rats with adenine-induced CKD, and tested the possible effects of lisinopril and/or curcumin thereon using several physiological, biochemical, histopathological and immunohistochemical parameters. Rats (either sedentary or subjected to SE) were randomly divided into several groups, and given for five weeks either normal food or food mixed with adenine (0.25% w/w) to induce CKD. Some of these groups were also concomitantly treated orally with curcumin (75 mg/kg), or lisinopril (10 mg/kg) and were subjected to moderate SE (45 min/day three days each week). Rats fed adenine showed the typical biochemical, histopathological signs of CKD such as elevations in blood pressure, urinary albumin / creatinine ratio, and plasma urea, creatinine, indoxyl sulfate and phosphorus. SE, curcumin or lisinopril, given singly, significantly ameliorated all the adenine-induced actions. Administering curcumin or lisinopril with SE improved the histopathology of the kidneys, a salutary effect not seen with SE alone. Combining SE to the nephroprotective agents’ curcumin or lisinopril might offer additional nephroprotection. PMID:28445490

  6. 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.

  7. Assessing the photoaging process at sun exposed and non-exposed skin using fluorescence lifetime spectroscopy

    NASA Astrophysics Data System (ADS)

    Saito Nogueira, Marcelo; Kurachi, Cristina

    2016-03-01

    Photoaging is the skin premature aging due to exposure to ultraviolet light, which damage the collagen, elastin and can induce alterations on the skin cells DNA, and, then, it may evolve to precancerous lesions, which are widely investigated by fluorescence spectroscopy and lifetime. The fluorescence spectra and fluorescence lifetime analysis has been presented as a technique of great potential for biological tissue characterization at optical diagnostics. The main targeted fluorophores are NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), which have free and bound states, each one with different average lifetimes. The average lifetimes for free and bound NADH and FAD change according to tissue metabolic alterations and may contribute to a non-invasive clinical investigation of injuries such as skin lesions. These lesions and the possible areas where they may develop can be interrogated using fluorescence lifetime spectroscopy taking into account the variability of skin phototypes and the changes related to melanin, collagen and elastin, endogenous fluorophores which have emissions that spectrally overlap to the NADH and FAD emission. The objective of this study is to assess the variation on fluorescence lifetimes of normal skin at sun exposed and non-exposed areas and associate this variation to the photoaging process.

  8. Rapid measurement of meat spoilage using fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Wu, Binlin; Dahlberg, Kevin; Gao, Xin; Smith, Jason; Bailin, Jacob

    2017-02-01

    Food spoilage is mainly caused by microorganisms, such as bacteria. In this study, we measure the autofluorescence in meat samples longitudinally over a week in an attempt to develop a method to rapidly detect meat spoilage using fluorescence spectroscopy. Meat food is a biological tissue, which contains intrinsic fluorophores, such as tryptophan, collagen, nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) etc. As meat spoils, it undergoes various morphological and chemical changes. The concentrations of the native fluorophores present in a sample may change. In particular, the changes in NADH and FAD are associated with microbial metabolism, which is the most important process of the bacteria in food spoilage. Such changes may be revealed by fluorescence spectroscopy and used to indicate the status of meat spoilage. Therefore, such native fluorophores may be unique, reliable and nonsubjective indicators for detection of spoiled meat. The results of the study show that the relative concentrations of all above fluorophores change as the meat samples kept in room temperature ( 19° C) spoil. The changes become more rapidly after about two days. For the meat samples kept in a freezer ( -12° C), the changes are much less or even unnoticeable over a-week-long storage.

  9. The Arabidopsis dwarf1 Mutant Is Defective in the Conversion of 24-Methylenecholesterol to Campesterol in Brassinosteroid Biosynthesis1

    PubMed Central

    Choe, Sunghwa; Dilkes, Brian P.; Gregory, Brian D.; Ross, Amanda S.; Yuan, Heng; Noguchi, Takahiro; Fujioka, Shozo; Takatsuto, Suguru; Tanaka, Atsushi; Yoshida, Shigeo; Tax, Frans E.; Feldmann, Kenneth A.

    1999-01-01

    Since the isolation and characterization of dwarf1-1 (dwf1-1) from a T-DNA insertion mutant population, phenotypically similar mutants, including deetiolated2 (det2), constitutive photomorphogenesis and dwarfism (cpd), brassinosteroid insensitive1 (bri1), and dwf4, have been reported to be defective in either the biosynthesis or the perception of brassinosteroids. We present further characterization of dwf1-1 and additional dwf1 alleles. Feeding tests with brassinosteroid-biosynthetic intermediates revealed that dwf1 can be rescued by 22α-hydroxycampesterol and downstream intermediates in the brassinosteroid pathway. Analysis of the endogenous levels of brassinosteroid intermediates showed that 24-methylenecholesterol in dwf1 accumulates to 12 times the level of the wild type, whereas the level of campesterol is greatly diminished, indicating that the defective step is in C-24 reduction. Furthermore, the deduced amino acid sequence of DWF1 shows significant similarity to a flavin adenine dinucleotide-binding domain conserved in various oxidoreductases, suggesting an enzymatic role for DWF1. In support of this, 7 of 10 dwf1 mutations directly affected the flavin adenine dinucleotide-binding domain. Our molecular characterization of dwf1 alleles, together with our biochemical data, suggest that the biosynthetic defect in dwf1 results in reduced synthesis of bioactive brassinosteroids, causing dwarfism. PMID:10069828

  10. Imaging Redox State in Mouse Muscles of Different Ages.

    PubMed

    Moon, Lily; Frederick, David W; Baur, Joseph A; Li, Lin Z

    2017-01-01

    Aging is the greatest risk factor for many diseases. Intracellular concentrations of nicotinamide adenine dinucleotide (NAD + ) and the NAD + -coupled redox state have been proposed to moderate many aging-related processes, yet the specific mechanisms remain unclear. The concentration of NAD + falls with age in skeletal muscle, yet there is no consensus on whether aging will increase or decrease the redox potential of NAD + /NADH. Oxidized flavin groups (Fp) (e.g. FAD, i.e., flavin adenine dinucleotide, contained in flavoproteins) and NADH are intrinsic fluorescent indicators of oxidation and reduction status of tissue, respectively. The redox ratio, i.e., the ratio of Fp to NADH, may be a surrogate indicator of the NAD + /NADH redox potential. In this study we used the Chance redox scanner (NADH/Fp fluorescence imaging at low temperature) to investigate the effect of aging on the redox state of mitochondria in skeletal muscles. The results showed that there are borderline significant differences in nominal concentrations of Fp and NADH, but not in the redox ratio s when comparing 3.5-month and 13-month old muscles of mice (n = 6). It may be necessary to increase the number of muscle samples and study mice of more advanced age.

  11. Non-Euclidean phasor analysis for quantification of oxidative stress in ex vivo human skin exposed to sun filters using fluorescence lifetime imaging microscopy

    NASA Astrophysics Data System (ADS)

    Osseiran, Sam; Roider, Elisabeth M.; Wang, Hequn; Suita, Yusuke; Murphy, Michael; Fisher, David E.; Evans, Conor L.

    2017-12-01

    Chemical sun filters are commonly used as active ingredients in sunscreens due to their efficient absorption of ultraviolet (UV) radiation. Yet, it is known that these compounds can photochemically react with UV light and generate reactive oxygen species and oxidative stress in vitro, though this has yet to be validated in vivo. One label-free approach to probe oxidative stress is to measure and compare the relative endogenous fluorescence generated by cellular coenzymes nicotinamide adenine dinucleotides and flavin adenine dinucleotides. However, chemical sun filters are fluorescent, with emissive properties that contaminate endogenous fluorescent signals. To accurately distinguish the source of fluorescence in ex vivo skin samples treated with chemical sun filters, fluorescence lifetime imaging microscopy data were processed on a pixel-by-pixel basis using a non-Euclidean separation algorithm based on Mahalanobis distance and validated on simulated data. Applying this method, ex vivo samples exhibited a small oxidative shift when exposed to sun filters alone, though this shift was much smaller than that imparted by UV irradiation. Given the need for investigative tools to further study the clinical impact of chemical sun filters in patients, the reported methodology may be applied to visualize chemical sun filters and measure oxidative stress in patients' skin.

  12. Electrocatalytic reaction of hydrogen peroxide and NADH based on poly(neutral red) and FAD hybrid film.

    PubMed

    Lin, Kuo Chiang; Lin, Yu Ching; Chen, Shen Ming

    2012-01-07

    A simple method to immobilize poly(neutral red) (PNR) and flavin adenine dinucleotide (FAD) hybrid film (PNR/FAD) by cyclic voltammetry is proposed. The PNR/FAD hybrid film can be easily prepared on an electrode surface involving electropolymerization of neutral red (NR) monomers and the electrostatic interaction between the positively charged PNR and the negatively charged FAD. It exhibits electroactive, stable, surface-confined, pH-dependent, nano-sized, and compatible properties. It provides good electrocatalytic properties to various species. It shows a sensitivity of 5.4 μA mM(-1) cm(-2) and 21.5 μA mM(-1) cm(-2) for hydrogen peroxide (H(2)O(2)) and nicotinamide adenine dinucleotide (NADH) with the linear range of 0.1 μM-39 mM and 5 × 10(-5) to 2.5 × 10(-4) M, respectively. It shows another linear range of 48.8-355.5 mM with the sensitivity of 12.3 μA mM(-1) cm(-2) for H(2)O(2). In particular, the PNR/FAD hybrid film has potential to replace some hemoproteins to be a cathode of biofuel cells and provide the biosensing system for glucose and ethanol. This journal is © The Royal Society of Chemistry 2012

  13. Non-Euclidean phasor analysis for quantification of oxidative stress in ex vivo human skin exposed to sun filters using fluorescence lifetime imaging microscopy.

    PubMed

    Osseiran, Sam; Roider, Elisabeth M; Wang, Hequn; Suita, Yusuke; Murphy, Michael; Fisher, David E; Evans, Conor L

    2017-12-01

    Chemical sun filters are commonly used as active ingredients in sunscreens due to their efficient absorption of ultraviolet (UV) radiation. Yet, it is known that these compounds can photochemically react with UV light and generate reactive oxygen species and oxidative stress in vitro, though this has yet to be validated in vivo. One label-free approach to probe oxidative stress is to measure and compare the relative endogenous fluorescence generated by cellular coenzymes nicotinamide adenine dinucleotides and flavin adenine dinucleotides. However, chemical sun filters are fluorescent, with emissive properties that contaminate endogenous fluorescent signals. To accurately distinguish the source of fluorescence in ex vivo skin samples treated with chemical sun filters, fluorescence lifetime imaging microscopy data were processed on a pixel-by-pixel basis using a non-Euclidean separation algorithm based on Mahalanobis distance and validated on simulated data. Applying this method, ex vivo samples exhibited a small oxidative shift when exposed to sun filters alone, though this shift was much smaller than that imparted by UV irradiation. Given the need for investigative tools to further study the clinical impact of chemical sun filters in patients, the reported methodology may be applied to visualize chemical sun filters and measure oxidative stress in patients' skin. (2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

  14. Ebselen Reversibly Inhibits Human Glutamate Dehydrogenase at the Catalytic Site.

    PubMed

    Jin, Yanhong; Li, Di; Lu, Shiying; Zhao, Han; Chen, Zhao; Hou, Wei; Ruan, Benfang Helen

    Human glutamate dehydrogenase (GDH) plays an important role in neurological diseases, tumor metabolism, and hyperinsulinism-hyperammonemia syndrome (HHS). However, there are very few inhibitors known for human GDH. Recently, Ebselen was reported to crosslink with Escherichia coli GDH at the active site cysteine residue (Cys321), but the sequence alignment showed that the corresponding residue is Ala329 in human GDH. To investigate whether Ebselen could be an inhibitor for human GDH, we cloned and expressed an N-terminal His-tagged human GDH in E. coli. The recombinant human GDH enzyme showed expected properties such as adenosine diphosphate activation and nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate dual recognition. Further, we developed a 2-(3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-tetrazol-3-ium-5-yl) benzenesulfonate sodium salt (EZMTT)-based assay for human GDH, which was highly sensitive and is suitable for high-throughput screening for potent GDH inhibitors. In addition, ForteBio binding assays demonstrated that Ebselen is a reversible active site inhibitor for human GDH. Since Ebselen is a multifunctional organoselenium compound in Phase III clinical trials for inflammation, an Ebselen-based GDH inhibitor might be valuable for future drug discovery for HHS patients.

  15. Fluorescence decay kinetics and imaging of NAD(P)H and flavins as metabolic indicators

    NASA Astrophysics Data System (ADS)

    Schneckenburger, Herbert; Koenig, Karsten

    1992-07-01

    The intrinsic fluorescence of various cell cultures in the blue and green spectral range has been attributed mainly to hydrated nicotinamide adenine dinucleotide (NADH) and flavin molecules. Their fluorescence decay curves were measured with subnanosecond resolution. The reduced coenzymes NADH and hydrated nicotinamide adenine dinucleotide phosphate NADPH, both showed a biexponential decay pattern in solution with similar time constants, but different relative intensities of the two components. They could thus be distinguished from one another as well as from their oxidized forms. The NADPH fluorescence of Saccharomyces cerevisiae was located within the cytoplasm and its organelles and was by about a factor of 4 higher for respiratory-deficient than for intact yeast strains. Intracellular flavin fluorescence showed a triexponential behavior--probably due to a superposition of protein-bound and free flavin molecules. The lifetime of the shortest component varied within the range of 0.20 to 0.50 ns between respiratory-deficient and intact yeast strains, and the relative intensity of this component was most pronounced for the intact strain DL1. Time- resolved fluorescence seems therefore to be an appropriate method of probe the function of the respiratory chain and--in the further step--to differentiate between various types of cells and tissues in medical diagnosis or environmental research.

  16. Catabolism of Phloroglucinol by the Rumen Anaerobe Coprococcus

    PubMed Central

    Patel, T. R.; Jure, K. G.; Jones, G. A.

    1981-01-01

    A rumen isolate, Coprococcus, sp. Pe15, was found to carry phloroglucinol reductase, which catalyzed the initial step in the breakdown of phloroglucinol. The organism uses phloroglucinol as the sole source of carbon and energy when grown in the absence of oxygen. Induced levels of enzyme were detected in cells grown either on phloroglucinol or on other carbon sources in the presence of limiting quantities of phloroglucinol. Although the organism is a strict anaerobe, the enzyme from anaerobically grown cells was insensitive to air. The partially purified enzyme required reduced nicotinamide adenine dinucleotide phosphate as an electron donor and was specific for phloroglucinol. However, partial enzyme activity (14 to 17%) was also detected in the presence of 2-methyl-1,4-naphthoquinone but not in the presence of several other phenolic compounds. The enzyme exhibited a higher affinity for phloroglucinol than for reduced nicotinamide adenine dinucleotide phosphate, with Km values of 3.0 × 10−5 M and 29.0 × 10−5 M, respectively. The optimum pH for maximal enzyme activity was 7.4, and the molecular weight of the native protein was about 130,000, as determined by the Sephadex gel filtration technique. PMID:16345897

  17. Blood modulates the kinetics of reactive oxygen release in pancreatic ischemia-reperfusion injury.

    PubMed

    Neeff, Hannes P; Sommer, Olaf; Meyer, Sebastian; Tinelli, Anja; Scholtes, Moritz; Hopt, Ulrich T; Drognitz, Oliver; von Dobschuetz, Ernst

    2012-10-01

    Reason for the unsuccessful use of antioxidants in transplantation might be the unknown kinetics of reactive oxygen species (ROS) release. In this study, we compared the kinetics of ROS release from rat pancreata in the presence and absence of blood. In vivo, ischemia-reperfusion injury (IRI) was induced in pancreata of male Wistar rats by occlusion of the arterial blood supply for 1 or 2 hours. In vitro, isolated pancreata were single-pass perfused with Krebs-Henseleit bicarbonate solution. Reactive oxygen species were quantified by electron spin resonance spectroscopy using CMH (1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine) as spin label. Thiols (glutathione), nicotinamide adenine dinucleotide phosphate-oxidase activity, myeloperoxidase activity, and adenosine triphosphate content were measured. During reperfusion, an increase in IRI-induced ROS in arterial blood was noted after 2 hours of warm ischemia. In sharp contrast, ROS release was immediate and short lived in blood-free perfused organs. The degree of tissue damage correlated with nicotinamide adenine dinucleotide phosphate-oxidase activity and adenosine triphosphate content. Antioxidative capacity of tissues was reduced. Electron spin resonance spectroscopy in conjunction with spin labels allows for the detection of ROS kinetics in pancreatic IRI. Reactive oxygen species kinetics are dependent on the length of the ischemic period and the presence or absence of blood.

  18. Human sperm NADH and NADPH diaphorase cytochemistry: correlation with sperm motility.

    PubMed

    Zini, A; O'Bryan, M K; Israel, L; Schlegel, P N

    1998-03-01

    We have examined the correlation between the retention of residual sperm cytoplasm and sperm motility in semen from men presenting for infertility evaluation. Semen samples (n = 12) were obtained from nonazoospermic men presenting for infertility evaluation at our institution. Samples were fractionated into high-, intermediate-, and low-density subpopulations by Percoll gradients in order to examine the correlation between the retention of residual sperm cytoplasm and sperm motility. Residual sperm cytoplasm retention was detected by cytochemical staining of sperm for nicotinamide adenine dinucleotide (NADH)- or nicotinamide adenine dinucleotide phosphate (NADPH)-dependent diaphorase activity. The different sperm subpopulations (low, intermediate, and high density) had significantly different percentages of sperm with droplet retention (analysis of variance, P < 0.05). Using either NADH or NADPH diaphorase staining as a marker of the cytoplasmic space, a significant negative correlation was observed between the percentage of sperm with residual cytoplasmic droplets and the percentage of motile sperm (r = -0.58 and -0.61, respectively, P < 0.05). Assessment of residual sperm cytoplasm retention is a simple diagnostic test. Although this test is of unproven value in the management of infertile men, this and other studies suggest that it may provide useful data on sperm function.

  19. Hydration properties of natural and synthetic DNA sequences with methylated adenine or cytosine bases in the R.DpnI target and BDNF promoter studied by molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Shanak, Siba; Helms, Volkhard

    2014-12-01

    Adenine and cytosine methylation are two important epigenetic modifications of DNA sequences at the levels of the genome and transcriptome. To characterize the differential roles of methylating adenine or cytosine with respect to their hydration properties, we performed conventional MD simulations and free energy perturbation calculations for two particular DNA sequences, namely the brain-derived neurotrophic factor (BDNF) promoter and the R.DpnI-bound DNA that are known to undergo methylation of C5-methyl cytosine and N6-methyl adenine, respectively. We found that a single methylated cytosine has a clearly favorable hydration free energy over cytosine since the attached methyl group has a slightly polar character. In contrast, capping the strongly polar N6 of adenine with a methyl group gives a slightly unfavorable contribution to its free energy of solvation. Performing the same demethylation in the context of a DNA double-strand gave quite similar results for the more solvent-accessible cytosine but much more unfavorable results for the rather buried adenine. Interestingly, the same demethylation reactions are far more unfavorable when performed in the context of the opposite (BDNF or R.DpnI target) sequence. This suggests a natural preference for methylation in a specific sequence context. In addition, free energy calculations for demethylating adenine or cytosine in the context of B-DNA vs. Z-DNA suggest that the conformational B-Z transition of DNA transition is rather a property of cytosine methylated sequences but is not preferable for the adenine-methylated sequences investigated here.

  20. Hydration properties of natural and synthetic DNA sequences with methylated adenine or cytosine bases in the R.DpnI target and BDNF promoter studied by molecular dynamics simulations.

    PubMed

    Shanak, Siba; Helms, Volkhard

    2014-12-14

    Adenine and cytosine methylation are two important epigenetic modifications of DNA sequences at the levels of the genome and transcriptome. To characterize the differential roles of methylating adenine or cytosine with respect to their hydration properties, we performed conventional MD simulations and free energy perturbation calculations for two particular DNA sequences, namely the brain-derived neurotrophic factor (BDNF) promoter and the R.DpnI-bound DNA that are known to undergo methylation of C5-methyl cytosine and N6-methyl adenine, respectively. We found that a single methylated cytosine has a clearly favorable hydration free energy over cytosine since the attached methyl group has a slightly polar character. In contrast, capping the strongly polar N6 of adenine with a methyl group gives a slightly unfavorable contribution to its free energy of solvation. Performing the same demethylation in the context of a DNA double-strand gave quite similar results for the more solvent-accessible cytosine but much more unfavorable results for the rather buried adenine. Interestingly, the same demethylation reactions are far more unfavorable when performed in the context of the opposite (BDNF or R.DpnI target) sequence. This suggests a natural preference for methylation in a specific sequence context. In addition, free energy calculations for demethylating adenine or cytosine in the context of B-DNA vs. Z-DNA suggest that the conformational B-Z transition of DNA transition is rather a property of cytosine methylated sequences but is not preferable for the adenine-methylated sequences investigated here.

  1. Proton-impact ionization cross sections of adenine measured at 0.5 and 2.0 MeV by electron spectroscopy

    NASA Astrophysics Data System (ADS)

    Iriki, Y.; Kikuchi, Y.; Imai, M.; Itoh, A.

    2011-11-01

    Double-differential ionization cross sections (DDCSs) of vapor-phase adenine molecules (C5H5N5) by 0.5- and 2.0-MeV proton impact have been measured by the electron spectroscopy method. Electrons ejected from adenine were analyzed by a 45∘ parallel-plate electrostatic spectrometer over an energy range of 1.0-1000 eV at emission angles from 15∘ to 165∘. Single-differential cross sections (SDCSs) and total ionization cross sections (TICSs) were also deduced. It was found from the Platzman plot, defined as SDCSs divided by the classical Rutherford knock-on cross sections per target electron, that the SDCSs at higher electron energies are proportional to the total number of valence electrons (50) of adenine, while those at low-energy electrons are highly enhanced due to dipole and higher-order interactions. The present results of TICS are in fairly good agreement with recent classical trajectory Monte Carlo calculations, and moreover, a simple analytical formula gives nearly equivalent cross sections in magnitude at the incident proton energies investigated.

  2. Kinetics and Thermodynamics of the Reaction between the (•)OH Radical and Adenine: A Theoretical Investigation.

    PubMed

    Milhøj, Birgitte O; Sauer, Stephan P A

    2015-06-18

    The accessibility of all possible reaction paths for the reaction between the nucleobase adenine and the (•)OH radical is investigated through quantum chemical calculations of barrier heights and rate constants at the ωB97X-D/6-311++G(2df,2pd) level with Eckart tunneling corrections. First the computational method is validated by considering the hydrogen abstraction from the heterocyclic N9 nitrogen in adenine as a test system. Geometries for all molecules in the reaction are optimized with four different DFT exchange-correlation functionals (B3LYP, BHandHLYP, M06-2X, and ωB97X-D), in combination with Pople and Dunning basis sets, all of which have been employed in similar investigations in the literature. Improved energies are obtained through single point calculations with CCSD(T) and the same basis sets, and reaction rate constants are calculated for all methods both without tunneling corrections and with the Wigner, Bell, and Eckart corrections. In comparison to CCSD(T)//BHandHLYP/aug-cc-pVTZ reference results, the ωB97X-D/6-311++G(2df,2pd) method combined with Eckart tunneling corrections provides a sensible compromise between accuracy and time. Using this method, all subreactions of the reaction between adenine and the (•)OH radical are investigated. The total rate constants for hydrogen abstraction and addition for adenine are predicted with this method to be 1.06 × 10(-12) and 1.10 × 10(-12) cm(3) molecules(-1) s(-1), respectively. Abstractions of H61 and H62 contribute the most, while only addition to the C8 carbon is found to be of any significance, in contrast to previous claims that addition is the dominant reaction pathway. The overall rate constant for the complete reaction is found to be 2.17 × 10(-12) cm(3) molecules(-1) s(-1), which agrees exceptionally well with experimental results.

  3. NAD(P)H-Independent Asymmetric C=C Bond Reduction Catalyzed by Ene Reductases by Using Artificial Co-substrates as the Hydrogen Donor

    PubMed Central

    Winkler, Christoph K; Clay, Dorina; Entner, Marcello; Plank, Markus; Faber, Kurt

    2014-01-01

    To develop a nicotinamide-independent single flavoenzyme system for the asymmetric bioreduction of C=C bonds, four types of hydrogen donor, encompassing more than 50 candidates, were investigated. Six highly potent, cheap, and commercially available co-substrates were identified that (under the optimized conditions) resulted in conversions and enantioselectivities comparable with, or even superior to, those obtained with traditional two-enzyme nicotinamide adenine dinucleotide phosphate (NAD(P)H)-recycling systems. PMID:24382795

  4. An Enzymatic Bioassay for Perchlorate

    DTIC Science & Technology

    2010-07-01

    redox active dye phenazine methosulfate (PMS) and nicotine adenine dinucleotide (NADH). By using a specific addition scheme and covering all...redox potentials determined from (Fultz and Durst, 1982)). The dye structures include an indole, a quinone, a bipyridinium, and two phenazine ...listed above, as well as 100 μM of the dye shown on the x-axis. As can be seen in figure 18, phenazine methosulfate (PMS, fourth from the left) is

  5. Native fluorescence spectroscopy of blood plasma of rats with experimental diabetes: identifying fingerprints of glucose-related metabolic pathways

    NASA Astrophysics Data System (ADS)

    Shirshin, Evgeny; Cherkasova, Olga; Tikhonova, Tatiana; Berlovskaya, Elena; Priezzhev, Alexander; Fadeev, Victor

    2015-05-01

    We present the results of a native fluorescence spectroscopy study of blood plasma of rats with experimental diabetes. It was shown that the fluorescence emission band shape at 320 nm excitation is the most indicative of hyperglycemia in the blood plasma samples. We provide the interpretation of this fact based on the changes in reduced nicotinamide adenine dinucleotide phosphate concentration due to glucose-related metabolic pathways and protein fluorescent cross-linking formation following nonenzymatic glycation.

  6. Spectroscopic study of intermolecular complexes between FAD and some β-carboline derivatives

    NASA Astrophysics Data System (ADS)

    Codoñer, Armando; Monzó, Isidro S.; Tomás, Francisco; Valero, Rosa

    The formation of molecular complexes between flavine adenine dinucleotide (FAD) and some β-carboline derivatives [antidepressant drugs that have a pronounced inhibition of monoamine oxidase (MAO)] has been studied by using electronic absorption and fluorescence spectroscopic methods. Thermodynamic parameters have been determined from the values of association constants for the molecular complexes at various temperatures. The influence of substituents in the β-carboline molecule on the stability of the complexes formed was also investigated.

  7. Defining efficient enzyme-cofactor pairs for bioorthogonal profiling of protein methylation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Islam, Kabirul; Chen, Yuling; Wu, Hong

    2013-11-18

    Protein methyltransferase (PMT)-mediated posttranslational modification of histone and nonhistone substrates modulates stability, localization, and interacting partners of target proteins in diverse cellular contexts. These events play critical roles in normal biological processes and are frequently deregulated in human diseases. In the course of identifying substrates of individual PMTs, bioorthogonal profiling of protein methylation (BPPM) has demonstrated its merits. In this approach, specific PMTs are engineered to process S-adenosyl-L-methionine (SAM) analogs as cofactor surrogates and label their substrates with distinct chemical modifications for target elucidation. Despite the proof-of-concept advancement of BPPM, few efforts have been made to explore its generality. Withmore » two cancer-relevant PMTs, EuHMT1 (GLP1/KMT1D) and EuHMT2 (G9a/KMT1C), as models, we defined the key structural features of engineered PMTs and matched SAM analogs that can render the orthogonal enzyme–cofactor pairs for efficient catalysis. Here we have demonstrated that the presence of sulfonium-β-sp 2 carbon and flexible, medium-sized sulfonium-δ-substituents are crucial for SAM analogs as BPPM reagents. The bulky cofactors can be accommodated by tailoring the conserved Y1211/Y1154 residues and nearby hydrophobic cavities of EuHMT1/2. Profiling proteome-wide substrates with BPPM allowed identification of >500 targets of EuHMT1/2 with representative targets validated using native EuHMT1/2 and SAM. This finding indicates that EuHMT1/2 may regulate many cellular events previously unrecognized to be modulated by methylation. The present work, therefore, paves the way to a broader application of the BPPM technology to profile methylomes of diverse PMTs and elucidate their downstream functions.« less

  8. 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.

  9. CD/MCD/VTVH-MCD Studies of Escherichia coli Bacterioferritin Support a Binuclear Iron Cofactor Site.

    PubMed

    Kwak, Yeonju; Schwartz, Jennifer K; Huang, Victor W; Boice, Emily; Kurtz, Donald M; Solomon, Edward I

    2015-12-01

    Ferritins and bacterioferritins (Bfrs) utilize a binuclear non-heme iron binding site to catalyze oxidation of Fe(II), leading to formation of an iron mineral core within a protein shell. Unlike ferritins, in which the diiron site binds Fe(II) as a substrate, which then autoxidizes and migrates to the mineral core, the diiron site in Bfr has a 2-His/4-carboxylate ligand set that is commonly found in diiron cofactor enzymes. Bfrs could, therefore, utilize the diiron site as a cofactor rather than for substrate iron binding. In this study, we applied circular dichroism (CD), magnetic CD (MCD), and variable-temperature, variable-field MCD (VTVH-MCD) spectroscopies to define the geometric and electronic structures of the biferrous active site in Escherichia coli Bfr. For these studies, we used an engineered M52L variant, which is known to eliminate binding of a heme cofactor but to have very minor effects on either iron oxidation or mineral core formation. We also examined an H46A/D50A/M52L Bfr variant, which additionally disrupts a previously observed mononuclear non-heme iron binding site inside the protein shell. The spectral analyses define a binuclear and an additional mononuclear ferrous site. The biferrous site shows two different five-coordinate centers. After O2 oxidation and re-reduction, only the mononuclear ferrous signal is eliminated. The retention of the biferrous but not the mononuclear ferrous site upon O2 cycling supports a mechanism in which the binuclear site acts as a cofactor for the O2 reaction, while the mononuclear site binds the substrate Fe(II) that, after its oxidation to Fe(III), migrates to the mineral core.

  10. A network analysis of cofactor-protein interactions for analyzing associations between human nutrition and diseases

    PubMed Central

    Scott-Boyer, Marie Pier; Lacroix, Sébastien; Scotti, Marco; Morine, Melissa J.; Kaput, Jim; Priami, Corrado

    2016-01-01

    The involvement of vitamins and other micronutrients in intermediary metabolism was elucidated in the mid 1900’s at the level of individual biochemical reactions. Biochemical pathways remain the foundational knowledgebase for understanding how micronutrient adequacy modulates health in all life stages. Current daily recommended intakes were usually established on the basis of the association of a single nutrient to a single, most sensitive adverse effect and thus neglect interdependent and pleiotropic effects of micronutrients on biological systems. Hence, the understanding of the impact of overt or sub-clinical nutrient deficiencies on biological processes remains incomplete. Developing a more complete view of the role of micronutrients and their metabolic products in protein-mediated reactions is of importance. We thus integrated and represented cofactor-protein interaction data from multiple and diverse sources into a multi-layer network representation that links cofactors, cofactor-interacting proteins, biological processes, and diseases. Network representation of this information is a key feature of the present analysis and enables the integration of data from individual biochemical reactions and protein-protein interactions into a systems view, which may guide strategies for targeted nutritional interventions aimed at improving health and preventing diseases. PMID:26777674

  11. A new microplatform based on titanium dioxide nanofibers/graphene oxide nanosheets nanocomposite modified screen printed carbon electrode for electrochemical determination of adenine in the presence of guanine.

    PubMed

    Arvand, Majid; Ghodsi, Navid; Zanjanchi, Mohammad Ali

    2016-03-15

    The current techniques for determining adenine have several shortcomings such as high cost, high time consumption, tedious pretreatment steps and the requirements for highly skilled personnel often restrict their use in routine analytical practice. This paper describes the development and utilization of a new nanocomposite consisting of titanium dioxide nanofibers (TNFs) and graphene oxide nanosheets (GONs) for screen printed carbon electrode (SPCE) modification. The synthesized GONs and TNFs were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR). The modified electrode (TNFs/GONs/SPCE) was used for electrochemical characterization of adenine. The TNFs/GONs/SPCE exhibited an increase in peak current and the electron transfer kinetics and decrease in the overpotential for the oxidation reaction of adenine. Using differential pulse voltammetry (DPV), the prepared sensor showed good sensitivity for determining adenine in two ranges from 0.1-1 and 1-10 μM, with a detection limit (DL) of 1.71 nM. Electrochemical studies suggested that the TNFs/GONs/SPCE provided a synergistic augmentation on the voltammetric behavior of electrochemical oxidation of adenine, which was indicated by the improvement of anodic peak current and a decrease in anodic peak potential. The amount of adenine in pBudCE4.1 plasmid was determined via the proposed sensor and the result was in good compatibility with the sequence data of pBudCE4.1 plasmid. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Adenine derivatives as phosphate-activating groups for the regioselective formation of 3',5'-linked oligoadenylates on montmorillonite: possible phosphate-activating groups for the prebiotic synthesis of RNA

    NASA Technical Reports Server (NTRS)

    Prabahar, K. J.; Ferris, J. P.

    1997-01-01

    Methyladenine and adenine N-phosphoryl derivatives of adenosine 5'-monophosphate (5'-AMP) and uridine 5'-monophosphate (5'-UMP) are synthesized, and their structures are elucidated. The oligomerization reactions of the adenine derivatives of 5'-phosphoramidates of adenosine on montmorillonite are investigated. 1-Methyladenine and 3-methyladenine derivatives on montmorillonite yielded oligoadenylates as long as undecamer, and the 2-methyladenine and adenine derivatives on montmorillonite yielded oligomers up to hexamers and pentamers, respectively. The 1-methyladenine derivative yielded linear, cyclic, and A5'ppA-derived oligonucleotides with a regioselectivity for the 3',5'-phosphodiester linkages averaging 84%. The effect of pKa and amine structure of phosphate-activating groups on the montmorillonite-catalyzed oligomerization of the 5'-phosphoramidate of adenosine are discussed. The binding and reaction of methyladenine and adenine N-phosphoryl derivatives of adenosine are described.

  13. Zhen-wu-tang ameliorates adenine-induced chronic renal failure in rats: regulation of the canonical Wnt4/beta-catenin signaling in the kidneys.

    PubMed

    La, Lei; Wang, Lili; Qin, Fei; Jiang, Jian; He, Songqi; Wang, Chunxia; Li, Yuhao

    2018-06-12

    Zhen-wu-tang (ZWT), composed of Radix Aconiti lateralis, Rhizoma Atractylodis macrocephalae, Poria, Radix Paeoniae alba and ginger, is a classic Chinese herbal formula for the treatment of chronic kidney diseases that may cause chronic renal failure (CRF). To better understand its clinical use, this study investigated the effects and underlying mechanisms of action of ZWT on CRF. CRF was induced by adenine. ZWT was given via an oral gavage method. The serum biochemical parameters were measured enzymatically or by ELISA. The kidneys were examined pathohistologically. The gene expression was analyzed by real time PCR and Western blot. Similar to the positive control losartan, ZWT extract inhibited adenine-induced increase in serum concentrations of creatinine, BUN and advanced oxidation protein products in rats. These effects were accompanied by attenuation of proteinuria and renal pathological changes and suppression of renal mRNA and protein overexpression of Collagen IV and fibronectin, two of the key components of fibrosis. Mechanistically, renal mRNA and protein expression of Wnt4, a Wnt signaling ligand, was increased in the adenine-treated group, compared to the vehicle-treated control. Consistently, Wnt4 downstream genes beta-catenin and Axin were also overexpressed. Treatment with ZWT extract and losartan suppressed adenine-stimulated overexpression of these mRNAs and proteins. The present results demonstrate that ZWT extract ameliorates adenine-induced CRF in rats by regulation of the canonical Wnt4/beta-catenin signaling in the kidneys. Our findings provide new insight into the underlying renoprotective mechanisms of the ancient formula. Copyright © 2017. Published by Elsevier B.V.

  14. Insect Cell-Derived Cofactors Become Fully Functional after Proteinase K and Heat Treatment for High-Fidelity Amplification of Glycosylphosphatidylinositol-Anchored Recombinant Scrapie and BSE Prion Proteins

    PubMed Central

    Imamura, Morikazu; Kato, Nobuko; Okada, Hiroyuki; Yoshioka, Miyako; Iwamaru, Yoshifumi; Shimizu, Yoshihisa; Mohri, Shirou; Yokoyama, Takashi; Murayama, Yuichi

    2013-01-01

    The central event in prion infection is the conformational conversion of host-encoded cellular prion protein (PrPC) into the pathogenic isoform (PrPSc). Diverse mammalian species possess the cofactors required for in vitro replication of PrPSc by protein-misfolding cyclic amplification (PMCA), but lower organisms, such as bacteria, yeasts, and insects, reportedly lack the essential cofactors. Various cellular components, such as RNA, lipids, and other identified cofactor molecules, are commonly distributed in both eukaryotes and prokaryotes, but the reasons for the absence of cofactor activity in lower organisms remain to be elucidated. Previously, we reported that brain-derived factors were necessary for the in vitro replication of glycosylphosphatidylinositol-anchored baculovirus-derived recombinant PrP (Bac-PrP). Here, we demonstrate that following protease digestion and heat treatment, insect cell lysates had the functional cofactor activity required for Bac-PrP replication by PMCA. Mammalian PrPSc seeds and Bac-PrPSc generated by PMCA using Bac-PrP and insect cell-derived cofactors showed similar pathogenicity and produced very similar lesions in the brains of inoculated mice. These results suggested that the essential cofactors required for the high-fidelity replication of mammalian PrPSc were present in the insect cells but that the cofactor activity was masked or inhibited in the native state. We suggest that not only RNA, but also DNA, are the key components of PMCA, although other cellular factors were necessary for the expression of the cofactor activity of nucleic acids. PMCA using only insect cell-derived substances (iPMCA) was highly useful for the ultrasensitive detection of PrPSc of some prion strains. PMID:24367521

  15. Structural Basis for Assembly of the MnIV/FeIII Cofactor in the Class Ic Ribonucleotide Reductase from Chlamydia trachomatis‡

    PubMed Central

    Dassama, Laura M.K.; Krebs, Carsten; Bollinger, J. Martin; Rosenzweig, Amy C.; Boal, Amie K.

    2013-01-01

    The class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (Ct) employs a MnIV/FeIII cofactor in each monomer of its β2 subunit to initiate nucleotide reduction. The cofactor forms by reaction of MnII/FeII-β2 with O2. Previously, in vitro cofactor assembly from apo β2 and divalent metal ions produced a mixture of two forms, with Mn in site 1 (MnIV/FeIII) or site 2 (FeIII/MnIV), of which the more active MnIV/FeIII product predominates. Here we have addressed the basis for metal site-selectivity by solving X-ray crystal structures of apo, MnII, and MnII/FeII complexes of Ct β2. A structure obtained anaerobically with equimolar MnII, FeII, and apo protein reveals exclusive incorporation of MnII in site 1 and FeII in site 2, in contrast to the more modest site-selectivity achieved previously. Site-specificity is controlled thermodynamically by the apo protein structure, as only minor adjustments of ligands occur upon metal binding. Additional structures imply that, by itself, MnII binds in either site. Together the structures are consistent with a model for in vitro cofactor assembly in which FeII specificity for site 2 drives assembly of the appropriately configured heterobimetallic center, provided that FeII is substoichiometric. This model suggests that use of an MnIV/FeIII cofactor in vivo could be an adaptation to FeII limitation. A 1.8 Å resolution model of the MnII/FeII-β2 complex reveals additional structural determinants for activation of the cofactor, including a proposed site for side-on (η2) addition of O2 to FeII and a short (3.2 Å) MnII-FeII interionic distance, promoting formation of the MnIV/FeIV activation intermediate. PMID:23924396

  16. Decoding a Signature-Based Model of Transcription Cofactor Recruitment Dictated by Cardinal Cis-Regulatory Elements in Proximal Promoter Regions

    PubMed Central

    Benner, Christopher; Hutt, Kasey R.; Stunnenberg, Rieka; Garcia-Bassets, Ivan

    2013-01-01

    Genome-wide maps of DNase I hypersensitive sites (DHSs) reveal that most human promoters contain perpetually active cis-regulatory elements between −150 bp and +50 bp (−150/+50 bp) relative to the transcription start site (TSS). Transcription factors (TFs) recruit cofactors (chromatin remodelers, histone/protein-modifying enzymes, and scaffold proteins) to these elements in order to organize the local chromatin structure and coordinate the balance of post-translational modifications nearby, contributing to the overall regulation of transcription. However, the rules of TF-mediated cofactor recruitment to the −150/+50 bp promoter regions remain poorly understood. Here, we provide evidence for a general model in which a series of cis-regulatory elements (here termed ‘cardinal’ motifs) prefer acting individually, rather than in fixed combinations, within the −150/+50 bp regions to recruit TFs that dictate cofactor signatures distinctive of specific promoter subsets. Subsequently, human promoters can be subclassified based on the presence of cardinal elements and their associated cofactor signatures. In this study, furthermore, we have focused on promoters containing the nuclear respiratory factor 1 (NRF1) motif as the cardinal cis-regulatory element and have identified the pervasive association of NRF1 with the cofactor lysine-specific demethylase 1 (LSD1/KDM1A). This signature might be distinctive of promoters regulating nuclear-encoded mitochondrial and other particular genes in at least some cells. Together, we propose that decoding a signature-based, expanded model of control at proximal promoter regions should lead to a better understanding of coordinated regulation of gene transcription. PMID:24244184

  17. Electron transfer flavoprotein domain II orientation monitored using double electron-electron resonance between an enzymatically reduced, native FAD cofactor, and spin labels.

    PubMed

    Swanson, Michael A; Kathirvelu, Velavan; Majtan, Tomas; Frerman, Frank E; Eaton, Gareth R; Eaton, Sandra S

    2011-03-01

    Human electron transfer flavoprotein (ETF) is a soluble mitochondrial heterodimeric flavoprotein that links fatty acid β-oxidation to the main respiratory chain. The crystal structure of human ETF bound to medium chain acyl-CoA dehydrogenase indicates that the flavin adenine dinucleotide (FAD) domain (αII) is mobile, which permits more rapid electron transfer with donors and acceptors by providing closer access to the flavin and allows ETF to accept electrons from at least 10 different flavoprotein dehydrogenases. Sequence homology is high and low-angle X-ray scattering is identical for Paracoccus denitrificans (P. denitrificans) and human ETF. To characterize the orientations of the αII domain of P. denitrificans ETF, distances between enzymatically reduced FAD and spin labels in the three structural domains were measured by double electron-electron resonance (DEER) at X- and Q-bands. An FAD to spin label distance of 2.8 ± 0.15 nm for the label in the FAD-containing αII domain (A210C) agreed with estimates from the crystal structure (3.0 nm), molecular dynamics simulations (2.7 nm), and rotamer library analysis (2.8 nm). Distances between the reduced FAD and labels in αI (A43C) were between 4.0 and 4.5 ± 0.35 nm and for βIII (A111C) the distance was 4.3 ± 0.15 nm. These values were intermediate between estimates from the crystal structure of P. denitrificans ETF and a homology model based on substrate-bound human ETF. These distances suggest that the αII domain adopts orientations in solution that are intermediate between those which are observed in the crystal structures of free ETF (closed) and ETF bound to a dehydrogenase (open). Copyright © 2011 The Protein Society.

  18. Electron transfer flavoprotein domain II orientation monitored using double electron-electron resonance between an enzymatically reduced, native FAD cofactor, and spin labels

    PubMed Central

    Swanson, Michael A; Kathirvelu, Velavan; Majtan, Tomas; Frerman, Frank E; Eaton, Gareth R; Eaton, Sandra S

    2011-01-01

    Human electron transfer flavoprotein (ETF) is a soluble mitochondrial heterodimeric flavoprotein that links fatty acid β-oxidation to the main respiratory chain. The crystal structure of human ETF bound to medium chain acyl-CoA dehydrogenase indicates that the flavin adenine dinucleotide (FAD) domain (αII) is mobile, which permits more rapid electron transfer with donors and acceptors by providing closer access to the flavin and allows ETF to accept electrons from at least 10 different flavoprotein dehydrogenases. Sequence homology is high and low-angle X-ray scattering is identical for Paracoccus denitrificans (P. denitrificans) and human ETF. To characterize the orientations of the αII domain of P. denitrificans ETF, distances between enzymatically reduced FAD and spin labels in the three structural domains were measured by double electron-electron resonance (DEER) at X- and Q-bands. An FAD to spin label distance of 2.8 ± 0.15 nm for the label in the FAD-containing αII domain (A210C) agreed with estimates from the crystal structure (3.0 nm), molecular dynamics simulations (2.7 nm), and rotamer library analysis (2.8 nm). Distances between the reduced FAD and labels in αI (A43C) were between 4.0 and 4.5 ± 0.35 nm and for βIII (A111C) the distance was 4.3 ± 0.15 nm. These values were intermediate between estimates from the crystal structure of P. denitrificans ETF and a homology model based on substrate-bound human ETF. These distances suggest that the αII domain adopts orientations in solution that are intermediate between those which are observed in the crystal structures of free ETF (closed) and ETF bound to a dehydrogenase (open). PMID:21308847

  19. Cofactor specificity motifs and the induced fit mechanism in class I ketol-acid reductoisomerases.

    PubMed

    Cahn, Jackson K B; Brinkmann-Chen, Sabine; Spatzal, Thomas; Wiig, Jared A; Buller, Andrew R; Einsle, Oliver; Hu, Yilin; Ribbe, Markus W; Arnold, Frances H

    2015-06-15

    Although most sequenced members of the industrially important ketol-acid reductoisomerase (KARI) family are class I enzymes, structural studies to date have focused primarily on the class II KARIs, which arose through domain duplication. In the present study, we present five new crystal structures of class I KARIs. These include the first structure of a KARI with a six-residue β2αB (cofactor specificity determining) loop and an NADPH phosphate-binding geometry distinct from that of the seven- and 12-residue loops. We also present the first structures of naturally occurring KARIs that utilize NADH as cofactor. These results show insertions in the specificity loops that confounded previous attempts to classify them according to loop length. Lastly, we explore the conformational changes that occur in class I KARIs upon binding of cofactor and metal ions. The class I KARI structures indicate that the active sites close upon binding NAD(P)H, similar to what is observed in the class II KARIs of rice and spinach and different from the opening of the active site observed in the class II KARI of Escherichia coli. This conformational change involves a decrease in the bending of the helix that runs between the domains and a rearrangement of the nicotinamide-binding site. © The Authors Journal Compilation © 2015 Biochemical Society.

  20. Cytosine methylation effects on the repair of O6-methylguanines within CG dinucleotides.

    PubMed

    Guza, Rebecca; Ma, Linan; Fang, Qingming; Pegg, Anthony E; Tretyakova, Natalia

    2009-08-21

    O(6)-alkyldeoxyguanine adducts induced by tobacco-specific nitrosamines are repaired by O(6)-alkylguanine DNA alkyltransferase (AGT), which transfers the O(6)-alkyl group from the damaged base to a cysteine residue within the protein. In the present study, a mass spectrometry-based approach was used to analyze the effects of cytosine methylation on the kinetics of AGT repair of O(6)-methyldeoxyguanosine (O(6)-Me-dG) adducts placed within frequently mutated 5'-CG-3' dinucleotides of the p53 tumor suppressor gene. O(6)-Me-dG-containing DNA duplexes were incubated with human recombinant AGT protein, followed by rapid quenching, acid hydrolysis, and isotope dilution high pressure liquid chromatography-electrospray ionization tandem mass spectrometry analysis of unrepaired O(6)-methylguanine. Second-order rate constants were calculated in the absence or presence of the C-5 methyl group at neighboring cytosine residues. We found that the kinetics of AGT-mediated repair of O(6)-Me-dG were affected by neighboring 5-methylcytosine ((Me)C) in a sequence-dependent manner. AGT repair of O(6)-Me-dG adducts placed within 5'-CG-3' dinucleotides of p53 codons 245 and 248 was hindered when (Me)C was present in both DNA strands. In contrast, cytosine methylation within p53 codon 158 slightly increased the rate of O(6)-Me-dG repair by AGT. The effects of (Me)C located immediately 5' and in the base paired position to O(6)-Me-dG were not additive as revealed by experiments with hypomethylated sequences. Furthermore, differences in dealkylation rates did not correlate with AGT protein affinity for cytosine-methylated and unmethylated DNA duplexes or with the rates of AGT-mediated nucleotide flipping, suggesting that (Me)C influences other kinetic steps involved in repair, e.g. the rate of alkyl transfer from DNA to AGT.

  1. Robust Production, Crystallization, Structure Determination, and Analysis of [Fe-S] Proteins: Uncovering Control of Electron Shuttling and Gating in the Respiratory Metabolism of Molybdopterin Guanine Dinucleotide Enzymes.

    PubMed

    Tsai, Chi-Lin; Tainer, John A

    2018-01-01

    [Fe-S] clusters are essential cofactors in all domains of life. They play many biological roles due to their unique abilities for electron transfer and conformational control. Yet, producing and analyzing Fe-S proteins can be difficult and even misleading if not done anaerobically. Due to unique redox properties of [Fe-S] clusters and their oxygen sensitivity, they pose multiple challenges and can lose enzymatic activity or cause their component proteins to be structurally disordered due to [Fe-S] cluster oxidation and loss in air. Here we highlight tested protocols and strategies enabling efficient and stable [Fe-S] protein production, purification, crystallization, X-ray diffraction data collection, and structure determination. From multiple high-resolution anaerobic crystal structures, we furthermore analyze exemplary data defining [Fe-S] clusters, substrate entry, and product exit for the functional oxidation states of type II molybdo-bis(molybdopterin guanine dinucleotide) (Mo-bisMGD) enzymes. Notably, these enzymes perform electron shuttling between quinone pools and specific substrates to catalyze respiratory metabolism. The identified structure-activity relationships for this enzyme class have broad implications germane to perchlorate environments on Earth and Mars extending to an alternative mechanism underlying metabolic origins for the evolution of the oxygen atmosphere. Integrated structural analyses of type II Mo-bisMGD enzymes unveil novel distinctive shared molecular mechanisms for dynamic control of substrate entry and product release gated by hydrophobic residues. Collective findings support a prototypic model for type II Mo-bisMGD enzymes including insights for a fundamental molecular mechanistic understanding of selectivity and regulation by a conformationally gated channel with general implications for [Fe-S] cluster respiratory enzymes. © 2018 Elsevier Inc. All rights reserved.

  2. Germination Requirements of Bacillus macerans Spores

    PubMed Central

    Sacks, L. E.; Thompson, P. A.

    1971-01-01

    2-Phenylacetamide is an effective germinant for spores of five strains of Bacillus macerans, particularly in the presence of fructose. Benzyl penicillin, the phenyl acetamide derivative of penicillin, and phenylacetic acid are also good germinants. l-Asparagine is an excellent germinant for four strains. α-Amino-butyric acid is moderately effective. Pyridoxine, pyridoxal, adenine, and 2,6-diaminopurine are potent germinants for NCA strain 7X1 only. d-Glucose is a powerful germinant for strain B-70 only. d-Fructose and d-ribose strongly potentiate germination induced by other germinants (except l-asparagine) but have only weak activity by themselves. Niacinamide and nicotinamide-adenine dinucleotide, inactive by themselves, are active in the presence of fructose or ribose. Effects of pH, ion concentration, and temperature are described. PMID:4251279

  3. Detection of Guanine and Adenine Using an Aminated Reduced Graphene Oxide Functional Membrane-Modified Glassy Carbon Electrode

    PubMed Central

    Li, Di; Yang, Xiao-Lu; Xiao, Bao-Lin; Geng, Fang-Yong; Hong, Jun; Sheibani, Nader; Moosavi-Movahedi, Ali Akbar

    2017-01-01

    A new electrochemical sensor based on a Nafion, aminated reduced graphene oxide and chitosan functional membrane-modified glassy carbon electrode was proposed for the simultaneous detection of adenine and guanine. Fourier transform-infrared spectrometry (FTIR), transmission electron microscopy (TEM), and electrochemical methods were utilized for the additional characterization of the membrane materials. The prepared electrode was utilized for the detection of guanine (G) and adenine (A). The anodic peak currents to G and A were linear in the concentrations ranging from 0.1 to 120 μM and 0.2 to 110 μM, respectively. The detection limits were found to be 0.1 μM and 0.2 μM, respectively. Moreover, the modified electrode could also be used to determine G and A in calf thymus DNA. PMID:28718793

  4. Fabrication of submicron proteinaceous structures by direct laser writing

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Serien, Daniela; Takeuchi, Shoji, E-mail: takeuchi@iis.u-tokyo.ac.jp; ERATO Takeuchi Biohybrid Innovation Project, Japan Science and Technology Agency, 4-6-1 Komaba, Meguro-ku, 153-8505 Tokyo

    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.

  5. Structures of NADH and CH[subscript 3]-H[subscript 4] Folate Complexes of Escherichia coli Methylenetetrahydrofolate Reductase Reveal a Spartan Strategy for a Ping-Pong Reaction

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pejchal, Robert; Sargeant, Ryan; Ludwig, Martha L.

    Methylenetetrahydrofolate reductases (MTHFRs; EC 1.7.99.5) catalyze the NAD(P)H-dependent reduction of 5,10-methylenetetrahydrofolate (CH{sub 2}-H{sub 4}folate) to 5-methyltetrahydrofolate (CH{sub 3}-H{sub 4}folate) using flavin adenine dinucleotide (FAD) as a cofactor. The initial X-ray structure of Escherichia coli MTHFR revealed that this 33-kDa polypeptide is a ({beta}{alpha}){sub 8} barrel that aggregates to form an unusual tetramer with only 2-fold symmetry. Structures of reduced enzyme complexed with NADH and of oxidized Glu28Gln enzyme complexed with CH{sub 3}-H{sub 4}folate have now been determined at resolutions of 1.95 and 1.85 {angstrom}, respectively. The NADH complex reveals a rare mode of dinucleotide binding; NADH adopts a hairpin conformationmore » and is sandwiched between a conserved phenylalanine, Phe223, and the isoalloxazine ring of FAD. The nicotinamide of the bound pyridine nucleotide is stacked against the si face of the flavin ring with C4 adjoining the N5 of FAD, implying that this structure models a complex that is competent for hydride transfer. In the complex with CH{sub 3}-H{sub 4}folate, the pterin ring is also stacked against FAD in an orientation that is favorable for hydride transfer. Thus, the binding sites for the two substrates overlap, as expected for many enzymes that catalyze ping-pong reactions, and several invariant residues interact with both folate and pyridine nucleotide substrates. Comparisons of liganded and substrate-free structures reveal multiple conformations for the loops {beta}2-{alpha}2 (L2), {beta}3-{alpha}3 (L3), and {beta}4-{alpha}4 (L4) and suggest that motions of these loops facilitate the ping-pong reaction. In particular, the L4 loop adopts a 'closed' conformation that allows Asp120 to hydrogen bond to the pterin ring in the folate complex but must move to an 'open' conformation to allow NADH to bind.« less

  6. Deconvoluting AMP-activated protein kinase (AMPK) adenine nucleotide binding and sensing

    PubMed Central

    Gu, Xin; Yan, Yan; Novick, Scott J.; Kovach, Amanda; Goswami, Devrishi; Ke, Jiyuan; Tan, M. H. Eileen; Wang, Lili; Li, Xiaodan; de Waal, Parker W.; Webb, Martin R.; Griffin, Patrick R.; Xu, H. Eric

    2017-01-01

    AMP-activated protein kinase (AMPK) is a central cellular energy sensor that adapts metabolism and growth to the energy state of the cell. AMPK senses the ratio of adenine nucleotides (adenylate energy charge) by competitive binding of AMP, ADP, and ATP to three sites (CBS1, CBS3, and CBS4) in its γ-subunit. Because these three binding sites are functionally interconnected, it remains unclear how nucleotides bind to individual sites, which nucleotides occupy each site under physiological conditions, and how binding to one site affects binding to the other sites. Here, we comprehensively analyze nucleotide binding to wild-type and mutant AMPK protein complexes by quantitative competition assays and by hydrogen-deuterium exchange MS. We also demonstrate that NADPH, in addition to the known AMPK ligand NADH, directly and competitively binds AMPK at the AMP-sensing CBS3 site. Our findings reveal how AMP binding to one site affects the conformation and adenine nucleotide binding at the other two sites and establish CBS3, and not CBS1, as the high affinity exchangeable AMP/ADP/ATP-binding site. We further show that AMP binding at CBS4 increases AMP binding at CBS3 by 2 orders of magnitude and reverses the AMP/ATP preference of CBS3. Together, these results illustrate how the three CBS sites collaborate to enable highly sensitive detection of cellular energy states to maintain the tight ATP homeostastis required for cellular metabolism. PMID:28615457

  7. Inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex by reduced nicotinamide adenine dinucleotide in the presence or absence of calcium ion and effect of adenosine 5'-diphosphate on reduced nicotinamide adenine dinucleotide inhibition.

    PubMed

    Lawlis, V B; Roche, T E

    1981-04-28

    Micromolar Ca2+ markedly reduces NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex [Lawlis, V. B., & Roche, T. E. (1980) Mol. Cell. Biochem. 32, 147-152]. Product inhibition patterns from initial velocity studies conducted at less than 10(-9) M or at 1.5 X 10(-5) M Ca2+ with NAD+, CoA, or alpha-ketoglutarate as the variable substrate showed that NADH was a noncompetitive inhibitor with respect to each of these substrates, except at high NAD+ concentrations, where reciprocal plots were nonlinear and the inhibition pattern for NADH vs. NAD+ changed from a noncompetitive to a competitive pattern. From slope and intercept replots, 2-fold to 12-fold higher inhibition constants were estimated for inhibition by NADH vs. the various substrates in the presence of 1.5 X 10(-5) M Ca2+ than for inhibition at less than 10(-9) M Ca2+. These inhibition patterns and the lack of an effect of Ca2+ on the inhibition of the dihydrolipoyl dehydrogenase component suggested that Ca2+-modulated NADH inhibition occurs at an allosteric site with competitive binding at the site by high levels of NAD+. Decarboxylation of alpha-keto[1-14C]glutarate by the resolved alpha-ketoglutarate dehydrogenase component was investigated in the presence of 5.0 mM glyoxylate which served as an efficient acceptor. NADH (0.2 mM) or 1.0 mM ATP inhibited the partial reaction whereas 15 muM Ca2+, 1.0 mM ADP, or 10 mM NAD+ stimulated the partial reaction and reduced NADH inhibition of this reaction. Thus these effectors alter the activity of the alpha-ketoglutarate dehydrogenase complex by binding at allosteric sites on the alpha-ketoglutarate dehydrogenase component. Inhibition by NADH over a wide range of NADH/NAD+ ratios was measured under conditions in which the level of alpha-ketoglutarate was adjusted to give matching control activities at less than 10(-9) M Ca2+ or 1.5 X 10(-5) M Ca2+ in either the presence or the absence of 1.6 mM ADP. These studies establish that both Ca2+ and ADP decreased NADH inhibition under conditions compensating for the effects of Ca2+ and ADP on S0.5 for alpha-ketoglutarate. ADP was particularly effective in reducing NADH inhibition; further studies are required to determine whether this occurs through binding of NADH and ADP at the same, overlapping, or interacting sites.

  8. Elucidation of new condition-dependent roles for fructose-1,6-bisphosphatase linked to cofactor balances

    PubMed Central

    Kilian, Stephanus G.; du Preez, James C.

    2017-01-01

    The cofactor balances in metabolism is of paramount importance in the design of a metabolic engineering strategy and understanding the regulation of metabolism in general. ATP, NAD+ and NADP+ balances are central players linking the various fluxes in central metabolism as well as biomass formation. NADP+ is especially important in the metabolic engineering of yeasts for xylose fermentation, since NADPH is required by most yeasts in the initial step of xylose utilisation, including the fast-growing Kluyveromyces marxianus. In this simulation study of yeast metabolism, the complex interplay between these cofactors was investigated; in particular, how they may affect the possible roles of fructose-1,6-bisphosphatase, the pentose phosphate pathway, glycerol production and the pyruvate dehydrogenase bypass. Using flux balance analysis, it was found that the potential role of fructose-1,6-bisphosphatase was highly dependent on the cofactor specificity of the oxidative pentose phosphate pathway and on the carbon source. Additionally, the excessive production of ATP under certain conditions might be involved in some of the phenomena observed, which may have been overlooked to date. Based on these findings, a strategy is proposed for the metabolic engineering of a future xylose-fermenting yeast for biofuel production. PMID:28542187

  9. The General Definition of the p97/Valosin-containing Protein (VCP)-interacting Motif (VIM) Delineates a New Family of p97 Cofactors*

    PubMed Central

    Stapf, Christopher; Cartwright, Edward; Bycroft, Mark; Hofmann, Kay; Buchberger, Alexander

    2011-01-01

    Cellular functions of the essential, ubiquitin-selective AAA ATPase p97/valosin-containing protein (VCP) are controlled by regulatory cofactors determining substrate specificity and fate. Most cofactors bind p97 through a ubiquitin regulatory X (UBX) or UBX-like domain or linear sequence motifs, including the hitherto ill defined p97/VCP-interacting motif (VIM). Here, we present the new, minimal consensus sequence RX5AAX2R as a general definition of the VIM that unites a novel family of known and putative p97 cofactors, among them UBXD1 and ZNF744/ANKZF1. We demonstrate that this minimal VIM consensus sequence is necessary and sufficient for p97 binding. Using NMR chemical shift mapping, we identified several residues of the p97 N-terminal domain (N domain) that are critical for VIM binding. Importantly, we show that cellular stress resistance conferred by the yeast VIM-containing cofactor Vms1 depends on the physical interaction between its VIM and the critical N domain residues of the yeast p97 homolog, Cdc48. Thus, the VIM-N domain interaction characterized in this study is required for the physiological function of Vms1 and most likely other members of the newly defined VIM family of cofactors. PMID:21896481

  10. Kinetics based reaction optimization of enzyme catalyzed reduction of formaldehyde to methanol with synchronous cofactor regeneration.

    PubMed

    Marpani, Fauziah; Sárossy, Zsuzsa; Pinelo, Manuel; Meyer, Anne S

    2017-12-01

    Enzymatic reduction of carbon dioxide (CO 2 ) to methanol (CH 3 OH) can be accomplished using a designed set-up of three oxidoreductases utilizing reduced pyridine nucleotide (NADH) as cofactor for the reducing equivalents electron supply. For this enzyme system to function efficiently a balanced regeneration of the reducing equivalents during reaction is required. Herein, we report the optimization of the enzymatic conversion of formaldehyde (CHOH) to CH 3 OH by alcohol dehydrogenase, the final step of the enzymatic redox reaction of CO 2 to CH 3 OH, with kinetically synchronous enzymatic cofactor regeneration using either glucose dehydrogenase (System I) or xylose dehydrogenase (System II). A mathematical model of the enzyme kinetics was employed to identify the best reaction set-up for attaining optimal cofactor recycling rate and enzyme utilization efficiency. Targeted process optimization experiments were conducted to verify the kinetically modeled results. Repetitive reaction cycles were shown to enhance the yield of CH 3 OH, increase the total turnover number (TTN) and the biocatalytic productivity rate (BPR) value for both system I and II whilst minimizing the exposure of the enzymes to high concentrations of CHOH. System II was found to be superior to System I with a yield of 8 mM CH 3 OH, a TTN of 160 and BPR of 24 μmol CH 3 OH/U · h during 6 hr of reaction. The study demonstrates that an optimal reaction set-up could be designed from rational kinetics modeling to maximize the yield of CH 3 OH, whilst simultaneously optimizing cofactor recycling and enzyme utilization efficiency. © 2017 Wiley Periodicals, Inc.

  11. Influence of gamma irradiation and benzyl adenine on keeping quality of custard apple fruits during storage.

    PubMed

    Chouksey, Swati; Singh, Alpana; Thakur, Rajendra Singh; Deshmukh, Reena

    2013-10-01

    The custard apple (Annona squamosa) fruits were procured from local market, irradiated with radiation doses 0, 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75 kGy and then treated with benzyl adenine (50 and 100 part per million) and stored at ambient temperature (25 ± 5 °C, Relative Humidity 90 ± 2%) for 12 days. The treated fruits were evaluated for sensory (viz; flavour, texture, internal and external colour) and chemical constituents (viz; Total Soluble Solids, titrable acidity, ascorbic acid, free soluble sugar, reducing sugar. non reducing sugar, carbohydrate) during storage. The study concluded that radiation dose of 1.5 kilo Gray along with 50 ppm benzyl adenine enhanced in shelf-life of custard apple fruits by 6 days at ambient temperature with good pulp texture, flavour, colour and nutritional quality as compared to control.

  12. Catalase in peroxidase clothing: Interdependent cooperation of two cofactors in the catalytic versatility of KatG.

    PubMed

    Njuma, Olive J; Ndontsa, Elizabeth N; Goodwin, Douglas C

    2014-02-15

    Catalase-peroxidase (KatG) is found in eubacteria, archaea, and lower eukaryotae. The enzyme from Mycobacterium tuberculosis has received the greatest attention because of its role in activation of the antitubercular pro-drug isoniazid, and the high frequency with which drug resistance stems from mutations to the katG gene. Generally, the catalase activity of KatGs is striking. It rivals that of typical catalases, enzymes with which KatGs share no structural similarity. Instead, catalatic turnover is accomplished with an active site that bears a strong resemblance to a typical peroxidase (e.g., cytochrome c peroxidase). Yet, KatG is the only member of its superfamily with such capability. It does so using two mutually dependent cofactors: a heme and an entirely unique Met-Tyr-Trp (MYW) covalent adduct. Heme is required to generate the MYW cofactor. The MYW cofactor allows KatG to leverage heme intermediates toward a unique mechanism for H2O2 oxidation. This review evaluates the range of intermediates identified and their connection to the diverse catalytic processes KatG facilitates, including mechanisms of isoniazid activation. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Muscle adenine nucleotide degradation during submaximal treadmill exercise to fatigue.

    PubMed

    Essén-Gustavsson, B; Gottlieb-Vedi, M; Lindholm, A

    1999-07-01

    The aim was to investigate metabolic response in muscle during submaximal treadmill exercise to fatigue, with a special emphasis on adenine nucleotide degradation products such as inosine monophosphate (IMP) in muscle and hypoxanthine, xanthine and uric acid in plasma. Five Standardbred trotters performed treadmill exercise on 2 occasions, once at 7 m/s and once at 10 m/s. Venous blood samples were taken at rest, during exercise and at the end of exercise. Muscle biopsies were taken before and after exercise and muscle temperature was measured before and after exercise. Running time differed among horses and was 48-58 min at 7 m/s and 10-15.5 min at 10 m/s. Both lactate and uric acid concentrations in plasma showed a gradual increase during exercise at both 7 and 10 m/s. At the end of exercise, values for uric acid were higher and values for lactate lower at 7 m/s compared with at 10 m/s. No marked changes were seen in plasma concentrations of hypoxanthine or xanthine with exercise. Muscle glycogen decreased after exercise at both 7 and 10 m/s with a marked depletion seen in some fibres. Muscle lactate concentrations increased after exercise at both 7 m/s and at 10 m/s. No significant changes were seen in adenosine triphosphate (ATP), ADP and AMP concentrations, whereas IMP concentrations increased after exercise at both 7 m/s and at 10 m/s. The results of this study indicate that AMP deamination occurs with submaximal exercise and that development of fatigue may be related to adenine nucleotide degradation in muscle.

  14. Combined molecular modelling and 3D-QSAR study for understanding the inhibition of NQO1 by heterocyclic quinone derivatives.

    PubMed

    López-Lira, Claudia; Alzate-Morales, Jans H; Paulino, Margot; Mella-Raipán, Jaime; Salas, Cristian O; Tapia, Ricardo A; Soto-Delgado, Jorge

    2018-01-01

    A combination of three-dimensional quantitative structure-activity relationship (3D-QSAR), and molecular modelling methods were used to understand the potent inhibitory NAD(P)H:quinone oxidoreductase 1 (NQO1) activity of a set of 52 heterocyclic quinones. Molecular docking results indicated that some favourable interactions of key amino acid residues at the binding site of NQO1 with these quinones would be responsible for an improvement of the NQO1 activity of these compounds. The main interactions involved are hydrogen bond of the amino group of residue Tyr128, π-stacking interactions with Phe106 and Phe178, and electrostatic interactions with flavin adenine dinucleotide (FADH) cofactor. Three models were prepared by 3D-QSAR analysis. The models derived from Model I and Model III, shown leave-one-out cross-validation correlation coefficients (q 2 LOO ) of .75 and .73 as well as conventional correlation coefficients (R 2 ) of .93 and .95, respectively. In addition, the external predictive abilities of these models were evaluated using a test set, producing the predicted correlation coefficients (r 2 pred ) of .76 and .74, respectively. The good concordance between the docking results and 3D-QSAR contour maps provides helpful information about a rational modification of new molecules based in quinone scaffold, in order to design more potent NQO1 inhibitors, which would exhibit highly potent antitumor activity. © 2017 John Wiley & Sons A/S.

  15. The NAMPT/E2F2/SIRT1 axis promotes proliferation and inhibits p53-dependent apoptosis in human melanoma cells.

    PubMed

    Zhao, Hailong; Tang, Weiwei; Chen, Xiaowen; Wang, Siyu; Wang, Xianyan; Xu, Haiyan; Li, Lijuan

    2017-11-04

    Melanoma is the most common primary malignant neoplasm in adults, causing more deaths than any other skin cancer, necessitating the development of new target-based approaches. Current evidence suggests SIRT1, the mammalian nicotinamide adenine dinucleotide (NAD + )-dependent protein deacetylase, and nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting NAD + biosynthetic enzyme, together comprise a novel systemic regulatory network to play a pivotal role in cell proliferation and apoptosis. Nevertheless, how the regulation of this cofactor interfaces with signal transduction network remains poorly understood in melanoma. Here, we report NAMPT is highly expressed in melanomaassociated with poor overall survival in patients. Pharmacological and genetic inhibition of NAMPT decreased NAD + levels and melanoma cell proliferation capacity, and NAMPT knockdown induced apoptosis through the activity of the tumor suppressor p53. Next, we demonstrate NAMPT regulates the transcription factor E2F family member 2 (E2F2) in the apoptosis process. Downstream, E2F2 control the mRNA and protein levels of SIRT1. Finally, we find NAMPT mediates the apoptosis resistance of melanoma cells through NAMPT-E2F2-SIRT1 axis, more than NAD + -driven transcriptional program. Accordingly, our results demonstrated that NAMPT is a prognostic marker in melanoma, and the identificationofNAMPT-E2F2-SIRT1 pathway establishes another link between NAMPT and apoptosis events in melanoma, with therapeutic implications for this deadly cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Therapeutic potential of Mucuna pruriens (Linn.) on ageing induced damage in dorsal nerve of the penis and its implication on erectile function: an experimental study using albino rats.

    PubMed

    Seppan, Prakash; Muhammed, Ibrahim; Mohanraj, Karthik Ganesh; Lakshmanan, Ganesh; Premavathy, Dinesh; Muthu, Sakthi Jothi; Wungmarong Shimray, Khayinmi; Sathyanathan, Sathya Bharathy

    2018-02-15

    To study the effect of ethanolic seed extract of Mucuna pruriens on damaged dorsal nerve of the penis (DNP) in aged rat in relation to penile erection. The rats were divided into four groups Young (3 months), Aged (24 - 28 months), Aged + M. pruriens, and Young + M. pruriens (200 mg/kg b.w/60 days) and were subjected to the hypophysial - gonadal axis, nerve conduction velocity (NCV), and penile reflex. DNP sections were stained with nitric oxide synthase (nNOS), nicotinamide adenine dinucleotide phosphate (NaDPH) diaphorase, androgen receptor (AR), and osmium tetroxide. Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) staining, electron microscopy(EM) and histometric analyses were done. Significant disturbance in hypophysial - gonadal axis was noted in aged rat. With reduced number of myelinated fibers, diameter, vacuolization, indentation of the myelin sheath, and degeneration. nNOS and its cofactor (NaDPH diaphorase) were reduced in aged rat DNP. NCV was slow in aged rats and concomitant poor penile reflex was also noted. AR showed reduced expression in aged rat DNP when compared to young and control groups. TUNEL positive cells were increased in aged rat DNP. These pathological changes were remarkably reduced or recovered in M. pruriens treated aged rats. The results indicate a multi-factorial therapeutic activity in penile innervations towards sustaining the penile erection in the presence of the extract in aged rats and justifying the claim of traditional usage.

  17. Structural basis of kynurenine 3-monooxygenase inhibition.

    PubMed

    Amaral, Marta; Levy, Colin; Heyes, Derren J; Lafite, Pierre; Outeiro, Tiago F; Giorgini, Flaviano; Leys, David; Scrutton, Nigel S

    2013-04-18

    Inhibition of kynurenine 3-monooxygenase (KMO), an enzyme in the eukaryotic tryptophan catabolic pathway (that is, kynurenine pathway), leads to amelioration of Huntington's-disease-relevant phenotypes in yeast, fruitfly and mouse models, as well as in a mouse model of Alzheimer's disease. KMO is a flavin adenine dinucleotide (FAD)-dependent monooxygenase and is located in the outer mitochondrial membrane where it converts l-kynurenine to 3-hydroxykynurenine. Perturbations in the levels of kynurenine pathway metabolites have been linked to the pathogenesis of a spectrum of brain disorders, as well as cancer and several peripheral inflammatory conditions. Despite the importance of KMO as a target for neurodegenerative disease, the molecular basis of KMO inhibition by available lead compounds has remained unknown. Here we report the first crystal structure of Saccharomyces cerevisiae KMO, in the free form and in complex with the tight-binding inhibitor UPF 648. UPF 648 binds close to the FAD cofactor and perturbs the local active-site structure, preventing productive binding of the substrate l-kynurenine. Functional assays and targeted mutagenesis reveal that the active-site architecture and UPF 648 binding are essentially identical in human KMO, validating the yeast KMO-UPF 648 structure as a template for structure-based drug design. This will inform the search for new KMO inhibitors that are able to cross the blood-brain barrier in targeted therapies against neurodegenerative diseases such as Huntington's, Alzheimer's and Parkinson's diseases.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hamill, Michael J.; Jost, Marco; Wong, Cintyu

    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 formore » 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.« less

  19. Membraneless enzymatic ethanol/O2 fuel cell: Transitioning from an air-breathing Pt-based cathode to a bilirubin oxidase-based biocathode

    NASA Astrophysics Data System (ADS)

    Aquino Neto, Sidney; Milton, Ross D.; Hickey, David P.; De Andrade, Adalgisa R.; Minteer, Shelley D.

    2016-08-01

    The bioelectrooxidation of ethanol was investigated in a fully enzymatic membraneless ethanol/O2 biofuel cell assembly using hybrid bioanodes containing multi-walled carbon nanotube (MWCNT)-decorated gold metallic nanoparticles with either a pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) enzyme or a nicotinamide adenine dinucleotide (NAD+)-dependent ADH enzyme. The biofuel cell anode was prepared with the PQQ-dependent enzyme and designed using either a direct electron transfer (DET) architecture or via a mediated electron transfer (MET) configuration through a redox polymer, 1,1‧-dimethylferrocene-modified linear polyethyleneimine (FcMe2-C3-LPEI). In the case of the bioanode containing the NAD+-dependent enzyme, only the mediated electron transfer mechanism was employed using an electropolymerized methylene green film to regenerate the NAD+ cofactor. Regardless of the enzyme being employed at the anode, a bilirubin oxidase-based biocathode prepared within a DET architecture afforded efficient electrocatalytic oxygen reduction in an ethanol/O2 biofuel cell. The power curves showed that DET-based bioanodes via the PQQ-dependent ADH still lack high current densities, whereas the MET architecture furnished maximum power density values as high as 226 ± 21 μW cm-2. Considering the complete membraneless enzymatic biofuel cell with the NAD+-dependent ADH-based bioanode, power densities as high as 111 ± 14 μW cm-2 were obtained. This shows the advantage of PQQ-dependent ADH for membraneless ethanol/O2 biofuel cell applications.

  20. Optical imaging of mitochondrial redox state in rodent model of retinitis pigmentosa

    NASA Astrophysics Data System (ADS)

    Maleki, Sepideh; Gopalakrishnan, Sandeep; Ghanian, Zahra; Sepehr, Reyhaneh; Schmitt, Heather; Eells, Janis; Ranji, Mahsa

    2013-01-01

    Oxidative stress (OS) and mitochondrial dysfunction contribute to photoreceptor cell loss in retinal degenerative disorders. The metabolic state of the retina in a rodent model of retinitis pigmentosa (RP) was investigated using a cryo-fluorescence imaging technique. The mitochondrial metabolic coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are autofluorescent and can be monitored without exogenous labels using optical techniques. The cryo-fluorescence redox imaging technique provides a quantitative assessment of the metabolism. More specifically, the ratio of the fluorescence intensity of these fluorophores (NADH/FAD), the NADH redox ratio (RR), is a marker of the metabolic state of the tissue. The NADH RR and retinal function were examined in an established rodent model of RP, the P23H rat compared to that of nondystrophic Sprague-Dawley (SD) rats. The NADH RR mean values were 1.11±0.03 in the SD normal and 0.841±0.01 in the P23H retina, indicating increased OS in the P23H retina. Electroretinographic data revealed a significant reduction in photoreceptor function in P23H animals compared to SD nozrmal rats. Thus, cryo-fluorescence redox imaging was used as a quantitative marker of OS in eyes from transgenic rats and demonstrated that alterations in the oxidative state of eyes occur during the early stages of RP.

  1. Optical imaging of tissue mitochondrial redox state in intact rat lungs in two models of pulmonary oxidative stress

    PubMed Central

    Sepehr, Reyhaneh; Staniszewski, Kevin; Maleki, Sepideh; Jacobs, Elizabeth R.; Audi, Said

    2012-01-01

    Abstract. Ventilation with enhanced fractions of O2 (hyperoxia) is a common and necessary treatment for hypoxemia in patients with lung failure, but prolonged exposure to hyperoxia causes lung injury. Ischemia-reperfusion (IR) injury of lung tissue is common in lung transplant or crush injury to the chest. These conditions are associated with apoptosis and decreased survival of lung tissue. The objective of this work is to use cryoimaging to evaluate the effect of exposure to hyperoxia and IR injury on lung tissue mitochondrial redox state in rats. The autofluorescent mitochondrial metabolic coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are electron carriers in ATP generation. These intrinsic fluorophores were imaged for rat lungs using low-temperature fluorescence imaging (cryoimaging). Perfused lungs from four groups of rats were studied: normoxia (control), control perfused with an mitochondrial complex IV inhibitor (potassium cyanide, KCN), rats exposed to hyperoxia (85% O2) for seven days, and from rats subjected to lung IR in vivo 24 hours prior to study. Each lung was sectioned sequentially in the transverse direction, and the images were used to reconstruct a three-dimensional (3-D) rendering. In KCN perfused lungs the respiratory chain was more reduced, whereas hyperoxic and IR lung tissue have a more oxidized respiratory chain than control lung tissue, consistent with previously measured mitochondrial dysfunction in both hyperoxic and IR lungs. PMID:22559688

  2. Ethanol and liver: Recent insights into the mechanisms of ethanol-induced fatty liver

    PubMed Central

    Liu, Jinyao

    2014-01-01

    Alcoholic fatty liver disease (AFLD), a potentially pathologic condition, can progress to steatohepatitis, fibrosis, and cirrhosis, leading to an increased probability of hepatic failure and death. Alcohol induces fatty liver by increasing the ratio of reduced form of nicotinamide adenine dinucleotide to oxidized form of nicotinamide adenine dinucleotide in hepatocytes; increasing hepatic sterol regulatory element-binding protein (SREBP)-1, plasminogen activator inhibitor (PAI)-1, and early growth response-1 activity; and decreasing hepatic peroxisome proliferator-activated receptor-α activity. Alcohol activates the innate immune system and induces an imbalance of the immune response, which is followed by activated Kupffer cell-derived tumor necrosis factor (TNF)-α overproduction, which is in turn responsible for the changes in the hepatic SREBP-1 and PAI-1 activity. Alcohol abuse promotes the migration of bone marrow-derived cells (BMDCs) to the liver and then reprograms TNF-α expression from BMDCs. Chronic alcohol intake triggers the sympathetic hyperactivity-activated hepatic stellate cell (HSC) feedback loop that in turn activates the HSCs, resulting in HSC-derived TNF-α overproduction. Carvedilol may block this feedback loop by suppressing sympathetic activity, which attenuates the progression of AFLD. Clinical studies evaluating combination therapy of carvedilol with a TNF-α inhibitor to treat patients with AFLD are warranted to prevent the development of alcoholic liver disease. PMID:25356030

  3. Mitochondrial respiratory complex I probed by delayed luminescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Baran, Irina; Ionescu, Diana; Privitera, Simona; Scordino, Agata; Mocanu, Maria Magdalena; Musumeci, Francesco; Grasso, Rosaria; Gulino, Marisa; Iftime, Adrian; Tofolean, Ioana Teodora; Garaiman, Alexandru; Goicea, Alexandru; Irimia, Ruxandra; Dimancea, Alexandru; Ganea, Constanta

    2013-12-01

    The role of mitochondrial complex I in ultraweak photon-induced delayed photon emission [delayed luminescence (DL)] of human leukemia Jurkat T cells was probed by using complex I targeting agents like rotenone, menadione, and quercetin. Rotenone, a complex I-specific inhibitor, dose-dependently increased the mitochondrial level of reduced nicotinamide adenine dinucleotide (NADH), decreased clonogenic survival, and induced apoptosis. A strong correlation was found between the mitochondrial levels of NADH and oxidized flavin mononucleotide (FMNox) in rotenone-, menadione- and quercetin-treated cells. Rotenone enhanced DL dose-dependently, whereas quercetin and menadione inhibited DL as well as NADH or FMNox. Collectively, the data suggest that DL of Jurkat cells originates mainly from mitochondrial complex I, which functions predominantly as a dimer and less frequently as a tetramer. In individual monomers, both pairs of pyridine nucleotide (NADH/reduced nicotinamide adenine dinucleotide phosphate) sites and flavin (FMN-a/FMN-b) sites appear to bind cooperatively their specific ligands. Enhancement of delayed red-light emission by rotenone suggests that the mean time for one-electron reduction of ubiquinone or FMN-a by the terminal Fe/S center (N2) is 20 or 284 μs, respectively. All these findings suggest that DL spectroscopy could be used as a reliable, sensitive, and robust technique to probe electron flow within complex I in situ.

  4. Protein kinase C epsilon regulates mitochondrial pools of Nampt and NAD following resveratrol and ischemic preconditioning in the rat cortex

    PubMed Central

    Morris-Blanco, Kahlilia C; Cohan, Charles H; Neumann, Jake T; Sick, Thomas J; Perez-Pinzon, Miguel A

    2014-01-01

    Preserving mitochondrial pools of nicotinamide adenine dinucleotide (NAD) or nicotinamide phosphoribosyltransferase (Nampt), an enzyme involved in NAD production, maintains mitochondrial function and confers neuroprotection after ischemic stress. However, the mechanisms involved in regulating mitochondrial-localized Nampt or NAD have not been defined. In this study, we investigated the roles of protein kinase C epsilon (PKCɛ) and AMP-activated protein kinase (AMPK) in regulating mitochondrial pools of Nampt and NAD after resveratrol or ischemic preconditioning (IPC) in the cortex and in primary neuronal-glial cortical cultures. Using the specific PKCɛ agonist ψɛRACK, we found that PKCɛ induced robust activation of AMPK in vitro and in vivo and that AMPK was required for PKCɛ-mediated ischemic neuroprotection. In purified mitochondrial fractions, PKCɛ enhanced Nampt levels in an AMPK-dependent manner and was required for increased mitochondrial Nampt after IPC or resveratrol treatment. Analysis of intrinsic NAD autofluorescence using two-photon microscopy revealed that PKCɛ modulated NAD in the mitochondrial fraction. Further assessments of mitochondrial NAD concentrations showed that PKCɛ has a key role in regulating the mitochondrial NAD+/nicotinamide adenine dinucleotide reduced (NADH) ratio after IPC and resveratrol treatment in an AMPK- and Nampt-dependent manner. These findings indicate that PKCɛ is critical to increase or maintain mitochondrial Nampt and NAD after pathways of ischemic neuroprotection in the brain. PMID:24667915

  5. Interactive response of photosynthetic characteristics in Haloxylon ammodendron and Hedysarum scoparium exposed to soil water and air vapor pressure deficits.

    PubMed

    Gong, Chunmei; Wang, Jiajia; Hu, Congxia; Wang, Junhui; Ning, Pengbo; Bai, Juan

    2015-08-01

    C4 plants possess better drought tolerance than C3 plants. However, Hedysarum scoparium, a C3 species, is dominant and widely distributed in the desert areas of northwestern China due to its strong drought tolerance. This study compared it with Haloxylon ammodendron, a C4 species, regarding the interactive effects of drought stress and different leaf-air vapor pressure deficits. Variables of interest included gas exchange, the activity levels of key C4 photosynthetic enzymes, and cellular anatomy. In both species, gas exchange parameters were more sensitive to high vapor pressure deficit than to strong water stress, and the net CO2 assimilation rate (An) was enhanced as vapor pressure deficits increased. A close relationship between An and stomatal conductance (gs) suggested that the species shared a similar response mechanism. In H. ammodendron, the activity levels of key C4 enzymes were higher, including those of phosphoenolpyruvate carboxylase (PEPC) and nicotinamide adenine dinucleotide phosphate-malate enzyme (NADP-ME), whereas in H. scoparium, the activity level of nicotinamide adenine dinucleotide-malate enzyme (NAD-ME) was higher. Meanwhile, H. scoparium utilized adaptive structural features, including a larger relative vessel area and a shorter distance from vein to stomata, which facilitated the movement of water. These findings implied that some C4 biochemical pathways were present in H. scoparium to respond to environmental challenges. Copyright © 2015. Published by Elsevier B.V.

  6. A second target of benzamide riboside: dihydrofolate reductase.

    PubMed

    Roussel, Breton; Johnson-Farley, Nadine; Kerrigan, John E; Scotto, Kathleen W; Banerjee, Debabrata; Felczak, Krzysztof; Pankiewicz, Krzysztof W; Gounder, Murugesan; Lin, HongXia; Abali, Emine Ercikan; Bertino, Joseph R

    2012-11-01

    Dihydrofolate reductase (DHFR) is an essential enzyme involved in de novo purine and thymidine biosynthesis. For several decades, selective inhibition of DHFR has proven to be a potent therapeutic approach in the treatment of various cancers including acute lymphoblastic leukemia, non-Hodgkin's lymphoma, osteogenic sarcoma, carcinoma of the breast, and head and neck cancer. Therapeutic success with DHFR inhibitor methotrexate (MTX) has been compromised in the clinic, which limits the success of MTX treatment by both acquired and intrinsic resistance mechanisms. We report that benzamide riboside (BR), via anabolism to benzamide adenine dinucleotide (BAD) known to potently inhibit inosine monophosphate dehydrogenase (IMPDH), also inhibits cell growth through a mechanism involving downregulation of DHFR protein. Evidence to support this second site of action of BR includes the finding that CCRF-CEM/R human T-cell lymphoblasic leukemia cells, resistant to MTX as a consequence of gene amplification and overexpression of DHFR, are more resistant to BR than are parental cells. Studies of the mechanism by which BR lowers DHFR showed that BR, through its metabolite BAD, reduced NADP and NADPH cellular levels by inhibiting nicotinamide adenine dinucleotide kinase (NADK). As consequence of the lack of NADPH, DHFR was shown to be destabilized. We suggest that, inhibition of NADK is a new approach to downregulate DHFR and to inhibit cell growth.

  7. PnuC and the utilization of the nicotinamide riboside analog 3-aminopyridine in Haemophilus influenzae.

    PubMed

    Sauer, Elizabeta; Merdanovic, Melisa; Mortimer, Anne Price; Bringmann, Gerhard; Reidl, Joachim

    2004-12-01

    The utilization pathway for the uptake of NAD and nicotinamide riboside was previously characterized for Haemophilus influenzae. We now report on the cellular location, topology, and substrate specificity of PnuC. pnuC of H. influenzae is only distantly related to pnuC of Escherichia coli and Salmonella enterica serovar Typhimurium. When E. coli PnuC was expressed in an H. influenzae pnuC mutant, it was able to take up only nicotinamide riboside and not nicotinamide mononucleotide. Therefore, we postulated that PnuC transporters in general possess specificity for nicotinamide riboside. Earlier studies showed that 3-aminopyridine derivatives (e.g., 3-aminopyridine adenine dinucleotide) are inhibitory for H. influenzae growth. By testing characterized strains with mutations in the NAD utilization pathway, we show that 3-aminopyridine riboside is inhibitory to H. influenzae and is taken up by the NAD-processing and nicotinamide riboside route. 3-Aminopyridine riboside is utilized effectively in a pnuC+ background. In addition, we demonstrate that 3-aminopyridine adenine dinucleotide resynthesis is produced by NadR. 3-Aminopyridine riboside-resistant H. influenzae isolates were characterized, and mutations in nadR could be detected. We also tested other species of the family Pasteurellaceae, Pasteurella multocida and Actinobacillus actinomycetemcomitans, and found that 3-aminopyridine riboside does not act as a growth inhibitor; hence, 3-aminopyridine riboside represents an anti-infective agent with a very narrow host range.

  8. Human Immunodeficiency Virus Immune Cell Receptors, Coreceptors, and Cofactors: Implications for Prevention and Treatment.

    PubMed

    Woodham, Andrew W; Skeate, Joseph G; Sanna, Adriana M; Taylor, Julia R; Da Silva, Diane M; Cannon, Paula M; Kast, W Martin

    2016-07-01

    In the last three decades, extensive research on human immunodeficiency virus (HIV) has highlighted its capability to exploit a variety of strategies to enter and infect immune cells. Although CD4(+) T cells are well known as the major HIV target, with infection occurring through the canonical combination of the cluster of differentiation 4 (CD4) receptor and either the C-C chemokine receptor type 5 (CCR5) or C-X-C chemokine receptor type 4 (CXCR4) coreceptors, HIV has also been found to enter other important immune cell types such as macrophages, dendritic cells, Langerhans cells, B cells, and granulocytes. Interestingly, the expression of distinct cellular cofactors partially regulates the rate in which HIV infects each distinct cell type. Furthermore, HIV can benefit from the acquisition of new proteins incorporated into its envelope during budding events. While several publications have investigated details of how HIV manipulates particular cell types or subtypes, an up-to-date comprehensive review on HIV tropism for different immune cells is lacking. Therefore, this review is meant to focus on the different receptors, coreceptors, and cofactors that HIV exploits to enter particular immune cells. Additionally, prophylactic approaches that have targeted particular molecules associated with HIV entry and infection of different immune cells will be discussed. Unveiling the underlying cellular receptors and cofactors that lead to HIV preference for specific immune cell populations is crucial in identifying novel preventative/therapeutic targets for comprehensive strategies to eliminate viral infection.

  9. Human Immunodeficiency Virus Immune Cell Receptors, Coreceptors, and Cofactors: Implications for Prevention and Treatment

    PubMed Central

    Woodham, Andrew W.; Skeate, Joseph G.; Sanna, Adriana M.; Taylor, Julia R.; Da Silva, Diane M.; Cannon, Paula M.

    2016-01-01

    Abstract In the last three decades, extensive research on human immunodeficiency virus (HIV) has highlighted its capability to exploit a variety of strategies to enter and infect immune cells. Although CD4+ T cells are well known as the major HIV target, with infection occurring through the canonical combination of the cluster of differentiation 4 (CD4) receptor and either the C-C chemokine receptor type 5 (CCR5) or C-X-C chemokine receptor type 4 (CXCR4) coreceptors, HIV has also been found to enter other important immune cell types such as macrophages, dendritic cells, Langerhans cells, B cells, and granulocytes. Interestingly, the expression of distinct cellular cofactors partially regulates the rate in which HIV infects each distinct cell type. Furthermore, HIV can benefit from the acquisition of new proteins incorporated into its envelope during budding events. While several publications have investigated details of how HIV manipulates particular cell types or subtypes, an up-to-date comprehensive review on HIV tropism for different immune cells is lacking. Therefore, this review is meant to focus on the different receptors, coreceptors, and cofactors that HIV exploits to enter particular immune cells. Additionally, prophylactic approaches that have targeted particular molecules associated with HIV entry and infection of different immune cells will be discussed. Unveiling the underlying cellular receptors and cofactors that lead to HIV preference for specific immune cell populations is crucial in identifying novel preventative/therapeutic targets for comprehensive strategies to eliminate viral infection. PMID:27410493

  10. Binding of p-mercaptobenzoic acid and adenine to gold-coated electroless etched silicon nanowires studied by surface-enhanced Raman scattering

    NASA Astrophysics Data System (ADS)

    Mohaček-Grošev, Vlasta; Gebavi, Hrvoje; Bonifacio, Alois; Sergo, Valter; Daković, Marko; Bajuk-Bogdanović, Danica

    2018-07-01

    Modern diagnostic tools ever aim to reduce the amount of analyte and the time needed for obtaining the result. Surface-enhanced Raman spectroscopy is a method that could satisfy both of these requirements, provided that for each analyte an adequate substrate is found. Here we demonstrate the ability of gold-sputtered silicon nanowires (SiNW) to bind p-mercaptobenzoic acid in 10-3, 10-4 and 10-5 M and adenine in 30 and 100 μM concentrations. Based on the normal mode analysis, presented here for the first time, the binding of p-mercaptobenzoic acid is deduced. The intensity enhancement of the 1106 cm-1 band is explained by involvement of the Csbnd S stretching deformation, and the appearance of the broad 300 cm-1 band attributed to Ssbnd Au stretching mode. Adenine SERS spectra demonstrate the existence of the 7H tautomer since the strongest band observed is at 736 cm-1. The adenine binding is likely to occur in several ways, because the number of observed bands in the 1200-1600 cm-1 interval exceeds the number of observed bands in the normal Raman spectrum of the free molecule.

  11. Co-factors Required for TLR7- and TLR9- dependent Innate Immune Responses

    PubMed Central

    Chiang, Chih-yuan; Engel, Alex; Opaluch, Amanda M.; Ramos, Irene; Maestre, Ana M.; Secundino, Ismael; De Jesus, Paul D.; Nguyen, Quy T.; Welch, Genevieve; Bonamy, Ghislain M.C.; Miraglia, Loren J.; Orth, Anthony P.; Nizet, Victor; Fernandez-Sesma, Ana; Zhou, Yingyao; Barton, Gregory M.; Chanda, Sumit K.

    2012-01-01

    SUMMARY Pathogens commonly utilize endocytic pathways to gain cellular access. The endosomal pattern recognition receptors TLR7 and TLR9 detect pathogen-encoded nucleic acids to initiate MyD88-dependent pro-inflammatory responses to microbial infection. Using genome-wide RNAi screening and integrative systems-based analysis we identify 190 co-factors required for TLR7- and TLR9-directed signaling responses. A set of co-factors were cross-profiled for their activities downstream of several immunoreceptors, and then functionally mapped based on the known architecture of NF-κB signaling pathways. Protein complexes and pathways involved in ubiquitin-protein ligase activities, sphingolipid metabolism, chromatin modifications, and ancient stress responses were found to modulate innate recognition of endosomal nucleic acids. Additionally, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) was characterized as necessary for ubiquitin-dependent TLR9 targeting to the endolysosome. Proteins and pathways identified here should prove useful in delineating strategies to manipulate innate responses for treatment of autoimmune disorders and microbial infection. PMID:22423970

  12. X-ray emission spectroscopy evidences a central carbon in the nitrogenase iron-molybdenum cofactor.

    PubMed

    Lancaster, Kyle M; Roemelt, Michael; Ettenhuber, Patrick; Hu, Yilin; Ribbe, Markus W; Neese, Frank; Bergmann, Uwe; DeBeer, Serena

    2011-11-18

    Nitrogenase is a complex enzyme that catalyzes the reduction of dinitrogen to ammonia. Despite insight from structural and biochemical studies, its structure and mechanism await full characterization. An iron-molybdenum cofactor (FeMoco) is thought to be the site of dinitrogen reduction, but the identity of a central atom in this cofactor remains unknown. Fe Kβ x-ray emission spectroscopy (XES) of intact nitrogenase MoFe protein, isolated FeMoco, and the FeMoco-deficient nifB protein indicates that among the candidate atoms oxygen, nitrogen, and carbon, it is carbon that best fits the XES data. The experimental XES is supported by computational efforts, which show that oxidation and spin states do not affect the assignment of the central atom to C(4-). Identification of the central atom will drive further studies on its role in catalysis.

  13. Dynamics of dipole- and valence bound anions in iodide-adenine binary complexes: A time-resolved photoelectron imaging and quantum mechanical investigation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Stephansen, Anne B.; King, Sarah B.; Li, Wei-Li

    2015-09-14

    Dipole bound (DB) and valence bound (VB) anions of binary iodide-adenine complexes have been studied using one-color and time-resolved photoelectron imaging at excitation energies near the vertical detachment energy. The experiments are complemented by quantum chemical calculations. One-color spectra show evidence for two adenine tautomers, the canonical, biologically relevant A9 tautomer and the A3 tautomer. In the UV-pump/IR-probe time-resolved experiments, transient adenine anions can be formed by electron transfer from the iodide. These experiments show signals from both DB and VB states of adenine anions formed on femto- and picosecond time scales, respectively. Analysis of the spectra and comparison withmore » calculations suggest that while both the A9 and A3 tautomers contribute to the DB signal, only the DB state of the A3 tautomer undergoes a transition to the VB anion. The VB anion of A9 is higher in energy than both the DB anion and the neutral, and the VB anion is therefore not accessible through the DB state. Experimental evidence of the metastable A9 VB anion is instead observed as a shape resonance in the one-color photoelectron spectra, as a result of UV absorption by A9 and subsequent electron transfer from iodide into the empty π-orbital. In contrast, the iodide-A3 complex constitutes an excellent example of how DB states can act as doorway state for VB anion formation when the VB state is energetically available.« less

  14. A hepcidin lowering agent mobilizes iron for incorporation into red blood cells in an adenine-induced kidney disease model of anemia in rats

    PubMed Central

    Sun, Chia Chi; Vaja, Valentina; Chen, Shanzhuo; Theurl, Igor; Stepanek, Aaron; Brown, Diane E.; Cappellini, Maria D.; Weiss, Guenter; Hong, Charles C.; Lin, Herbert Y.; Babitt, Jodie L.

    2013-01-01

    Background Anemia is a common complication of chronic kidney disease (CKD) that negatively impacts the quality of life and is associated with numerous adverse outcomes. Excess levels of the iron regulatory hormone hepcidin are thought to contribute to anemia in CKD patients by decreasing iron availability from the diet and from body stores. Adenine treatment in rats has been proposed as an animal model of anemia of CKD with high hepcidin levels that mirrors the condition in human patients. Methods We developed a modified adenine-induced kidney disease model with a higher survival rate than previously reported models, while maintaining persistent kidney disease and anemia. We then tested whether the small molecule bone morphogenetic protein (BMP) inhibitor LDN-193189, which was previously shown to lower hepcidin levels in rodents, mobilized iron into the plasma and improved iron-restricted erythropoiesis in this model. Results Adenine-treated rats exhibited increased hepatic hepcidin mRNA, decreased serum iron, increased spleen iron content, low hemoglobin (Hb) and inappropriately low erythropoietin (EPO) levels relative to the degree of anemia. LDN-193189 administration to adenine-treated rats lowered hepatic hepcidin mRNA, mobilized stored iron into plasma and increased Hb content of reticulocytes. Conclusions Our data suggest that hepcidin lowering agents may provide a new therapeutic strategy to improve iron availability for erythropoiesis in CKD. PMID:23345622

  15. Structural insights into the neuroprotective-acting carbonyl reductase Sniffer of Drosophila melanogaster.

    PubMed

    Sgraja, Tanja; Ulschmid, Julia; Becker, Katja; Schneuwly, Stephan; Klebe, Gerhard; Reuter, Klaus; Heine, Andreas

    2004-10-01

    In vivo studies with the fruit-fly Drosophila melanogaster have shown that the Sniffer protein prevents age-dependent and oxidative stress-induced neurodegenerative processes. Sniffer is a NADPH-dependent carbonyl reductase belonging to the enzyme family of short-chain dehydrogenases/reductases (SDRs). The crystal structure of the homodimeric Sniffer protein from Drosophila melanogaster in complex with NADP+ has been determined by multiple-wavelength anomalous dispersion and refined to a resolution of 1.75 A. The observed fold represents a typical dinucleotide-binding domain as detected for other SDRs. With respect to the cofactor-binding site and the region referred to as substrate-binding loop, the Sniffer protein shows a striking similarity to the porcine carbonyl reductase (PTCR). This loop, in both Sniffer and PTCR, is substantially shortened compared to other SDRs. In most enzymes of the SDR family this loop adopts a well-defined conformation only after substrate binding and remains disordered in the absence of any bound ligands or even if only the dinucleotide cofactor is bound. In the structure of the Sniffer protein, however, the conformation of this loop is well defined, although no substrate is present. Molecular modeling studies provide an idea of how binding of substrate molecules to Sniffer could possibly occur.

  16. Three-dimensional structure of holo 3 alpha,20 beta-hydroxysteroid dehydrogenase: a member of a short-chain dehydrogenase family.

    PubMed Central

    Ghosh, D; Weeks, C M; Grochulski, P; Duax, W L; Erman, M; Rimsay, R L; Orr, J C

    1991-01-01

    The x-ray structure of a short-chain dehydrogenase, the bacterial holo 3 alpha,20 beta-hydroxysteroid dehydrogenase (EC 1.1.1.53), is described at 2.6 A resolution. This enzyme is active as a tetramer and crystallizes with four identical subunits in the asymmetric unit. It has the alpha/beta fold characteristic of the dinucleotide binding region. The fold of the rest of the subunit, the quaternary structure, and the nature of the cofactor-enzyme interactions are, however, significantly different from those observed in the long-chain dehydrogenases. The architecture of the postulated active site is consistent with the observed stereospecificity of the enzyme and the fact that the tetramer is the active form. There is only one cofactor and one substrate-binding site per subunit; the specificity for both 3 alpha- and 20 beta-ends of the steroid results from the binding of the steroid in two orientations near the same cofactor at the same catalytic site. Images PMID:1946424

  17. Panning for SNuRMs: using cofactor profiling for the rational discovery of selective nuclear receptor modulators.

    PubMed

    Kremoser, Claus; Albers, Michael; Burris, Thomas P; Deuschle, Ulrich; Koegl, Manfred

    2007-10-01

    Drugs that target nuclear receptors are clinically, as well as commercially, successful. Their widespread use, however, is limited by an inherent propensity of nuclear receptors to trigger beneficial, as well as adverse, pharmacological effects upon drug activation. Hence, selective drugs that display reduced adverse effects, such as the selective estrogen receptor modulator (SERM) Raloxifene, have been developed by guidance through classical cell culture assays and animal trials. Full agonist and selective modulator nuclear receptor drugs, in general, differ by their ability to recruit certain cofactors to the receptor protein. Hence, systematic cofactor profiling is advancing into an approach for the rationally guided identification of selective NR modulators (SNuRMs) with improved therapeutic ratio.

  18. Parvovirus B19 DNA CpG Dinucleotide Methylation and Epigenetic Regulation of Viral Expression

    PubMed Central

    Bonvicini, Francesca; Manaresi, Elisabetta; Di Furio, Francesca; De Falco, Luisa; Gallinella, Giorgio

    2012-01-01

    CpG DNA methylation is one of the main epigenetic modifications playing a role in the control of gene expression. For DNA viruses whose genome has the ability to integrate in the host genome or to maintain as a latent episome, a correlation has been found between the extent of DNA methylation and viral quiescence. No information is available for Parvovirus B19, a human pathogenic virus, which is capable of both lytic and persistent infections. Within Parvovirus B19 genome, the inverted terminal regions display all the characteristic signatures of a genomic CpG island; therefore we hypothesised a role of CpG dinucleotide methylation in the regulation of viral genome expression. The analysis of CpG dinucleotide methylation of Parvovirus B19 DNA was carried out by an aptly designed quantitative real-time PCR assay on bisulfite-modified DNA. The effects of CpG methylation on the regulation of viral genome expression were first investigated by transfection of either unmethylated or in vitro methylated viral DNA in a model cell line, showing that methylation of viral DNA was correlated to lower expression levels of the viral genome. Then, in the course of in vitro infections in different cellular environments, it was observed that absence of viral expression and genome replication were both correlated to increasing levels of CpG methylation of viral DNA. Finally, the presence of CpG methylation was documented in viral DNA present in bioptic samples, indicating the occurrence and a possible role of this epigenetic modification in the course of natural infections. The presence of an epigenetic level of regulation of viral genome expression, possibly correlated to the silencing of the viral genome and contributing to the maintenance of the virus in tissues, can be relevant to the balance and outcome of the different types of infection associated to Parvovirus B19. PMID:22413013

  19. Multiple Decay Mechanisms and 2D-UV Spectroscopic Fingerprints of Singlet Excited Solvated Adenine-Uracil Monophosphate.

    PubMed

    Li, Quansong; Giussani, Angelo; Segarra-Martí, Javier; Nenov, Artur; Rivalta, Ivan; Voityuk, Alexander A; Mukamel, Shaul; Roca-Sanjuán, Daniel; Garavelli, Marco; Blancafort, Lluís

    2016-05-23

    The decay channels of singlet excited adenine uracil monophosphate (ApU) in water are studied with CASPT2//CASSCF:MM potential energy calculations and simulation of the 2D-UV spectroscopic fingerprints with the aim of elucidating the role of the different electronic states of the stacked conformer in the excited state dynamics. The adenine (1) La state can decay without a barrier to a conical intersection with the ground state. In contrast, the adenine (1) Lb and uracil S(U) states have minima that are separated from the intersections by sizeable barriers. Depending on the backbone conformation, the CT state can undergo inter-base hydrogen transfer and decay to the ground state through a conical intersection, or it can yield a long-lived minimum stabilized by a hydrogen bond between the two ribose rings. This suggests that the (1) Lb , S(U) and CT states of the stacked conformer may all contribute to the experimental lifetimes of 18 and 240 ps. We have also simulated the time evolution of the 2D-UV spectra and provide the specific fingerprint of each species in a recommended probe window between 25 000 and 38 000 cm(-1) in which decongested, clearly distinguishable spectra can be obtained. This is expected to allow the mechanistic scenarios to be discerned in the near future with the help of the corresponding experiments. Our results reveal the complexity of the photophysics of the relatively small ApU system, and the potential of 2D-UV spectroscopy to disentangle the photophysics of multichromophoric systems. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  20. The Na+-Translocating NADH:Quinone Oxidoreductase Enhances Oxidative Stress in the Cytoplasm of Vibrio cholerae

    PubMed Central

    Muras, Valentin; Dogaru-Kinn, Paul; Minato, Yusuke; Häse, Claudia C.

    2016-01-01

    ABSTRACT We searched for a source of reactive oxygen species (ROS) in the cytoplasm of the human pathogen Vibrio cholerae and addressed the mechanism of ROS formation using the dye 2′,7′-dichlorofluorescein diacetate (DCFH-DA) in respiring cells. By comparing V. cholerae strains with or without active Na+-translocating NADH:quinone oxidoreductase (Na+-NQR), this respiratory sodium ion redox pump was identified as a producer of ROS in vivo. The amount of cytoplasmic ROS detected in V. cholerae cells producing variants of Na+-NQR correlated well with rates of superoxide formation by the corresponding membrane fractions. Membranes from wild-type V. cholerae showed increased superoxide production activity (9.8 ± 0.6 μmol superoxide min−1 mg−1 membrane protein) compared to membranes from the mutant lacking Na+-NQR (0.18 ± 0.01 μmol min−1 mg−1). Overexpression of plasmid-encoded Na+-NQR in the nqr deletion strain resulted in a drastic increase in the formation of superoxide (42.6 ± 2.8 μmol min−1 mg−1). By analyzing a variant of Na+-NQR devoid of quinone reduction activity, we identified the reduced flavin adenine dinucleotide (FAD) cofactor of cytoplasmic NqrF subunit as the site for intracellular superoxide formation in V. cholerae. The impact of superoxide formation by the Na+-NQR on the virulence of V. cholerae is discussed. IMPORTANCE In several studies, it was demonstrated that the Na+-NQR in V. cholerae affects virulence in a yet unknown manner. We identified the reduced FAD cofactor in the NADH-oxidizing NqrF subunit of the Na+-NQR as the site of superoxide formation in the cytoplasm of V. cholerae. Our study provides the framework to understand how reactive oxygen species formed during respiration could participate in the regulated expression of virulence factors during the transition from aerobic to microaerophilic (intestinal) habitats. This hypothesis may turn out to be right for many other pathogens which, like V. cholerae, depend on