WorldWideScience

Sample records for carbon-oxygen lyases

  1. Selenocysteine Lyase.

    Science.gov (United States)

    Stadtman, Thressa C

    2004-12-01

    Selenocysteine is a naturally occurring analog of cysteine in which the sulfur atom of the latter is replaced with selenium. This seleno-amino acid occurs as a specific component of various selenoproteins and selenium-dependent enzymes. Incorporation of selenocysteine into these proteins occurs cotranslationally as directed by the UGA codon. For this process, a special tRNA having an anticodon complimentary to UGA, tRNASec, is utilized. In Escherichia coli and related bacteria, this tRNA first is amino acylated with serine, and the seryl-tRNASec is converted to selenocysteyl-tRNASec. The specific incorporation of selenocysteine into proteins directed by the UGA codon depends on the synthesis of selenocysteyl-tRNASec. Included in the selenium delivery protein category are rhodaneses that mobilize selenium from inorganic sources and NIFS-like proteins that liberate elemental selenium from selenocysteine. The NIFS protein from Azotobacter vinelandii was found to serve as an efficient catalyst in vitro for delivery of selenium from free selenocysteine to Escherichia coli selenophosphate synthetase for selenophosphate formation. The widespread distribution of selenocysteine lyase in numerous bacterial species was reported and the bacterial enzymes, like the pig liver enzyme, required pyridoxal phosphate as cofactor. Three NIFS-like genes were isolated from E. coli by Esaki and coworkers and the expressed gene products were isolated and characterized. One of these NIFS-like proteins also exhibited a high preference for selenocysteine over cysteine. M. vannielii, an anaerobic methane-producing organism, that grows in a mineral medium containing formate as sole organic carbon source, synthesizes several specific selenoenzymes required for growth and energy production under these conditions. PMID:26443359

  2. Small Scale Variations in Carbon Oxygen Ratio

    International Nuclear Information System (INIS)

    The aim of the research reported here is the development of a methodology for the measurement of small scale variations in chemical elements concentrations, in particular of carbon - oxygen ratio. Knowledge of the C/O ratio is of importance to many problems in various fields. Here we single out the application in obtaining important information about the oil fields. The most fundamental reservoir parameters - oil, gas and water content - are critical factors in determining how each oil field should be developed. It is well established that carbon to oxygen ratio log yields accurate and repeatable data that can be used to identify and monitor reserves depletion. Recent improvements in neutron generator and gamma detector technologies resulted in small devices which allowed through-tubing measurements. Although the ratio of carbon and oxygen yields is a measure of the amount of oil around the tool it should be realized that a carbon signal can originate from several sources including the borehole, the cement behind the casing, the formation rock and the formation fluid. In order to evaluate these contributions individually we are proposing the modification of the neutron generator by insertion of segmented associated alpha particle detector. From the measurement of time of flight spectra (alpha particle detector - start signal; gamma ray detector - stop signal) it would be possible to determine the location of gamma ray producing voxel and in such a way to determine radial variations in several chemical elements concentrations, in particular of carbon to oxygen ratio. (authors)

  3. Protein Crystal Isocitrate Lyase

    Science.gov (United States)

    1998-01-01

    The comparison of protein crystal, Isocitrate Lyase earth-grown (left) and space-grown (right). This is a target enzyme for fungicides. A better understanding of this enzyme should lead to the discovery of more potent fungicides to treat serious crop diseases such as rice blast; it regulates the flow of metabolic intermediates required for cell growth. Principal Investigator is Larry DeLucas.

  4. Remnant evolution after a carbon-oxygen white dwarf merger

    NARCIS (Netherlands)

    S.C. Yoon; P. Podsiadlowski; S. Rosswog

    2007-01-01

    We systematically explore the evolution of the merger of two carbon-oxygen (CO) white dwarfs. The dynamical evolution of a 0.9Msolar + 0.6Msolar CO white dwarf merger is followed by a 3D smoothed particle hydrodynamics (SPH) simulation. The calculation uses a state-of-the-art e

  5. A EUTECTIC IN CARBON-OXYGEN WHITE DWARFS ?

    OpenAIRE

    Stevenson, D.

    1980-01-01

    When the interior of a white dwarf begins to freeze, the coexisting solid and liquid phases will have different compositions in general. Two models for the carbon-oxygen phase diagram are described. In the more realistic model, a pronounced eutetic is predicted and the solid phase is either pure carbon or pure oxygen. The model predicts that a white dwarf begins to freeze later in its evolution and then cools more slowly.

  6. Regulatory Citrate Lyase Mutants of Salmonella typhimurium

    OpenAIRE

    Kulla, Hans G.

    1983-01-01

    Citrate lyase, the key enzyme of anaerobic citrate catabolism, could not be deleted from Salmonella typhimurium. The only class of mutants found had a mode of covalent regulation that strongly resembled the Escherichia coli system: citrate lyase was only active, i.e., acetylated, when a cosubstrate was present.

  7. Regulatory citrate lyase mutants of Salmonella typhimurium.

    Science.gov (United States)

    Kulla, H G

    1983-01-01

    Citrate lyase, the key enzyme of anaerobic citrate catabolism, could not be deleted from Salmonella typhimurium. The only class of mutants found had a mode of covalent regulation that strongly resembled the Escherichia coli system: citrate lyase was only active, i.e., acetylated, when a cosubstrate was present. PMID:6336740

  8. Rhamnogalacturonan lyase reveals a unique three-domain modular structure for polysaccharide lyase family 4

    DEFF Research Database (Denmark)

    McDonough, Michael A.; Kadirvelraj, Renuka; Harris, Pernille;

    2004-01-01

    Rhamnogalacturonan lyase (RG-lyase) specifically recognizes and cleaves alpha-1,4 glycosidic bonds between L-rhamnose and D-galacturonic acids in the backbone of rhamno galacturonan-I, a major component of the plant cell wall polysaccharide, pectin. The three-dimensional structure of RG-lyase from...... Aspergillus aculeatus has been determined to 1.5 Angstrom resolution representing the first known structure from polysaccharide lyase family 4 and of an enzyme with this catalytic specificity. The 508-amino acid polypeptide displays a unique arrangement of three distinct modular domains. Each domain shows...... structural homology to non-catalytic domains from other carbohydrate active enzymes....

  9. Enhancing RGI lyase thermostability by targeted single point mutations

    DEFF Research Database (Denmark)

    Silva, Inês R.; Larsen, Dorte Møller; Jers, Carsten; Derkx, Patrick; Meyer, Anne S.; Mikkelsen, Jørn Dalgaard

    2013-01-01

    Rhamnogalacturonan I lyase (RGI lyase) (EC 4.2.2.-) catalyzes the cleavage of rhamnogalacturonan I in pectins by β-elimination. In this study the thermal stability of a RGI lyase (PL 11) originating from Bacillus licheniformis DSM 13/ATCC14580 was increased by a targeted protein engineering...

  10. (PCG) Protein Crystal Growth Isocitrate Lyase

    Science.gov (United States)

    1989-01-01

    (PCG) Protein Crystal Growth Isocitrate Lyase. Target enzyme for fungicides. A better understanding of this enzyme should lead to the discovery of more potent fungicides to treat serious crop diseases such as rice blast. It regulates the flow of metabolic intermediates required for cell growth. Principal Investigator for STS-26 was Charles Bugg.

  11. Pulsations of massive ZZ Ceti stars with carbon/oxygen and oxygen/neon cores

    OpenAIRE

    Corsico, A. H.; Garcia-Berro, E.; L. G. Althaus; Isern, J.

    2004-01-01

    We explore the adiabatic pulsational properties of massive white dwarf stars with hydrogen-rich envelopes and oxygen/neon and carbon/oxygen cores. To this end, we compute the cooling of massive white dwarf models for both core compositions taking into account the evolutionary history of the progenitor stars and the chemical evolution caused by time-dependent element diffusion. In particular, for the oxygen/neon models, we adopt the chemical profile resulting from repeated carbon-burning shell...

  12. Xylella fastidiosa esterase rather than hydroxynitrile lyase.

    Science.gov (United States)

    Torrelo, Guzman; Ribeiro de Souza, Fayene Zeferino; Carrilho, Emanuel; Hanefeld, Ulf

    2015-03-01

    In 2009, we reported that the product of the gene SCJ21.16 (XFa0032) from Xylella fastidiosa, a xylem-restricted plant pathogen that causes a range of diseases in several important crops, encodes a protein (XfHNL) with putative hydroxynitrile lyase activity. Sequence analysis and activity tests indicated that XfHNL exhibits an α/β-hydrolase fold and could be classified as a member of the family of FAD-independent HNLs. Here we provide a more detailed sequence analysis and new experimental data. Using pure heterologously expressed XfHNL we show that this enzyme cannot catalyse the cleavage/synthesis of mandelonitrile and that this protein is in fact a non-enantioselective esterase. Homology modelling and ligand docking simulations were used to study the active site and support these results. This finding could help elucidate the common ancestor of esterases and hydroxynitrile lyases with an α/β -hydrolase fold. PMID:25684099

  13. Evidence of histidine phosphorylation in isocitrate lyase from Escherichia coli

    International Nuclear Information System (INIS)

    Escherichia coli isocitrate lyase can be phosphorylated in vitro in an ATP-dependent reaction. Partially purified extracts were incubated with γ-32P-ATP and analyzed by two-dimensional polyacrylamide gel electrophoresis followed by a Western blot and autoradiography. Radioactivity was associated with the lyase only when blotting was performed under alkaline conditions. This suggests that phosphate groups are attached to the lyase via an acid-labile P-N bond rather than a more stable P-O bond. Treatment of the lyase with diethyl pyrocarbonate, a histidine modifying agent, blocks incorporation of 32P-phosphate. Treatment with phosphoramidate, a histidine phosphorylating agent, alters the isoelectric point of the lyase suggesting that the enzyme can be phosphorylated at histidine residues. Loss of catalytic activity after treatment with potato acid phosphatase indicates that isocitrate lyase activity may be modulated by phosphorylation

  14. A New Member of Family 11 Polysaccharide Lyase, Rhamnogalacturonan Lyase (CtRGLf) from Clostridium thermocellum.

    Science.gov (United States)

    Dhillon, Arun; Fernandes, Vania O; Dias, Fernando M V; Prates, José A M; Ferreira, Luis M A; Fontes, Carlos M G A; Centeno, M S J; Goyal, Arun

    2016-04-01

    A thermostable, alkaline rhamnogalacturonan lyase (RG lyase) CtRGLf, of family 11 polysaccharide lyase from Clostridium thermocellum was cloned, expressed, purified and biochemically characterised. Both, the full-length CtRGLf (80 kDa) protein and its truncated derivative CtRGL (63.9 kDa) were expressed as soluble proteins and displayed maximum activity against rhamnogalacturonan I (RG I). CtRGLf showed maximum activity at 70 °C, while CtRGL at 60 °C. Both enzymes showed maximum activity at pH 8.5. CtRGLf and CtRGL do not show higher activity on substrates with high β-D-galactopyranose (D-Galp) substitution, this catalytic property deviates from that of some earlier characterised RG lyases which prefer substrates with high D-Galp substitution. The enzyme activity of CtRGLf and CtRGL was enhanced by 1.5 and 1.3 fold, respectively, in the presence of 3 mM of Ca(2+) ions. The TLC analysis of the degraded products of RG I, released by the action of CtRGLf and CtRGL revealed the production of RG oligosaccharides as major products confirming their endolytic activity. PMID:26921189

  15. A Parameter-Space Study of Carbon-Oxygen White Dwarf Mergers

    CERN Document Server

    Zhu, Chenchong; van Kerkwijk, Marten; Wadsley, James

    2012-01-01

    (Abbrev.) The merger of two carbon-oxygen white dwarfs (WDs) can lead to a spectacular transient--an SN Ia or AIC--or the formation of a carbon star or massive, rapidly rotating WD. Simulations of mergers have shown that the outcome strongly depends on whether the WDs are similar or dissimilar in mass. In the similar-mass case, both WDs merge fully and the remnant is hot throughout, while in the dissimilar case, the more massive, denser WD remains cold and essentially intact, with the disrupted lower mass one wrapped around it in a hot envelope and disk. In order to determine what constitutes "similar in mass" and more generally how the properties of the merger remnant depend on the input masses, we simulated unsynchronized carbon-oxygen WD mergers for a large range of masses using smoothed-particle hydrodynamics. Generally, we find that the properties of the remnants vary smoothly as a function of the two masses, with the remnant structure determined primarily by the ratio of the central densities of the two...

  16. Studies of reductive elimination reactions to form carbon-oxygen bonds from Pt(IV) complexes.

    Science.gov (United States)

    Williams, B S; Goldberg, K I

    2001-03-21

    The platinum(IV) complexes fac-L(2)PtMe(3)(OR) (L(2) = bis(diphenylphosphino)ethane, o-bis(diphenylphosphino)benzene, R = carboxyl, aryl; L = PMe(3), R = aryl) undergo reductive elimination reactions to form carbon-oxygen bonds and/or carbon-carbon bonds. The carbon-oxygen reductive elimination reaction produces either methyl esters or methyl aryl ethers (anisoles) and L(2)PtMe(2), while the carbon-carbon reductive elimination reaction affords ethane and L(2)PtMe(OR). Choice of reaction conditions allows the selection of either type of coupling over the other. A detailed mechanistic study of the reductive elimination reactions supports dissociation of the OR(-) ligand as the initial step for the C-O bond formation reaction. This is followed by a nucleophilic attack of OR(-) upon a methyl group bound to the Pt(IV) cation to produce the products MeOR and L(2)PtMe(2). C-C reductive elimination proceeds from L(2)PtMe(3)(OR) by initial L (L = PMe(3)) or OR(-) (L(2) = dppe, dppbz) dissociation, followed by C-C coupling from the resulting five-coordinate intermediate. Our studies demonstrate that both C-C and C-O reductive elimination reactions from Pt(IV) are more facile in polar solvents, in the presence of Lewis acids, and for OR(-) groups that contain electron withdrawing substituents. PMID:11456927

  17. An atomistic vision of the Mass Action Law: Prediction of carbon/oxygen defects in silicon

    International Nuclear Information System (INIS)

    We introduce an atomistic description of the kinetic Mass Action Law to predict concentrations of defects and complexes. We demonstrate in this paper that this approach accurately predicts carbon/oxygen related defect concentrations in silicon upon annealing. The model requires binding and migration energies of the impurities and complexes, here obtained from density functional theory (DFT) calculations. Vacancy-oxygen complex kinetics are studied as a model system during both isochronal and isothermal annealing. Results are in good agreement with experimental data, confirming the success of the methodology. More importantly, it gives access to the sequence of chain reactions by which oxygen and carbon related complexes are created in silicon. Beside the case of silicon, the understanding of such intricate reactions is a key to develop point defect engineering strategies to control defects and thus semiconductors properties

  18. An atomistic vision of the Mass Action Law: Prediction of carbon/oxygen defects in silicon

    Energy Technology Data Exchange (ETDEWEB)

    Brenet, G.; Timerkaeva, D.; Caliste, D.; Pochet, P. [CEA, INAC-SP2M, Atomistic Simulation Laboratory, F-38000 Grenoble (France); Univ. Grenoble Alpes, INAC-SP2M, L-Sim, F-38000 Grenoble (France); Sgourou, E. N.; Londos, C. A. [University of Athens, Solid State Physics Section, Panepistimiopolis Zografos, Athens 157 84 (Greece)

    2015-09-28

    We introduce an atomistic description of the kinetic Mass Action Law to predict concentrations of defects and complexes. We demonstrate in this paper that this approach accurately predicts carbon/oxygen related defect concentrations in silicon upon annealing. The model requires binding and migration energies of the impurities and complexes, here obtained from density functional theory (DFT) calculations. Vacancy-oxygen complex kinetics are studied as a model system during both isochronal and isothermal annealing. Results are in good agreement with experimental data, confirming the success of the methodology. More importantly, it gives access to the sequence of chain reactions by which oxygen and carbon related complexes are created in silicon. Beside the case of silicon, the understanding of such intricate reactions is a key to develop point defect engineering strategies to control defects and thus semiconductors properties.

  19. Thermodynamic functions, freezing transition, and phase diagram of dense carbon-oxygen mixtures in white dwarfs

    International Nuclear Information System (INIS)

    Equations of state for dense carbon-oxygen (C-O) binary-ionic mixtures (BIM's) appropriate to the interiors of white dwarfs are investigated through Monte Carlo simulations, by solution of relevant integral equations and variational calculations in the density-functional formalism. It is thereby shown that the internal energies of the C-O BIM solids and fluids both obey precisely the linear mixing formulas. We then present an accurate calculation of the phase diagram associated with freezing transitions in such BIM materials, resulting in a novel prediction of an azeotropic diagram. Discontinuities of the mass density across the azeotropic phase boundaries are evaluated numerically for application to a study of white-dwarf evolution

  20. Energy-dependent inactivation of citrate lyase in Enterobacter aerogenes.

    Science.gov (United States)

    Kulla, H; Gottschalk, G

    1977-12-01

    Enterobacter aerogenes was grown in continous culture with ammonia as the growth-limiting substrate, and changes in citrate lyase and citrate synthase activities were monitored after growth shifts from anaerobic growth on citrate to aerobic growth on citrate, aerobic growth on glucose, anaerobic growth on glucose, and anaerobic growth on glucose plus nitrate. Citrate lyase was inactivated during aerobic growth on glucose and during anaerobic growth with glucose plus nitrate. Inactivation did not occur during anaerobic growth on glucose, and as a result of the simultaneous presence of citrate lyase and citrate synthase, growth difficulties were observed. Citrate lyase inactivation consisted of deacetylation of the enzyme. The corresponding deacetylase could not be demonstrated in cell extracts, and it is concluded that, as in a number of other inactivations, electron transport to oxygen or nitrate was required for inactivation. PMID:924971

  1. Pectin Lyase Activity in a Penicillium italicum Strain

    OpenAIRE

    Alaña, Aitor; Alkorta, Itziar; Domínguez, Juan B.; Llama, Maria J.; Serra, Juan L.

    1990-01-01

    An extracellular pectin lyase (PNL) [poly-(methoxygalacturonide)lyase; EC 4.2.2.10] produced by Penicillium italicum CECT 2294 grown on a surface bran (natural medium) or in a submerged (synthetic medium) culture was investigated. Both culture filtrates showed macerating activity at low pH on cucumber, potato, and orange tissues. The physicochemical properties of the enzyme obtained from both culture methods were identical, as well as its catalytic properties, which were assayed by different ...

  2. The hydroxynitrile lyase from almond: crystal structure and mechanistical studies

    International Nuclear Information System (INIS)

    Cyanogenesis is a defense process of several thousand plant species. Hydroxynitrile lyase (HNL), a key enzyme of this process, cleaves a cyanohydrin precursor into hydrocyanic acid and the corresponding aldehyde or ketone. The reverse reaction constitutes an important tool in industrial biocatalysis. Different classes of hydroxynitrile lyases have convergently evolved from FAD-dependent oxidoreductases, α/β hydrolases and alcohol dehydrogenases. The FAD-dependent hydroxynitrile lyases (FAD-HNLs) carry a flavin cofactor whose redox properties appear to be unimportant for catalysis. The high resolution crystal structure of the hydroxynitrile lyase from almond (Prunus amygdalus), PaHNL1, has been determined and constitutes the first 3D structure of an FAD-HNL. The overall fold and the architecture of the active site region showed that PaHNL1 belongs to the glucose-methanol-choline-oxidoreductase family, with closest structural similarity to glucose oxidase. There is strong evidence from the sequence and the reaction product that FAD-dependent hydroxynitrile lyases have evolved from an aryl alcohol oxidizing precursor. Structures of PaHNL1 in complex with its natural substrate mandelonitrile and the competitive inhibitor benzyl alcohol provided insight into the residues involved in catalysis and a mechanism without participation of the cofactor could be suggested. Although the catalytic residues differ between the α/β-hydrolase-type HNLs and PaHNL1, common general features relevant for hydroxynitrile lyase activity could be proposed. (author)

  3. Carbon-Oxygen-Neon mass nuclei in super-strong magnetic fields

    CERN Document Server

    Stein, Martin; Sedrakian, Armen; Reinhard, P -G

    2016-01-01

    The properties of carbon, oxygen and neon nuclei in strong magnetic fields $B\\simeq 10^{17}\\,$G are studied in the context of strongly magnetized neutron stars and white dwarfs. The Sky3D code is extended to incorporate the interaction of nucleons with the magnetic field and is utilized to solve the time-independent Hartree-Fock equations with a Skyrme interaction on a Cartesian 3D grid. The numerical solutions demonstrate a number of phenomena, which include a splitting of the energy levels of spin up and down nucleons, spontaneous rearrangment of energy levels in $^{16}O$ at a critical field, which leads to jump-like increase of magnetization and proton current in this nucleus, evolution of the intrinsically deformed $^{20}Ne$ nucleus towards a more spherical shape under increasing field strength. Many of the numerical features can be understood within a simple analytical model based on the occupation by the nucleons of the lowest states of harmonic oscillator in a magnetic field.

  4. The effect of oxygen fugacity on the solubility of carbon-oxygen fluids in basaltic melt

    Science.gov (United States)

    Pawley, Alison R.; Holloway, John R.; Mcmillan, Paul F.

    1992-01-01

    The solubility of CO2-CO fluids in a midocean ridge basalt have been measured at 1200 C, 500-1500 bar, and oxygen fugacities between NNO and NNO-4. In agreement with results of previous studies, the results reported here imply that, at least at low pressures, CO2 dissolves in basaltic melt only in the form of carbonate groups. The dissolution reaction is heterogeneous, with CO2 molecules in the fluid reacting directly with reactive oxygens in the melt to produce CO3(2-). CO, on the other hand, is insoluble, dissolving neither as carbon, molecular CO, nor CO3(2-). It is shown that, for a given pressure and temperature, the concentration of dissolved carbon-bearing species in basaltic melt in equilibrium with a carbon-oxygen fluid is proportional to the mole fraction of CO2 in the fluid, which is a function of fO2. At low pressures CO2 solubility is a linear function of CO2 fugacity at constant temperatures.

  5. Population synthesis of triple systems in the context of mergers of carbon-oxygen white dwarfs

    CERN Document Server

    Hamers, Adrian S; Claeys, Joke S W; Nelemans, Gijs

    2013-01-01

    Hierarchical triple systems are common among field stars yet their long-term evolution is poorly understood theoretically. In such systems Kozai cycles can be induced in the inner binary system during which the inner orbit eccentricity and the inclination between both binary orbits vary periodically. These cycles, combined with tidal friction and gravitational wave emission, can significantly affect the inner binary evolution. To investigate these effects quantitatively we perform a population synthesis study of triple systems and focus on evolutionary paths that lead to mergers of carbon-oxygen (CO) white dwarfs (WDs), which constitute an important candidate progenitor channel for type Ia supernovae (SNe Ia). We approach this problem by Monte Carlo sampling from observation-based distributions of systems within the primary mass range 1.0 - 6.5 M_Sun and inner orbit semi-major axes a_1 and eccentricities e_1 satisfying a_1 (1-e_1^2) > 12 AU, i.e. non-interacting in the absence of a tertiary component. We evol...

  6. Supernova SN 2011fe from an exploding carbon-oxygen white dwarf star.

    Science.gov (United States)

    Nugent, Peter E; Sullivan, Mark; Cenko, S Bradley; Thomas, Rollin C; Kasen, Daniel; Howell, D Andrew; Bersier, David; Bloom, Joshua S; Kulkarni, S R; Kandrashoff, Michael T; Filippenko, Alexei V; Silverman, Jeffrey M; Marcy, Geoffrey W; Howard, Andrew W; Isaacson, Howard T; Maguire, Kate; Suzuki, Nao; Tarlton, James E; Pan, Yen-Chen; Bildsten, Lars; Fulton, Benjamin J; Parrent, Jerod T; Sand, David; Podsiadlowski, Philipp; Bianco, Federica B; Dilday, Benjamin; Graham, Melissa L; Lyman, Joe; James, Phil; Kasliwal, Mansi M; Law, Nicholas M; Quimby, Robert M; Hook, Isobel M; Walker, Emma S; Mazzali, Paolo; Pian, Elena; Ofek, Eran O; Gal-Yam, Avishay; Poznanski, Dovi

    2011-12-15

    Type Ia supernovae have been used empirically as 'standard candles' to demonstrate the acceleration of the expansion of the Universe even though fundamental details, such as the nature of their progenitor systems and how the stars explode, remain a mystery. There is consensus that a white dwarf star explodes after accreting matter in a binary system, but the secondary body could be anything from a main-sequence star to a red giant, or even another white dwarf. This uncertainty stems from the fact that no recent type Ia supernova has been discovered close enough to Earth to detect the stars before explosion. Here we report early observations of supernova SN 2011fe in the galaxy M101 at a distance from Earth of 6.4 megaparsecs. We find that the exploding star was probably a carbon-oxygen white dwarf, and from the lack of an early shock we conclude that the companion was probably a main-sequence star. Early spectroscopy shows high-velocity oxygen that slows rapidly, on a timescale of hours, and extensive mixing of newly synthesized intermediate-mass elements in the outermost layers of the supernova. A companion paper uses pre-explosion images to rule out luminous red giants and most helium stars as companions to the progenitor. PMID:22170680

  7. Properties of Carbon-Oxygen White Dwarfs From Monte Carlo Stellar Models

    CERN Document Server

    Fields, C E; Petermann, I; Iliadis, C; Timmes, F X

    2016-01-01

    We investigate properties of carbon-oxygen white dwarfs with respect to the composite uncertainties in the reaction rates using the stellar evolution toolkit, Modules for Experiments in Stellar Astrophysics (MESA) and the probability density functions in the reaction rate library STARLIB. These are the first Monte Carlo stellar evolution studies that use complete stellar models. Focusing on 3 M$_{\\odot}$ models evolved from the pre main-sequence to the first thermal pulse, we survey the remnant core mass, composition, and structure properties as a function of 26 STARLIB reaction rates covering hydrogen and helium burning using a Principal Component Analysis and Spearman Rank-Order Correlation. Relative to the arithmetic mean value, we find the width of the 95\\% confidence interval to be $\\Delta M_{{\\rm 1TP}}$ $\\approx$ 0.019 M$_{\\odot}$ for the core mass at the first thermal pulse, $\\Delta$$t_{\\rm{1TP}}$ $\\approx$ 12.50 Myr for the age, $\\Delta \\log(T_{{\\rm c}}/{\\rm K}) \\approx$ 0.013 for the central temperat...

  8. Unexpected carbon-oxygen bond cleavage of THF promoted by guanidinate titanium complex/lithium diisopropylamide: Synthesis and crystal structure

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yong; WANG Mei; YAO Yingming; SHEN Qi

    2005-01-01

    An unexpected carbon-oxygen bond cleavage of THF (THF = tetrahydrofuran) promoted by guanidinate titanium complex was described. Guanidinate lithium [Pr2iNC(NCy)2]Li (Cy = cyclohexyl) formed in situ reacted with TiCl4(THF)2 in a 2:1 molar ratio to produce the guanidinate titanium chloride [Pr2iNC(NCy)2]2TiCl2 (1) in good yield. The reaction of [Pr2iNC(NCy)2]2TiCl2 with lithium diisopropylamide in THF afforded an unexpected [Pr2iNC(NCy)2]2Ti(OBun)2 (2), which was formed by the cleavage of carbon-oxygen bond of THF. Complexes 1 and 2 were fully characterized by elemental analysis, NMR and IR spectroscopies, and X-ray crystal structure determination for complex 2.

  9. Thermodynamics of the hydrogen-carbon-oxygen-tungsten system, as applied to the manufacture of tungsten and tungsten carbide

    International Nuclear Information System (INIS)

    The thermodynamics of the quaternary hydrogen-carbon oxygen-tungsten system and its binary and ternary sub-systems are reviewed. Published thermodynamic data are evaluated, and expression for free energies of formation are chosen. These expressions are integrated with and equilibrium-calculating algorithm, producing a powerful tool for understanding and improving the manufacture of tungsten and tungsten carbide. Three examples are presented: reduction/carburization of tungstic oxide with hydrogen, carbon, and methane. (author)

  10. Hydrodynamical evolution of merging carbon-oxygen white dwarfs: their pre-supernova structure and observational counterparts

    OpenAIRE

    Tanikawa, Ataru; Nakasato, Naohito; Sato, Yushi; Nomoto, Ken'ichi; Maeda, Keiichi; Hachisu, Izumi

    2015-01-01

    We perform smoothed particle hydrodynamics (SPH) simulations for merging binary carbon-oxygen (CO) white dwarfs (WDs) with masses of $1.1$ and $1.0$ $M_\\odot$, until the merger remnant reaches a dynamically steady state. Using these results, we assess whether the binary could induce a thermonuclear explosion, and whether the explosion could be observed as a type Ia supernova (SN Ia). We investigate three explosion mechanisms: a helium-ignition following the dynamical merger (`helium-ignited v...

  11. Crystallization and preliminary X-ray analysis of the rhamnogalacturonan lyase YesW from Bacillus subtilis strain 168, a member of polysaccharide lyase family 11

    International Nuclear Information System (INIS)

    The crystallization and preliminary X-ray characterization of the polysaccharide lyase family 11 rhamnogalacturonan lyase are presented. Rhamnogalacturonan lyases degrade rhamnogalacturonan I, a major component of pectin, through a β-elimination reaction. YesW from Bacillus subtilis strain 168 is a novel rhamnogalacturonan lyase classified into polysaccharide lyase family 11 (PL-11). The enzyme was crystallized at 293 K using the sitting-drop vapour-diffusion method with 2-methyl-2,4-pentanediol (MPD) as a precipitant. Preliminary X-ray analysis revealed that the YesW crystals belong to space group P21 and diffract to 2.40 Å resolution, with unit-cell parameters a = 56.7, b = 105.6, c = 101.4 Å, β = 94.9°. This is the first report on the crystallization and preliminary X-ray analysis of a family PL-11 rhamnogalacturonan lyase

  12. Polysaccharide Lyase: Molecular Cloning, Sequencing, and Overexpression of the Xanthan Lyase Gene of Bacillus sp. Strain GL1

    OpenAIRE

    Hashimoto, Wataru; Miki, Hikaru; Tsuchiya, Noriaki; Nankai, Hirokazu; Murata, Kousaku

    2001-01-01

    When grown on xanthan as a carbon source, the bacterium Bacillus sp. strain GL1 produces extracellular xanthan lyase (75 kDa), catalyzing the first step of xanthan depolymerization (H. Nankai, W. Hashimoto, H. Miki, S. Kawai, and K. Murata, Appl. Environ. Microbiol. 65:2520–2526, 1999). A gene for the lyase was cloned, and its nucleotide sequence was determined. The gene contained an open reading frame consisting of 2,793 bp coding for a polypeptide with a molecular weight of 99,308. The poly...

  13. Priming ammonia lyases and aminomutases for industrial and therapeutic applications

    NARCIS (Netherlands)

    Heberling, Matthew M.; Wu, Bian; Bartsch, Sebastian; Janssen, Dick B.; Truppo, Matthew D.; Turner, Nicholas J.

    2013-01-01

    Ammonia lyases (AL) and aminomutases (AM) are emerging in green synthetic routes to chiral amines and an AL is being explored as an enzyme therapeutic for treating phenylketonuria and cancer. Although the restricted substrate range of the wild-type enzymes limits their widespread application, the no

  14. Cystathionine .gamma.-lyase: Clinical, metabolic, genetic, and structural studies

    Czech Academy of Sciences Publication Activity Database

    Kraus, J. P.; Hašek, Jindřich; Kožich, V.; Collard, R.; Venezia, S.; Janošíková, B.; Wang, J.; Stabler, S. P.; Allen, R. H.; Jakobs, C.; Finn, C. T.; Chien, Y. H.; Hwu, W. L.; Hegele, R. A.; Mudd, S. H.

    2009-01-01

    Roč. 97, č. 4 (2009), s. 250-259. ISSN 1096-7192 R&D Projects: GA ČR GA305/07/1073 Institutional research plan: CEZ:AV0Z40500505 Keywords : cystathionine gamma-lyase * cystathioninuria * hypercystathioninemia Subject RIV: CD - Macromolecular Chemistry Impact factor: 2.897, year: 2009

  15. Cultivable Alginate Lyase-Excreting Bacteria Associated with the Arctic Brown Alga Laminaria

    Directory of Open Access Journals (Sweden)

    Yu-Zhong Zhang

    2012-11-01

    Full Text Available Although some alginate lyases have been isolated from marine bacteria, alginate lyases-excreting bacteria from the Arctic alga have not yet been investigated. Here, the diversity of the bacteria associated with the brown alga Laminaria from the Arctic Ocean was investigated for the first time. Sixty five strains belonging to nine genera were recovered from six Laminaria samples, in which Psychrobacter (33/65, Psychromonas (10/65 and Polaribacter (8/65 were the predominant groups. Moreover, 21 alginate lyase-excreting strains were further screened from these Laminaria-associated bacteria. These alginate lyase-excreting strains belong to five genera. Psychromonas (8/21, Psedoalteromonas (6/21 and Polaribacter (4/21 are the predominant genera, and Psychrobacter, Winogradskyella, Psychromonas and Polaribacter were first found to produce alginate lyases. The optimal temperatures for the growth and algiante lyase production of many strains were as low as 10–20 °C, indicating that they are psychrophilic bacteria. The alginate lyases produced by 11 strains showed the highest activity at 20–30 °C, indicating that these enzymes are cold-adapted enzymes. Some strians showed high levels of extracellular alginate lyase activity around 200 U/mL. These results suggest that these algiante lyase-excreting bacteria from the Arctic alga are good materials for studying bacterial cold-adapted alginate lyases.

  16. Crystallization and preliminary X-ray analysis of an exotype alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, a member of polysaccharide lyase family 15

    International Nuclear Information System (INIS)

    The crystallization and preliminary X-ray characterization of a family PL-15 exotype alginate lyase are presented. Almost all alginate lyases depolymerize alginate in an endolytical fashion via a β-elimination reaction. The alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, consisting of 776 amino-acid residues, is a novel exotype alginate lyase classified into polysaccharide lyase family 15. The enzyme was crystallized at 293 K by sitting-drop vapour diffusion with polyethylene glycol 4000 as a precipitant. Preliminary X-ray analysis showed that the Atu3025 crystal belonged to space group P21 and diffracted to 2.8 Å resolution, with unit-cell parameters a = 107.7, b = 108.3, c = 149.5 Å, β = 91.5°

  17. A hierarchical classification of polysaccharide lyases for glycogenomics.

    Science.gov (United States)

    Lombard, Vincent; Bernard, Thomas; Rancurel, Corinne; Brumer, Harry; Coutinho, Pedro M; Henrissat, Bernard

    2010-12-15

    Carbohydrate-active enzymes face huge substrate diversity in a highly selective manner using only a limited number of available folds. They are therefore subjected to multiple divergent and convergent evolutionary events. This and their frequent modularity render their functional annotation in genomes difficult in a number of cases. In the present paper, a classification of polysaccharide lyases (the enzymes that cleave polysaccharides using an elimination instead of a hydrolytic mechanism) is shown thoroughly for the first time. Based on the analysis of a large panel of experimentally characterized polysaccharide lyases, we examined the correlation of various enzyme properties with the three levels of the classification: fold, family and subfamily. The resulting hierarchical classification, which should help annotate relevant genes in genomic efforts, is available and constantly updated at the Carbohydrate-Active Enzymes Database (http://www.cazy.org). PMID:20925655

  18. Isolation of Protoplasts from Undaria pinnatifida by Alginate Lyase Digestion

    Institute of Scientific and Technical Information of China (English)

    HU Xiaoke; JIANG Xiaolu; GUAN Huashi

    2003-01-01

    The aim of this study is to isolate protoplasts from Undaria pinnatifida. Protoplasts of the alga were isolated enzymatically by using alginate lyase, which was prepared by fermenting culture of a strain Vibrio sp. 510. Monofacterial method was applied for optimizing digestion condition. The optimum condition for protoplast preparation is enzymatic digestion at 28 ℃ for 2 h using alginate lyase at the concentration of 213.36 U (8 mL) every 0.5 g fresh thalline with NaCl 50 and at the shaking speed of 150 r min-1 during digestion. The protoplast yield can reach 2.62 + 0.09 million per 0.5 g fresh leave under the optimum condition. The enzyme activity is inhibited by Ca2+ and slightly enhanced by Fe2+ and Mn2+ at concentrations of 0.05, 0.08 and 0.10 molL-1.

  19. Isolation of protoplasts from undaria pinnatifida by alginate lyase digestion

    Science.gov (United States)

    Xiaoke, Hu; Xiaolu, Jiang; Huashi, Guan

    2003-04-01

    The aim of this study is to isolate protoplasts from Undaria pinnatifida. Protoplasts of the alga were isolated enzymatically by using alginate lyase, which was prepared by fermenting culture of a strain Vibrio sp. 510. Monofacterial method was applied for optimizing digestion condition. The optimum condition for protoplast preparation is enzymatic digestion at 28°C for 2h using alginate lyase at the concentration of 213.36 U (8 mL) every 0.5g fresh thalline with NaCl 50 and at the shaking speed of 150 r min-1 during digestion. The protoplast yield can reach 2.62±0.09 million per 0.5 g fresh leave under the optimum condition. The enzyme activity is inhibited by Ca2+ and slightly enhanced by Fe2+ and Mn2+ at concentrations of 0.05, 0.08 and 0.10 mol L-1.

  20. ATP citrate lyase knockdown impacts cancer stem cells in vitro

    OpenAIRE

    Hanai, J-i; Doro, N; Seth, P; Sukhatme, V P

    2013-01-01

    ATP citrate lyase (ACL) knockdown (KD) causes tumor suppression and induces differentiation. We have previously reported that ACL KD reverses epithelial–mesenchymal transition (EMT) in lung cancer cells. Because EMT is often associated with processes that induce stemness, we hypothesized that ACL KD impacts cancer stem cells. By assessing tumorsphere formation and expression of stem cell markers, we showed this to be the case in A549 cells, which harbor a Ras mutation, and in two other non-sm...

  1. Truth and consequences of sphingosine-1-phosphate lyase

    OpenAIRE

    Aguilar, Ana; Saba, Julie D.

    2011-01-01

    Sphingosine phosphate lyase (SPL) is an intracellular enzyme responsible for the irreversible catabolism of the lipid signaling molecule sphingosine-1-phosphate (S1P). SPL catalyzes the cleavage of S1P resulting in the formation of hexadecenal and ethanolamine phosphate. S1P functions as a ligand for a family of ubiquitously expressed G protein-coupled receptors that mediate autocrine and paracrine signals controlling cell migration, proliferation and programmed cell death pathways. S1P has a...

  2. HISTIDINE BIOTRANSFORMATION MEDIATED BY L-HISTIDINE-AMMONIA-LYASE

    OpenAIRE

    Borisova, G.; Bessonova, O.

    2013-01-01

    Kinetics of the metabolism of the heterocyclic amino acid histidine exposed to the L-histidine ammonia-lyase enzyme has been investigated and the technology of extraction of histidine biotransformation products (urocanic acid and ammonia) from casein hydrolyzates enabling the subsequent use of these hydrolyzates as a milk protein concentrate for the production of specialized dietary products for the nutrition of histidinemia patients has been developed.

  3. Function analysis of cystathionine gamma-lyase mutants

    Czech Academy of Sciences Publication Activity Database

    Hašek, Jindřich

    2009-01-01

    Roč. 16, 1a (2009), b5. ISSN 1211-5894. [Discussions in Structural Molecular Biology /7./. 12.03.2009-14.03.2009, Nové Hrady] R&D Projects: GA ČR GA305/07/1073 Institutional research plan: CEZ:AV0Z40500505 Keywords : cystathionine gamma-lyase * medical treatment * vitamin B6 Subject RIV: CD - Macromolecular Chemistry

  4. Comparative characterization of three bacterial exo-type alginate lyases.

    Science.gov (United States)

    Hirayama, Makoto; Hashimoto, Wataru; Murata, Kousaku; Kawai, Shigeyuki

    2016-05-01

    Alginate, a major acidic polysaccharide in brown macroalgae, has attracted attention as a carbon source for production of ethanol and other chemical compounds. Alginate is monomerized by exo-type alginate lyase into an unsaturated uronate; thus, this enzyme is critical for the saccharification and utilization of alginate. Although several exo-type alginate lyases have been characterized independently, their activities were not assayed under the same conditions or using the same unit definition, making it difficult to compare enzymatic properties or to select the most suitable enzyme for saccharification of alginate. In this study, we characterized the three bacterial exo-type alginate lyases under the same conditions: A1-IV of Sphingomonas sp. strain A1, Atu3025 of Agrobacterium tumefaciens, and Alg17c of Saccharophagus degradans. A1-IV had the highest specific activity as well as the highest productivity of uronate, whereas Alg17c had the lowest activity and productivity. Only dialyzed Atu3025 and Alg17c were tolerant to freezing. Alg17c exhibited a remarkable halotolerance, which may be advantageous for monomerization of alginate from marine brown algae. Thus, each enzyme exhibited particular desirable and undesirable properties. Our results should facilitate further utilization of the promising polysaccharide alginate. PMID:26827758

  5. Utilization of Aspergillus oryzae to produce pectin lyase from various agro-industrial residues

    OpenAIRE

    Safia Koser; Zahid Anwar; Zafar Iqbal; Awais Anjum; Tahir Aqil; Sajid Mehmood; Muhammad Irshad

    2014-01-01

    The present study was aimed to investigate the culture influence on pectin lyase production potential of fungal strain Aspergillus oryzae. The enzyme profile of A. oryzae showed highest activity of pectin lyase after 3rd day of incubation on lemon peel waste under solid state fermentation conditions. To induce the pectin lyase synthesis capability of A. oryzae at optimal level various culture variables including physical and nutritional parameters were optimized by adopting classical optimiza...

  6. SN 2010LP—A TYPE IA SUPERNOVA FROM A VIOLENT MERGER OF TWO CARBON-OXYGEN WHITE DWARFS

    International Nuclear Information System (INIS)

    SN 2010lp is a subluminous Type Ia supernova (SN Ia) with slowly evolving lightcurves. Moreover, it is the only subluminous SN Ia observed so far that shows narrow emission lines of [O I] in late-time spectra, indicating unburned oxygen close to the center of the ejecta. Most explosion models for SNe Ia cannot explain the narrow [O I] emission. Here, we present hydrodynamic explosion and radiative transfer calculations showing that the violent merger of two carbon-oxygen white dwarfs of 0.9 and 0.76 M ☉ adequately reproduces the early-time observables of SN 2010lp. Moreover, our model predicts oxygen close to the center of the explosion ejecta, a pre-requisite for narrow [O I] emission in nebular spectra as observed in SN 2010lp

  7. Supernovae of types I and II and the neutrino mechanism of thermonuclear explosion of carbon-oxygen degenerated stellar cores

    International Nuclear Information System (INIS)

    The hydrodynamic process of thermonuclear explosion of hydrostatic equilibrium carbon-oxygen degenerated star cores with mass Msub(c)=1.40Msub(Sun) and different values of the central density rhosub(c) within the interval 2x10910 g/cm3 is studied. The initial temperature distribution has been determined by the preceding thermal stage of explosion. The calculations successively include the kinetics of thermonuclear matter burning, kinetics of β processes, and the neutrino energy losses. Consideration for the neutrino mechanism of heating and carbon ignition made it possible to obtain two characteristic versions of the explosion development in numerical hydrodynamic calculations: a) at 2x1099 g/cm3-disruption of the whole star with either complete or partial burning of the carbon and a 1050-1051 erg kinetic energy; b) at 9x10910 g/cm3-collapse of the stellar core into a neutron star with a partial outburst of the outer envelope with a small kinetic energy 1049-1050 erg. The paper puts forward and treats in detail a hypothesis on the first version of explosion corresponding to a Supernovae of type II and the second one, supplemented by some mechanism of slow energy release into the envelope from the formed neutron star, corresponding to a Supernovae of type I. Based on the proposed hypothesis a satisfactory agreement with the observed masses and energies os the Supernovae envelope, their light curves and spectra, as well as with the data on their chemical composition has been obtained. An important point in this agreement is the assumption on presupernovae of type I beina almost bare compact carbon-oxygen stellar cores and presupernovae of type II being red supergiants. Most probable, the evolution of presupernovae of type I takes place in close binaries, whereas that of presupernovae of type II seems practically not to differ from the evolution of a single star

  8. Alfalfa contains substantial 9-hydroperoxide lyase activity and a 3Z:2E-enal isomerase

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Noordermeer, M.A.; Veldink, G.A.

    1999-01-01

    Fatty acid hydroperoxides formed by lipoxygenase can be cleaved by hydroperoxide lyase resulting in the formation of short-chain aldehydes and omega-oxo acids. Plant hydroperoxide lyases use 13- or 9-hydroperoxy linoleic and linolenic acid as substrates. Alfalfa (Medicago sativa L.) has been reporte

  9. Overexpression of isocitrate lyase-glyoxylate bypass influence on metabolism in Aspergillus niger

    DEFF Research Database (Denmark)

    Meijer, Susan Lisette; Otero, José Manuel; Olivares Hernandez, Roberto;

    2009-01-01

    In order to improve the production of succinate and malate by the filamentous fungus Aspergillus niger the activity of the glyoxylate bypass pathway was increased by over-expression of the isocitrate lyase (icl) gene. The hypothesis was that when isocitrate lyase was up-regulated the flux towards...

  10. Structure and mechanism of the phycobiliprotein lyase CpcT.

    Science.gov (United States)

    Zhou, Wei; Ding, Wen-Long; Zeng, Xiao-Li; Dong, Liang-Liang; Zhao, Bin; Zhou, Ming; Scheer, Hugo; Zhao, Kai-Hong; Yang, Xiaojing

    2014-09-26

    Pigmentation of light-harvesting phycobiliproteins of cyanobacteria requires covalent attachment of open-chain tetrapyrroles, bilins, to the apoproteins. Thioether formation via addition of a cysteine residue to the 3-ethylidene substituent of bilins is mediated by lyases. T-type lyases are responsible for attachment to Cys-155 of phycobiliprotein β-subunits. We present crystal structures of CpcT (All5339) from Nostoc (Anabaena) sp. PCC 7120 and its complex with phycocyanobilin at 1.95 and 2.50 Å resolution, respectively. CpcT forms a dimer and adopts a calyx-shaped β-barrel fold. Although the overall structure of CpcT is largely retained upon chromophore binding, arginine residues at the opening of the binding pocket undergo major rotameric rearrangements anchoring the propionate groups of phycocyanobilin. Based on the structure and mutational analysis, a reaction mechanism is proposed that accounts for chromophore stabilization and regio- and stereospecificity of the addition reaction. At the dimer interface, a loop extending from one subunit partially shields the opening of the phycocyanobilin binding pocket in the other subunit. Deletion of the loop or disruptions of the dimer interface significantly reduce CpcT lyase activity, suggesting functional relevance of the dimer. Dimerization is further enhanced by chromophore binding. The chromophore is largely buried in the dimer, but in the monomer, the 3-ethylidene group is accessible for the apophycobiliprotein, preferentially from the chromophore α-side. Asp-163 and Tyr-65 at the β- and α-face near the E-configured ethylidene group, respectively, support the acid-catalyzed nucleophilic Michael addition of cysteine 155 of the apoprotein to an N-acylimmonium intermediate proposed by Grubmayr and Wagner (Grubmayr, K., and Wagner, U. G. (1988) Monatsh. Chem. 119, 965-983). PMID:25074932

  11. Structural insights into the bacterial carbon - phosphorus lyase machinery

    DEFF Research Database (Denmark)

    Seweryn, Paulina; Van, Lan Bich; Kjeldgaard, Morten;

    2015-01-01

    Phosphorus is required for all life and microorganisms can extract it from their environment through several metabolic pathways. When phosphate is in limited supply, some bacteria are able to use phosphonate compounds, which require specialized enzymatic machinery to break the stable carbon......–phosphorus (C–P) bond. Despite its importance, the details of how this machinery catabolizes phosphonates remain unknown. Here we determine the crystal structure of the 240-kilodalton Escherichia coli C–P lyase core complex (PhnG–PhnH–PhnI–PhnJ; PhnGHIJ), and show that it is a two-fold symmetric hetero...

  12. Cloning and expression of isocitrate lyase from human round worm Strongyloides stercoralis

    Directory of Open Access Journals (Sweden)

    Siddiqui A.A.

    2000-09-01

    Full Text Available A full length cDNA (1463 bp encoding isocitrate lyase (EC 4.1.3.1 of Strongyloides stercoralis is described. The nucleotide sequence of this insert identified a cDNA coding for the isocitrate lyase. The conceptually translated amino acid sequence of the open reading frame for S. stercoralis isocitrate lyase encodes a 450 amino acid residue protein with an apparent molecular weight of 50 kDa and a predicted pl of 6.39. The sequence is 69 % A/T, reflecting a characteristic A/T codon bias of S. stercoralis. The amino acid sequence of S. stercoralis isocitrate lyase is compared with bifunctional glyoxylate cycle protein of Caenorhabditis elegans and isocitrate lyases from Chlamydomonas reinhardtii and Myxococcus xanthus. The full length cDNA of S. stercoralis was expressed in pRSET vector and bacteriophage T7 promoter based expression system. S. stercoralis lyase recombinant protein, purified via immobilized metal affinity chromatography, showed a molecular mass of 50 kDa on polyacrylamide gels. The role of isocitrate lyase in the glyoxylate cycle and energy metabolism of S. stercoralis is also discussed.

  13. Hydrodynamical evolution of merging carbon-oxygen white dwarfs: their pre-supernova structure and observational counterparts

    CERN Document Server

    Tanikawa, Ataru; Sato, Yushi; Nomoto, Ken'ichi; Maeda, Keiichi; Hachisu, Izumi

    2015-01-01

    We perform smoothed particle hydrodynamics (SPH) simulations for merging binary carbon-oxygen (CO) white dwarfs (WDs) with masses of $1.1$ and $1.0$ $M_\\odot$, until the merger remnant reaches a dynamically steady state. Using these results, we assess whether the binary could induce a thermonuclear explosion, and whether the explosion could be observed as a type Ia supernova (SN Ia). We investigate three explosion mechanisms: a helium-ignition following the dynamical merger (`helium-ignited violent merger model'), a carbon-ignition (`carbon-ignited violent merger model'), and an explosion following the formation of the Chandrasekhar mass WD (`Chandrasekhar mass model'). An explosion of the helium-ignited violent merger model is possible, while we predict that the resulting SN ejecta are highly asymmetric since its companion star is fully intact at the time of the explosion. The carbon-ignited violent merger model can also lead to an explosion. However, the envelope of the exploding WD spreads out to $\\sim 0.1...

  14. The importance of four histidine residues in isocitrate lyase from Escherichia coli.

    OpenAIRE

    Diehl, P; McFadden, B A

    1994-01-01

    By site-directed mutagenesis, substitutions were made for His-184 (H-184), H-197, H-266, and H-306 in Escherichia coli isocitrate lyase. Of these changes, only mutations of H-184 and H-197 appreciably reduced enzyme activity. Mutation of H-184 to Lys, Arg, or Leu resulted in an inactive isocitrate lyase, and mutation of H-184 to Gln resulted in an enzyme with 0.28% activity. Nondenaturing polyacrylamide gel electrophoresis demonstrated that isocitrate lyase containing the Lys, Arg, Gln, and L...

  15. A novel gene encoding xanthan lyase of Paenibacillus alginolyticus strain XL-1

    OpenAIRE

    Ruijssenaars, H.J.; Hartmans, S.; Verdoes, J.C.

    2000-01-01

    Xanthan-modifying enzymes are powerful tools in studying structure-function relationships of this polysaccharide. One of these modifying enzymes is xanthan lyase, which removes the terminal side chain residue of xanthan. In this paper, the cloning and sequencing of the first xanthan lyase-encoding gene is described, i.e., the xalA gene, encoding pyruvated mannose-specific xanthan lyase of Paenibacillus alginolyticus XL-1. The xalA gene encoded a 100,823-Da protein, including a 36-amino-acid s...

  16. A Pyruvated Mannose-Specific Xanthan Lyase Involved in Xanthan Degradation by Paenibacillus alginolyticus XL-1

    OpenAIRE

    Ruijssenaars, Harald J.; de Bont, Jan A. M.; Hartmans, Sybe

    1999-01-01

    The xanthan-degrading bacterium Paenibacillus alginolyticus XL-1, isolated from soil, degrades approximately 28% of the xanthan molecule and appears to leave the backbone intact. Several xanthan-degrading enzymes were excreted during growth on xanthan, including xanthan lyase. Xanthan lyase production was induced by xanthan and inhibited by glucose and low-molecular-weight enzymatic degradation products from xanthan. A xanthan lyase with a molecular mass of 85 kDa and a pI of 7.9 was purified...

  17. Structural Insights Into The Bacterial Carbon-Phosphorus Lyase Machinery

    DEFF Research Database (Denmark)

    Brodersen, Ditlev Egeskov

    Phosphonate compounds act as a nutrient source for some microorganisms when phosphate is limiting but require a specialised enzymatic machinery due to the presence of the highly stable carbon-phosphorus bond. Despite the fundamental importance to microbial metabolism, the details of how the prote......Phosphonate compounds act as a nutrient source for some microorganisms when phosphate is limiting but require a specialised enzymatic machinery due to the presence of the highly stable carbon-phosphorus bond. Despite the fundamental importance to microbial metabolism, the details of how...... the reaction. Our results delineate the overall architecture of the carbon-phosphorus lyase machinery and give detailed insight into the mechanism underlying microbial phosphonate breakdown....

  18. Structure of methionine γ-lyase from Clostridium sporogenes.

    Science.gov (United States)

    Revtovich, Svetlana; Anufrieva, Natalya; Morozova, Elena; Kulikova, Vitalia; Nikulin, Alexey; Demidkina, Tatyana

    2016-01-01

    Methionine γ-lyase (MGL) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the γ-elimination reaction of L-methionine. The enzyme is a promising target for therapeutic intervention in some anaerobic pathogens and has attracted interest as a potential cancer treatment. The crystal structure of MGL from Clostridium sporogenes has been determined at 2.37 Å resolution. The fold of the protein is similar to those of homologous enzymes from Citrobacter freundii, Entamoeba histolytica, Pseudomonas putida and Trichomonas vaginalis. A comparison of these structures revealed differences in the conformation of two flexible regions of the N- and C-terminal domains involved in the active-site architecture. PMID:26750487

  19. High cerebral guanidinoacetate and variable creatine concentrations in argininosuccinate synthetase and lyase deficiency : Implications for treatment?

    NARCIS (Netherlands)

    van Spronsen, F. J.; Reijngoud, D. J.; Verhoeven, N. M.; Soorani-Lunsing, R. J.; Jakobs, C.; Sijens, P. E.

    2006-01-01

    Cerebral creatine and guanidinoacetate and blood and urine metabolites were studied in four patients with argininosuccinate synthetase (ASS) or argininosuccinate lyase (ASL) deficiency receiving large doses of arginine. Urine and blood metabolites varied largely. Cerebral guanidinoacetate was increa

  20. Isocitrate lyase and the glyoxylate cycle. Progress report, February 15, 1989--February 15, 1990

    Energy Technology Data Exchange (ETDEWEB)

    McFadden, B.A.

    1990-12-31

    Active site modifications of isocitrate lyase (icl) from Escherichia coli are described. In addition directed mutagenesis of icl gene are detailed aimed at varying the charge yet conserving the structure of the enzymes active site.

  1. Synthesis of novel 21-trifluoropregnane steroids: inhibitors of 17 alpha-hydroxylase/17,20-lyase (17 alpha-lyase).

    Science.gov (United States)

    Njar, V C; Klus, G T; Johnson, H H; Brodie, A M

    1997-06-01

    Novel 21-trifluoropregnenolone (6), 21-trifluoroprogesterone (7) and related compounds 4a and 8 have been synthesized in high yields from 3 beta-acetoxyandrost-5-ene-17 beta-carbaldehyde (3). The key reaction was the conversion of 3 into the 21-trifluoromethyl-20-alcohol as a diastereomeric mixture (4) by trifluoromethyltrimethylsilane (TMS-CF3) in the presence of tetrabutylammonium fluoride (TBAF). All compounds, including 6 and 7, were unambiguously characterized by IR, 1H and 19F NMR, high-resolution mass spectrometry (HRMS), and elemental analysis. On this basis, we concluded that the only report of an earlier synthesis of 6 and 7 is erroneous. Enzyme inhibition studies showed that 20 xi-hydroxy-21-trifluoropregn-4-en-3-one (8) is a potent inhibitor (IC50 value = 0.6 microM) of rat 17 alpha-hydroxylase/17,20-lyase. PMID:9185294

  2. Oxygen aggregation kinetics, thermal donors and carbon-oxygen defect formation in silicon containing carbon and tin

    International Nuclear Information System (INIS)

    Localized vibrational mode spectroscopy measurements on Czochralski silicon (Cz-Si) samples subjected to isothermal annealing at 450 °C are reported. First, we studied the effect of carbon (C) and tin (Sn) isovalent dopants on the aggregation kinetics of oxygen. It is determined that the reduction rate of oxygen is described by the Johnson-Mehl-Avrami equation in accordance with previous reports. The activation energy related with the reaction rate constant of the process is calculated to increase from Cz-Si, to C-doped Cz-Si (CCz-Si), to Sn-doped Cz-Si contained C (SnCz-Si). This is attributed to the presence of the isovalent dopants that may impact both the kinetics of the oxygen atoms and also may lead to the formation of other oxygen-related clusters. Second, we studied the effect of Sn on the formation and evolution of carbon-oxygen (C-O) defects. It was determined that the presence of Sn suppresses the formation of the C-O defects as indicated by the reduction in the strength of the 683, 626, and 586 cm−1 well-known bands of CsOi defect. The phenomenon is attributed to the association of Sn with C atoms that may prevent the pairing of O with C. Third, we investigated the effect of C and Sn on the formation of thermal donors (TDs). Regarding carbon our results verified previous reports that carbon suppresses the formation of TDs. Interestingly, when both C and Sn are present in Si, very weak bands of TDs were observed, although it is known that Sn alone suppress their formation. This may be attributed to the competing strains of C and Sn in the Si lattice

  3. Oxygen aggregation kinetics, thermal donors and carbon-oxygen defect formation in silicon containing carbon and tin

    Energy Technology Data Exchange (ETDEWEB)

    Angeletos, T.; Sgourou, E. N.; Andrianakis, A.; Diamantopoulou, A.; Londos, C. A. [Solid State Section, Physics Department, University of Athens, Panepistimiopolis, Zografos, 157 84 Athens (Greece); Chroneos, A. [Faculty of Engineering and Computing, Coventry University, Priory Street, Coventry CV1 5FB (United Kingdom); Department of Materials, Imperial College London, London SW7 2BP (United Kingdom)

    2015-07-07

    Localized vibrational mode spectroscopy measurements on Czochralski silicon (Cz-Si) samples subjected to isothermal annealing at 450 °C are reported. First, we studied the effect of carbon (C) and tin (Sn) isovalent dopants on the aggregation kinetics of oxygen. It is determined that the reduction rate of oxygen is described by the Johnson-Mehl-Avrami equation in accordance with previous reports. The activation energy related with the reaction rate constant of the process is calculated to increase from Cz-Si, to C-doped Cz-Si (CCz-Si), to Sn-doped Cz-Si contained C (SnCz-Si). This is attributed to the presence of the isovalent dopants that may impact both the kinetics of the oxygen atoms and also may lead to the formation of other oxygen-related clusters. Second, we studied the effect of Sn on the formation and evolution of carbon-oxygen (C-O) defects. It was determined that the presence of Sn suppresses the formation of the C-O defects as indicated by the reduction in the strength of the 683, 626, and 586 cm{sup −1} well-known bands of C{sub s}O{sub i} defect. The phenomenon is attributed to the association of Sn with C atoms that may prevent the pairing of O with C. Third, we investigated the effect of C and Sn on the formation of thermal donors (TDs). Regarding carbon our results verified previous reports that carbon suppresses the formation of TDs. Interestingly, when both C and Sn are present in Si, very weak bands of TDs were observed, although it is known that Sn alone suppress their formation. This may be attributed to the competing strains of C and Sn in the Si lattice.

  4. The Pectin Lyases in Arabidopsis thaliana: Evolution, Selection and Expression Profiles

    OpenAIRE

    Cao, Jun

    2012-01-01

    Pectin lyases are a group of enzymes that are thought to contribute to many biological processes, such as the degradation of pectin. However, until this study, no comprehensive study incorporating phylogeny, chromosomal location, gene duplication, gene organization, functional divergence, adaptive evolution, expression profiling and functional networks has been reported for Arabidopsis. Sixty-seven pectin lyase genes have been identified, and most of them possess signal sequences targeting th...

  5. In vivo synthesis of histidine by a cloned histidine ammonia-lyase in Escherichia coli.

    OpenAIRE

    Fuchs, R L; Kane, J F

    1985-01-01

    Histidine ammonia-lyase catalyzes the first step in histidine catabolism, the deamination of histidine to urocanate and ammonia. In vitro experiments have shown that histidine ammonia-lyase also can catalyze the reverse (amination) reaction, histidine synthesis, relatively efficiently under extreme reaction conditions (4 M NH4OH, pH 10). An Escherichia coli hisB deletion strain was transformed with a pBR322 derivative plasmid (pCB101) containing the entire Klebsiella aerogenes histidine utili...

  6. The Active Site of Oligogalacturonate Lyase Provides Unique Insights into Cytoplasmic Oligogalacturonate β-Elimination*

    OpenAIRE

    Abbott, D. Wade; Gilbert, Harry J.; Boraston, Alisdair B.

    2010-01-01

    Oligogalacturonate lyases (OGLs; now also classified as pectate lyase family 22) are cytoplasmic enzymes found in pectinolytic members of Enterobacteriaceae, such as the enteropathogen Yersinia enterocolitica. OGLs utilize a β-elimination mechanism to preferentially catalyze the conversion of saturated and unsaturated digalacturonate into monogalacturonate and the 4,5-unsaturated monogalacturonate-like molecule, 5-keto-4-deoxyuronate. To provide mechanistic insights into the specificity of th...

  7. Effect of Culture Conditions on the Production of Tyrosine Phenol-Lyase by Erwinia herbicola

    OpenAIRE

    Para, G. M.; Baratti, J. C.

    1984-01-01

    The effect of environmental parameters on the growth and the tyrosine phenol-lyase content of Erwinia herbicola was investigated. On mineral medium containing glycerol, l-tyrosine increased the enzyme content 23-fold. When the l-tyrosine was also the carbon source, bacterial growth was 300 times greater than the basal level. On a rich medium, tyrosine phenol-lyase production was strongly dependent on pH and aeration. Catabolite repression and induction both probably control enzyme content.

  8. Utilization of Aspergillus oryzae to produce pectin lyase from various agro-industrial residues

    Directory of Open Access Journals (Sweden)

    Safia Koser

    2014-07-01

    Full Text Available The present study was aimed to investigate the culture influence on pectin lyase production potential of fungal strain Aspergillus oryzae. The enzyme profile of A. oryzae showed highest activity of pectin lyase after 3rd day of incubation on lemon peel waste under solid state fermentation conditions. To induce the pectin lyase synthesis capability of A. oryzae at optimal level various culture variables including physical and nutritional parameters were optimized by adopting classical optimization technique. Therefore, through fermentation process optimization the production of pectin lyase was substantially induced up to the level of 875 U/mL, when fermentation medium of lemon peel waste inoculated with 5 mL spore suspension of A. oryzae. The optimal fermentation conditions for maximum pectin lyase yield were as: optimum pH 5, 70% moisture level and incubated at 40 °C in addition with 1% sterile glucose solution as readily available carbon source and 0.2% yeast extract as an inexpensive nitrogen supplement (1%. The results obtained in current investigation so far demonstrated that culture conditions have great influence on the pectin lyase production potential of A. oryzae.

  9. Three Alginate Lyases from Marine Bacterium Pseudomonas fluorescens HZJ216: Purification and Characterization

    Energy Technology Data Exchange (ETDEWEB)

    Liyan, Li [Ocean University of China, Qingdao, PRC; Jiang, Xiaolu [Ocean University of China, Qingdao, PRC; Wang, Peng [Ocean University of China, Qingdao, PRC; Guan, Huashi [Ocean University of China, Qingdao, PRC; Guo, Hong [ORNL

    2010-01-01

    Three alginate lyases (A, B, and C) from an alginate-degrading marine bacterium strain HZJ216 isolated from brown seaweed in the Yellow Sea of China and identified preliminarily as Pseudomonas fluorescens are purified, and their biochemical properties are described. Molecular masses of the three enzymes are determined by SDS-PAGE to be 60.25, 36, and 23 kDa with isoelectric points of 4, 4.36, and 4.59, respectively. Investigations of these enzymes at different pH and temperatures show that they are most active at pH 7.0 and 35 C. Alginate lyases A and B are stable in the pH range of 5.0 9.0, while alginate lyase C is stable in the pH range of 5.0 7.0. Among the metal ions tested, additions of Na+, K+, and Mg2+ ions can enhance the enzyme activities while Fe2+, Fe3+, Ba2+, and Zn2+ ions show inhibitory effects. The substrate specificity results demonstrate that alginate lyase C has the specificity for G block while alginate lyases A and B have the activities for both M and G blocks. It is the first report about extracellular alginate lyases with high alginate-degrading activity from P. fluorescens.

  10. Optimization of culturing condition and medium composition for the production of alginate lyase by a marine Vibrio sp. YKW-34

    Science.gov (United States)

    Fu, Xiaoting; Lin, Hong; Kim, Sang Moo

    2008-02-01

    Carbohydrases secreted by marine Vibrio sp. YKW-34 with strong Laminaria cell wall degrading ability were screened, and among them alginate lyase was found to be dominant. The effects of medium composition and culturing condition on the production of alginate lyase by marine Vibrio sp. YKW-34 in flask were investigated in this study. In the culture medium of marine broth, no alginate lyase was produced. The activity of the alginate lyase, after being induced, reached 5 UmL-1. The best inoculum volume and inoculum age were 10% and 12 h, respectively. The optimal temperature for alginate lyase production was 25°C. The fermentation medium was composed of 0.5% of Laminaria powder and 0.2% of KNO3 with an initial acidity of pH 8.0. Alginate could induce alginate lyase production but not as efficiently as Laminaria powder did. The addition of fucoidan, cellulose and glucose had negative effect on the alginate lyase production. Other kinds of nitrogen sources, such as yeast extract, beef extract and peptone, had positive effect on the growth of the microorganism and negative effect on alginate lyase production. In addition, the time course of alginate lyase production under the optimized condition was described. The optimal harvest time was 48 h.

  11. Production and Purification of a Novel Xanthan Lyase from a Xanthan-Degrading Microbacterium sp. Strain XT11

    Directory of Open Access Journals (Sweden)

    Fan Yang

    2014-01-01

    Full Text Available A xanthan lyase was produced and purified from the culture supernatant of an excellent xanthan-modifying strain Microbacterium sp. XT11. Xanthan lyase was induced by xanthan but was inhibited by its structural monomer glucose. Its production by strain XT11 is much higher than that by all other reported strains. The purified xanthan lyase has a molecular mass of 110 kDa and a specific activity of 28.2 U/mg that was much higher than that of both Paenibacillus and Bacillus lyases. It was specific on the pyruvated mannosyl residue in the intact xanthan molecule, but about 50% lyase activity remained when xanthan was partially depyruvated. Xanthan lyase was optimally active at pH 6.0–6.5 and 40°C and alkali-tolerant at a high pH value of 11.0. The metal ions including K+, Ca2+, Na+, Mg2+, Mn2+, and Li+ strongly stimulated xanthan lyase activity but ions Zn2+ and Cu2+ were its inhibitor. Xanthan lyase should be a novel enzyme different from the other xanthan lyases ever reported.

  12. Production and Purification of a Novel Xanthan Lyase from a Xanthan-Degrading Microbacterium sp. Strain XT11

    OpenAIRE

    Fan Yang; Lan Yang; Xiaoyu Guo; Xue Wang; Lili Li; Zhicheng Liu; Wei Wang(College of William and Mary); Xianzhen Li

    2014-01-01

    A xanthan lyase was produced and purified from the culture supernatant of an excellent xanthan-modifying strain Microbacterium sp. XT11. Xanthan lyase was induced by xanthan but was inhibited by its structural monomer glucose. Its production by strain XT11 is much higher than that by all other reported strains. The purified xanthan lyase has a molecular mass of 110 kDa and a specific activity of 28.2 U/mg that was much higher than that of both Paenibacillus and Bacillus lyases. It was specifi...

  13. Genomic Characterization of Phenylalanine Ammonia Lyase Gene in Buckwheat.

    Directory of Open Access Journals (Sweden)

    Karthikeyan Thiyagarajan

    Full Text Available Phenylalanine Ammonia Lyase (PAL gene which plays a key role in bio-synthesis of medicinally important compounds, Rutin/quercetin was sequence characterized for its efficient genomics application. These compounds possessing anti-diabetic and anti-cancer properties and are predominantly produced by Fagopyrum spp. In the present study, PAL gene was sequenced from three Fagopyrum spp. (F. tataricum, F. esculentum and F. dibotrys and showed the presence of three SNPs and four insertion/deletions at intra and inter specific level. Among them, the potential SNP (position 949th bp G>C with Parsimony Informative Site was selected and successfully utilised to individuate the zygosity/allelic variation of 16 F. tataricum varieties. Insertion mutations were identified in coding region, which resulted the change of a stretch of 39 amino acids on the putative protein. Our Study revealed that autogamous species (F. tataricum has lower frequency of observed SNPs as compared to allogamous species (F. dibotrys and F. esculentum. The identified SNPs in F. tataricum didn't result to amino acid change, while in other two species it caused both conservative and non-conservative variations. Consistent pattern of SNPs across the species revealed their phylogenetic importance. We found two groups of F. tataricum and one of them was closely related with F. dibotrys. Sequence characterization information of PAL gene reported in present investigation can be utilized in genetic improvement of buckwheat in reference to its medicinal value.

  14. Genomic Characterization of Phenylalanine Ammonia Lyase Gene in Buckwheat.

    Science.gov (United States)

    Thiyagarajan, Karthikeyan; Vitali, Fabio; Tolaini, Valentina; Galeffi, Patrizia; Cantale, Cristina; Vikram, Prashant; Singh, Sukhwinder; De Rossi, Patrizia; Nobili, Chiara; Procacci, Silvia; Del Fiore, Antonella; Antonini, Alessandro; Presenti, Ombretta; Brunori, Andrea

    2016-01-01

    Phenylalanine Ammonia Lyase (PAL) gene which plays a key role in bio-synthesis of medicinally important compounds, Rutin/quercetin was sequence characterized for its efficient genomics application. These compounds possessing anti-diabetic and anti-cancer properties and are predominantly produced by Fagopyrum spp. In the present study, PAL gene was sequenced from three Fagopyrum spp. (F. tataricum, F. esculentum and F. dibotrys) and showed the presence of three SNPs and four insertion/deletions at intra and inter specific level. Among them, the potential SNP (position 949th bp G>C) with Parsimony Informative Site was selected and successfully utilised to individuate the zygosity/allelic variation of 16 F. tataricum varieties. Insertion mutations were identified in coding region, which resulted the change of a stretch of 39 amino acids on the putative protein. Our Study revealed that autogamous species (F. tataricum) has lower frequency of observed SNPs as compared to allogamous species (F. dibotrys and F. esculentum). The identified SNPs in F. tataricum didn't result to amino acid change, while in other two species it caused both conservative and non-conservative variations. Consistent pattern of SNPs across the species revealed their phylogenetic importance. We found two groups of F. tataricum and one of them was closely related with F. dibotrys. Sequence characterization information of PAL gene reported in present investigation can be utilized in genetic improvement of buckwheat in reference to its medicinal value. PMID:26990297

  15. Genomic Characterization of Phenylalanine Ammonia Lyase Gene in Buckwheat

    Science.gov (United States)

    Thiyagarajan, Karthikeyan; Vitali, Fabio; Tolaini, Valentina; Galeffi, Patrizia; Cantale, Cristina; Vikram, Prashant; Singh, Sukhwinder; De Rossi, Patrizia; Nobili, Chiara; Procacci, Silvia; Del Fiore, Antonella; Antonini, Alessandro; Presenti, Ombretta; Brunori, Andrea

    2016-01-01

    Phenylalanine Ammonia Lyase (PAL) gene which plays a key role in bio-synthesis of medicinally important compounds, Rutin/quercetin was sequence characterized for its efficient genomics application. These compounds possessing anti-diabetic and anti-cancer properties and are predominantly produced by Fagopyrum spp. In the present study, PAL gene was sequenced from three Fagopyrum spp. (F. tataricum, F. esculentum and F. dibotrys) and showed the presence of three SNPs and four insertion/deletions at intra and inter specific level. Among them, the potential SNP (position 949th bp G>C) with Parsimony Informative Site was selected and successfully utilised to individuate the zygosity/allelic variation of 16 F. tataricum varieties. Insertion mutations were identified in coding region, which resulted the change of a stretch of 39 amino acids on the putative protein. Our Study revealed that autogamous species (F. tataricum) has lower frequency of observed SNPs as compared to allogamous species (F. dibotrys and F. esculentum). The identified SNPs in F. tataricum didn’t result to amino acid change, while in other two species it caused both conservative and non-conservative variations. Consistent pattern of SNPs across the species revealed their phylogenetic importance. We found two groups of F. tataricum and one of them was closely related with F. dibotrys. Sequence characterization information of PAL gene reported in present investigation can be utilized in genetic improvement of buckwheat in reference to its medicinal value. PMID:26990297

  16. Phenylalanine ammonia-lyase through evolution: A bioinformatic approach

    Directory of Open Access Journals (Sweden)

    Shiva Hemmati

    2015-03-01

    Full Text Available Phenylalanine ammonia-lyase (PAL is the first entry enzyme of the phenylpropanoid pathway that converts phenylalanine to cinnamic acid which is the precursor of various secondary metabolites. PAL is recently formulated for phenylketonuric patients in pegylated forms; therefore, screening a PAL with the highest affinity to the substrate is of a great importance. PAL exists in all higher plants and some fungi and few bacteria. Ancestors of land plants have been adopted by evolving metabolic pathways. A multi-gene family encodes PAL by gene duplication events in most plants. In this study, the taxonomic distribution and phylogeny of pal gene found in land plants, fungi and bacteria have been analyzed. It seems that the ancestor of plants acquired a pal gene via horizontal gene transfer in symbioses with bacteria and fungi. Gymnosperms have kept a diverse set of pal genes that arose from gene duplication events. In angiosperms, after the divergence of dicotyledons from monocots, pal genes were duplicated many times. The close paralogues of pal genes in some species indicate expansion of gene families after the divergence in plant pal gene evolution. Interestingly, some of the plant pals clustered by species in a way that pals within one species are more closely related to each other than to homologs in the other species which indicates this duplication event occurred more recently.

  17. Novel Proton MR Spectroscopy Findings in Adenylosuccinate Lyase Deficiency

    Science.gov (United States)

    Zulfiqar, Maria; Lin, Doris D.M.; Van der Graaf, Marinette; Barker, Peter B.; Fahrner, Jill A.; Marie, Sandrine; Morava, Eva; De Boer, Lonneke; Willemsen, Michel A.A.P; Vining, Eileen; Horská, Alena; Engelke, Udo; Wevers, Ron A.; Maegawa, Gustavo H.B.

    2016-01-01

    Adenylosuccinate lyase (ADSL) deficiency is a rare inborn error of metabolism resulting in accumulation of metabolites including succinylaminoimidazole carboxamide riboside (SAICAr) and succinyladenosine (S-Ado) in the brain and other tissues. Patients with ADSL have progressive psychomotor retardation, neonatal seizures, global developmental delay, hypotonia, and autistic features, although variable clinical manifestations may make the initial diagnosis challenging. Two cases of the severe form of the disease are reported here: an 18-month-old boy with global developmental delay, intractable neonatal seizures, progressive cerebral atrophy, and marked hypomyelination, and a 3-month-old girl presenting with microcephaly, neonatal seizures, and marked psychomotor retardation. In both patients in vivo proton magnetic resonance spectroscopy (MRS) showed the presence of S-Ado signal at 8.3 ppm, consistent with a prior report. Interestingly, SAICAr signal was also detectable at 7.5 ppm in affected white matter, which has not been reported in vivo before. A novel splice-site mutation, c.IVS12 + 1/G > C, in the ADSL gene was identified in the second patient. Our findings confirm the utility of in vivo proton MRS in suggesting a specific diagnosis of ADSL deficiency, and also demonstrate an additional in vivo resonance (7.5 ppm) of SAICAr in the cases of severe disease. PMID:23055421

  18. Altered Fermentative Metabolism in Chlamydomonas reinhardtii Mutants Lacking Pyruvate Formate Lyase and Both Pyruvate Formate Lyase and Alcohol Dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Catalanotti, C.; Dubini, A.; Subramanian, V.; Yang, W. Q.; Magneschi, L.; Mus, F.; Seibert, M.; Posewitz, M. C.; Grossman, A. R.

    2012-02-01

    Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H2 production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. To further probe the system, we generated a double mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H2 production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism.

  19. Post-merger evolution of carbon-oxygen + helium white dwarf binaries and the origin of R Coronae Borealis and extreme helium stars

    OpenAIRE

    Zhang, Xianfei; Jeffery, C. Simon; Chen, Xuefei; Han, Zhanwen

    2014-01-01

    Orbital decay by gravitational-wave radiation will cause some close-binary white dwarfs (WDs) to merge within a Hubble time. The results from previous hydrodynamical WD-merger simulations have been used to guide calculations of the post-merger evolution of carbon-oxygen + helium (CO+He) WD binaries. Our models include the formation of a hot corona in addition to a Keplerian disk. We introduce a 'destroyeddisk' model to simulate the effect of direct disk ingestion into the expanding envelope. ...

  20. Structure of putative 4-amino-4-deoxychorismate lyase from Thermus thermophilus HB8

    International Nuclear Information System (INIS)

    The putative 4-amino-4-deoxychorismate lyase (TTHA0621) from T. thermophilus HB8 was cloned, overexpressed, purified and crystallized. Its crystal structure was determined by a combination of SAD and molecular-replacement methods and was refined to 1.93 Å resolution. The pyridoxal 5′-phosphate-dependent enzyme 4-amino-4-deoxychorismate lyase converts 4-amino-4-deoxychorismate to p-aminobenzoate and pyruvate in one of the crucial steps in the folate-biosynthesis pathway. The primary structure of the hypothetical protein TTHA0621 from Thermus thermophilus HB8 suggests that TTHA0621 is a putative 4-amino-4-deoxychorismate lyase. Here, the crystal structure of TTHA0621 is reported at 1.93 Å resolution. The asymmetric unit contained four NCS molecules related by 222 noncrystallographic symmetry, in which the formation of intact dimers may be functionally important. The cofactor pyridoxal 5′-phosphate (PLP) binds to the protein in the large cleft formed by the N-terminal and C-terminal domains of TTHA0621. The high structural similarity and the conservation of the functional residues in the catalytic region compared with 4-amino-4-deoxychorismate lyase (PabC; EC 4.1.3.38) from Escherichia coli suggest that the TTHA0621 protein may also possess 4-amino-4-deoxychorismate lyase activity

  1. Structural insights into the loss of catalytic competence in pectate lyase activity at low pH

    DEFF Research Database (Denmark)

    Ali, Salyha; Søndergaard, Chresten Rauff; Teixeira, Susana;

    2015-01-01

    Pectate lyase, a family 1 polysaccharide lyase, catalyses cleavage of the α-1,4 linkage of the polysaccharide homogalacturonan via an anti β-elimination reaction. In the Michaelis complex two calcium ions bind between the C6 carboxylate of the d-galacturonate residue and enzyme aspartates at the...... organise the Michaelis complex....

  2. Probing the structure of glucan lyases – the lytic members of GH31 - by sequence analysis, circular dichroism and proteolysis

    DEFF Research Database (Denmark)

    Ernst, Heidi; Lo Leggio, Leila; Yu, Shukun;

    2005-01-01

    Glucan lyase (GL) is a polysaccharide lyase with unique characteristics. It is involved in an alternative pathway for the degradation of alpha-glucans, the anhydrofructose pathway. Sequence similarity suggests that this lytic enzyme belongs to glycoside hydrolase family 31, for which until very r...

  3. Properties of a Mg2+ independent isocitrate lyase from gamma irradiated preclimacteric banana (Musa cavendishii)

    International Nuclear Information System (INIS)

    Isocitrate lyase (EC 4.1, 3.1) was purified seventy fold from gamma irradiated banana pulp tissue acetone powder. It showed an optimum pH of 6.0, and the Ksub(m) value for DL-isocitrate was 0.8 mM. Among the various metabolic inhibitors, oxaloacetate was found to be the most potent and its inhibition was competitive. The enzyme activity was not dependent on externally added Mg2+. The Mg2+ content of the purified enzyme was 10-12 ng/mg protein. A method for the detection of the two multiple forms of isocitrate lyase presented in this preparation was developed using 2,4-dinitrophenylhydrazine as detecting agent for glyoxylate formed during the isocitrate lyase reaction. (author)

  4. Gene deletion strategy to examine the involvement of the two chondroitin lyases in Flavobacterium columnare virulence.

    Science.gov (United States)

    Li, Nan; Qin, Ting; Zhang, Xiao Lin; Huang, Bei; Liu, Zhi Xin; Xie, Hai Xia; Zhang, Jin; McBride, Mark J; Nie, Pin

    2015-11-01

    Flavobacterium columnare is an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. The F. johnsoniae ompA promoter (PompA) was fused to sacB to construct a counterselectable marker for F. columnare. F. columnare carrying PompA-sacB failed to grow on media containing 10% sucrose. A suicide vector carrying PompA-sacB was constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes, cslA and cslB, were deleted. The ΔcslA and ΔcslB mutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (ΔcslA ΔcslB) was completely deficient in chondroitin lyase activity. Cells of F. columnare wild-type strain G4 and of the chondroitin lyase-deficient ΔcslA ΔcslB mutant exhibited similar levels of virulence toward grass carp in single-strain infections. Coinfections, however, revealed a competitive advantage for the wild type over the chondroitin lyase mutant. The results indicate that chondroitin lyases are not essential virulence factors of F. columnare but may contribute to the ability of the pathogen to compete and cause disease in natural infections. The gene deletion method developed in this study may be employed to investigate the virulence factors of this bacterium and may have wide application in many other members of the phylum Bacteroidetes. PMID:26253667

  5. Isocitrate lyase and the glyoxylate cycle. Progress report, July 1, 1988--February 15, 1989

    Energy Technology Data Exchange (ETDEWEB)

    McFadden, B.A.

    1989-12-31

    Studies on the structure, regulation and catalytic function of isocitrate lyase are reported. This catalyzes the first unique step i the glyoxylate cycle. In this cycle, lipids are converted to carbohydrates in a process which contributes to microbial growth on fatty aids and to the growth of oil-rich seedlings and animal embryos. These studies will provide basic information about isocitrate lyase. The function of this enzyme is vital to microbial growth (on fatty acids) and to the growth of varied plant seedlings and their subsequent utilization of solar energy.

  6. Purification and Characterization of Alginate Lyase from Marine Vibrio sp. YWA

    Institute of Scientific and Technical Information of China (English)

    Yuan-Hong WANG; Guang-Li YU; Xin-Min WANG; Zhi-Hua LV; Xia ZHAO; Zhi-Hong WU; Wei-Shang JI

    2006-01-01

    Extracellular alginate lyase secreted by marine Vibrio sp. YWA, isolated from decayed Laminaria japonica, was purified by a combination of ammonium sulfate precipitation and diethylaminoethyl that the molecular mass of alginate lyase was approximately 62.5 kDa, with an optimal pH and temperature at pH 7.0 and 25 ℃C, respectively. Km was e enzyme was enhanced by EDTA and Zn2+, but inhibited by Ba2+.The substrates specificity analysis shows that it was specific for hydrolyzing poly-β-D-1,4-mannuronate in alginate

  7. Xanthan Lyase of Bacillus sp. Strain GL1 Liberates Pyruvylated Mannose from Xanthan Side Chains

    OpenAIRE

    Hashimoto, Wataru; Miki, Hikaru; Tsuchiya, Noriaki; Nankai, Hirokazu; Murata, Kousaku

    1998-01-01

    When the bacterium Bacillus sp. strain GL1 was grown in a medium containing xanthan as the carbon source, the viscosity of the medium decreased in association with growth, showing that the bacterium had xanthan-depolymerizing enzymes. One of the xanthan-depolymerizing enzymes (xanthan lyase) was present in the medium and was found to be induced by xanthan. The xanthan lyase purified from the culture fluid was a monomer with a molecular mass of 75 kDa, and was most active at pH 5.5 and 50°C. T...

  8. Reduced phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities and lignin synthesis in wheat grown under low pressure sodium lamps

    Science.gov (United States)

    Guerra, D.; Anderson, A. J.; Salisbury, F. B.

    1985-01-01

    Wheat (Triticum aestivum L. cv Fremont) grown in hydroponic culture under 24-hour continuous irradiation at 560 to 580 micromoles per square meter per second from either metalhalide (MH), high pressure sodium (HPS), or low pressure sodium (LPS) lamps reached maturity in 70 days. Grain yields were similar under all three lamps, although LPS-grown plants lodged at maturity. Phenylalanine ammonia-lyase (PAL) and a tyrosine ammonia lyase (TAL) with lesser activity were detected in all extracts of leaf, inflorescence, and stem. Ammonia-lyase activities increased with age of the plant, and plants grown under the LPS lamp displayed PAL and TAL activities lower than wheat cultured under MH and HPS radiation. Greenhouse solar-grown wheat had the highest PAL and TAL activities. Lignin content of LPS-grown wheat was also significantly reduced from that of plants grown under MH or HPS lamps or in the greenhouse, showing a correlation with the reduced PAL and TAL activities. Ratios of far red-absorbing phytochrome to total phytochrome were similar for all three lamps, but the data do not yet warrant a conclusion about specific wavelengths missing from the LPS lamps that might have induced PAL and TAL activities in plants under the other lamps.

  9. Thermodynamic Analysis on Ferrum-carbon-oxygen Equilibrium During Steelmaking%炼钢过程铁碳氧平衡热力学解析

    Institute of Scientific and Technical Information of China (English)

    万雪峰

    2015-01-01

    Based on the thermodynamic analysis on the product of carbon content and oxygen content at the blowing end in converter and the deep decarburization by RH refining together with the actual production data, the issue on the ferrum-carbon-oxygen equilibrium during the whole steelmaking process was discussed. It is concluded that the product of carbon content and oxygen content at the blowing end in converter does not remain unchanged, but it varies as environmental conditions are changing. Both interaction coefficients of other components and temperatures have a little influence on the product of carbon content and oxygen content. However the partial pressure resulted from the carbon-oxygen equilibrium has prominent influence on the product. So the con-tent of carbon in the carbon-oxygen equilibrium can reach to 10×10-6 when the partial pressure is 1 kPa. In the normal temperature range of converter tapping, the turning point of iron-removing and carbon-preserving in molten steel will occur as the content of carbon is 0.034% while corre-spondently the content of ferric oxide in slag needs only 11%.%通过对转炉终点碳氧积及RH深脱碳的热力学分析,结合实际生产数据,探讨了炼钢过程铁碳氧平衡问题。得出转炉终点碳氧积并非固定,而是随外界条件变化而变化;其他组元活度相互作用系数及温度对碳氧积影响很小,但CO平衡分压影响显著,当CO平衡分压达1 kPa时即可实现平衡碳含量10×10-6;在正常转炉出钢温度范围,碳含量处于0.034%时,钢液出现“脱铁保碳”转折点,与之相平衡的渣中氧化铁含量仅需11%。

  10. The management of pregnancy and delivery in 3-hydroxy-3-methylglutaryl-CoA lyase deficiency.

    Science.gov (United States)

    Pipitone, Angela; Raval, Donna B; Duis, Jessica; Vernon, Hilary; Martin, Regina; Hamosh, Ada; Valle, David; Gunay-Aygun, Meral

    2016-06-01

    3-hydroxy-3-methylglutaric (HMG)-CoA lyase is required for ketogenesis and leucine degradation. Patients with HMG-CoA lyase deficiency typically present with hypoketotic hypoglycemia and metabolic acidosis, which can be fatal if untreated. The patient is a 28-year-old female with HMG-CoA lyase deficiency who presented at 4 weeks gestation for prenatal care. Protein intake as well as carnitine supplementation were gradually increased to support maternal and fetal demands up to 65 g per day for protein and 80 mg/kg/day for carnitine. Fetal growth was appropriate. At 36 5/7 weeks, she presented with spontaneous rupture of membranes. Twice maintenance 10% glucose-containing intravenous fluids were initiated. During labor, vomiting and metabolic acidosis developed. Delivery was by cesarean. Preeclampsia developed postpartum. The patient recovered well and was discharged home on postpartum day 5. Stress of pregnancy and labor and delivery can lead to metabolic decompensation in HMG-CoA lyase deficiency. Patients should be monitored closely by a biochemical geneticist, dietitian, and high-risk obstetrician at a tertiary care center during their pregnancy. Fasting should be avoided. Intravenous 10% glucose-containing fluids should be provided to prevent catabolism and metabolic decompensation during labor and delivery. © 2016 Wiley Periodicals, Inc. PMID:26997609

  11. Characterization of alginate lyase activity on liquid, gelled, and complexed states of alginate.

    Science.gov (United States)

    Breguet, Véronique; von Stockar, Urs; Marison, Ian W

    2007-01-01

    A study of alginate lyase was carried out to determine if this enzyme could be used to remove alginate present in the core of alginate/poly-L-lysine (AG/PLL) microcapsules in order to maximize cell growth and colonization. A complete kinetic study was undertaken, which indicated an optimal activity of the enzyme at pH 7-8, 50 degrees C, in the presence of Ca2+. The buffer, not the ionic strength, influenced the alginate degradation rate. Alginate lyase was also shown to be active on gelled forms of alginate, as well as on the AG/PLL complex constituting the membrane of microcapsules. Batch cultures of CHO cells in the presence of alginate showed a decrease of the growth rate by a factor of 2, although the main metabolic flux rates were not modified. The addition of alginate lyase to cell culture medium increased the doubling time 5-7-fold and decreased the protein production rate, although cell viability was not affected. The addition of enzyme to medium containing alginate did not improve growth conditions. This suggests that alginate lyase is probably not suitable for hydrolysis of microcapsules in the presence of cells, in order to achieve high cell density and high productivity. However, the high activity may be useful for releasing cells from alginate beads or AG/PLL microcapsules. PMID:17691813

  12. Structural Insights into Substrate Specificity and the anti beta-Elimination Mechanism of Pectate Lyase

    DEFF Research Database (Denmark)

    Seyedarabi, A.; To, T.T.; Ali, S.;

    2010-01-01

    Pectate lyases harness anti beta-elimination chemistry to cleave the alpha-1,4 linkage in the homogalacturonan region of plant cell Wall pectin. We have studied the binding of five pectic oligosaccharides to Bacillus subtilis pectate Iyase in crystals of the inactive enzyme in which the catalytic...

  13. Alteration of the Diastereoselectivity of 3-Methylaspartate Ammonia Lyase by Using Structure-Based Mutagenesis

    NARCIS (Netherlands)

    Raj, Hans; Weiner, Barbara; Puthan Veetil, Vinod; Reis, Carlos R.; Quax, Wim J.; Janssen, Dick B.; Feringa, Ben L.; Poelarends, Gerrit J.

    2009-01-01

    3-Methylaspartate ammonia-lyase (MAL) catalyzes the reversible amination of mesaconate to give both (2S,3S)-3-methylaspartic acid and (2S,3R)-3-methylaspartic acid as products. The deamination mechanism of MAL is likely to involve general base catalysis, in which a catalytic base abstracts the C3 pr

  14. Biocatalytic Enantioselective Synthesis of N-Substituted Aspartic Acids by Aspartate Ammonia Lyase

    NARCIS (Netherlands)

    Weiner, Barbara; Poelarends, Gerrit J.; Janssen, Dick B.; Feringa, Ben L.

    2008-01-01

    The gene encoding aspartate ammonia lyase (aspB) from Bacillus sp. YM55-1 has been cloned and overexpressed, and the recombinant enzyme containing a C-terminal His6 tag has been purified to homogeneity and subjected to kinetic characterization. Kinetic studies have shown that the His6 tag does not a

  15. Gene deletion of cytosolic ATP: citrate lyase leads to altered organic acid production in Aspergillus niger

    DEFF Research Database (Denmark)

    Meijer, Susan Lisette; Nielsen, Michael Lynge; Olsson, Lisbeth;

    2009-01-01

    factory platform for production of chemicals. Using molecular biology techniques, this study focused on metabolic engineering of A. niger to manipulate its organic acid production in the direction of succinic acid. The gene target for complete gene deletion was cytosolic ATP: citrate lyase (acl), which...

  16. Metabolism of β-valine via a CoA-dependent ammonia lyase pathway

    NARCIS (Netherlands)

    Otzen, Marleen; Crismaru, Ciprian G.; Postema, Christiaan P.; Wijma, Hein J.; Heberling, Matthew M.; Szymanski, Wiktor; de Wildeman, Stefaan; Janssen, Dick B.

    2015-01-01

    Pseudomonas species strain SBV1 can rapidly grow on medium containing β-valine as a sole nitrogen source. The tertiary amine feature of β-valine prevents direct deamination reactions catalyzed by aminotransferases, amino acid dehydrogenases, and amino acid oxidases. However, lyase- or aminomutase-me

  17. Novel Pectate Lyase Genes of Heterodera glycines Play Key Roles in the Early Stage of Parasitism

    Science.gov (United States)

    Peng, Huan; Cui, Jiangkuan; Long, Haibo; Huang, Wenkun; Kong, Lingan; Liu, Shiming; He, Wenting; Hu, Xianqi; Peng, Deliang

    2016-01-01

    Pectate lyases are known to play a key role in pectin degradation by catalyzing the random cleavage of internal polymer linkages (endo-pectinases). In this paper, four novel cDNAs, designated Hg-pel-3, Hg-pel-4, Hg-pel-6 and Hg-pel-7, that encode pectate lyases were cloned and characterized from the soybean cyst nematode, Heterodera glycines. The predicted protein sequences of HG-PEL-3, HG-PEL-4 and HG-PEL-6 differed significantly in both their amino acid sequences and their genomic structures from other pectate lyases of H. glycines (HG-PEL-1, HG-PEL-2 and HG-PEL-7). A phylogenetic study revealed that the pectate lyase proteins of H. glycines are clustered into distinct clades and have distinct numbers and positioning of introns, which suggests that the pectate lyase genes of H. glycines may have evolved from at least two ancestral genes. A Southern blot analysis revealed that multiple Hg-pel-6-like genes were present in the H. glycines genome. In situ hybridization showed that four novel pectate lyases (Hg-pel-3, Hg-pel-4, Hg-pel-6 and Hg-pel-7) were actively transcribed in the subventral esophageal gland cells. A semi-quantitative RT-PCR assay supported the finding that the expression of these genes was strong in the egg, pre-parasitic second-stage juvenile (J2) and early parasitic J2 stages and that it declined in further developmental stages of the nematode. This expression pattern suggests that these proteins play a role in the migratory phase of the nematode life cycle. Knocking down Hg-pel-6 using in vitro RNA interference resulted in a 46.9% reduction of the number of nematodes that invaded the plants and a 61.5% suppression of the development of H. glycines females within roots compared to the GFP-dsRNA control. Plant host-derived RNAi induced the silencing of the Hg-pel-6gene, which significantly reduced the nematode infection levels at 7 Days post inoculation (dpi). Similarly, this procedure reduced the number of female adults at 40 dpi, which suggests

  18. Pectate lyase PelI of Erwinia chrysanthemi 3937 belongs to a new family.

    Science.gov (United States)

    Shevchik, V E; Robert-Baudouy, J; Hugouvieux-Cotte-Pattat, N

    1997-12-01

    Erwinia chrysanthemi 3937 secretes five major isoenzymes of pectate lyases encoded by the pel4, pelB, pelC, pelD, and pelE genes and a set of secondary pectate lyases, two of which, pelL and pelZ, have been already identified. We cloned the pelI gene, encoding a ninth pectate lyase of E. chrysanthemi 3937. The pelI reading frame is 1,035 bases long, corresponding to a protein of 344 amino acids including a typical amino-terminal signal sequence of 19 amino acids. The purified mature PelI protein has an isoelectric point of about 9 and an apparent molecular mass of 34 kDa. PelI has a preference for partially methyl esterified pectin and presents an endo-cleaving activity with an alkaline pH optimum and an absolute requirement for Ca2+ ions. PelI is an extracellular protein secreted by the Out secretory pathway of E. chrysanthemi. The PelI protein is very active in the maceration of plant tissues. A pelI mutant displayed reduced pathogenicity on chicory leaves, but its virulence did not appear to be affected on potato tubers or Saintpaulia ionantha plants. The pelI gene constitutes an independent transcriptional unit. As shown for the other pel genes, the transcription of pelI is dependent on various environmental conditions. It is induced by pectic catabolic products and affected by growth phase, oxygen limitation, temperature, nitrogen starvation, and catabolite repression. Regulation of pelI expression appeared to be dependent on the three repressors of pectinase synthesis, KdgR, PecS, and PecT, and on the global activator of sugar catabolism, cyclic AMP receptor protein. A functional KdgR binding site was identified close to the putative pelI promoter. Analysis of the amino acid sequence of PelI revealed high homology with a pectate lyase from Erwinia carotovora subsp. carotovora (65% identity) and low homology with pectate lyases of the phytopathogenic fungus Nectria haematococca (Fusarium solani). This finding indicates that PelI belongs to pectate lyase class

  19. Purification and Characterization of a Unique Pectin Lyase from Aspergillus giganteus Able to Release Unsaturated Monogalacturonate during Pectin Degradation

    OpenAIRE

    Danielle Biscaro Pedrolli; Eleonora Cano Carmona

    2014-01-01

    A pectin lyase, named PLIII, was purified to homogeneity from the culture filtrate of Aspergillus giganteus grown in submerged culture containing orange peel waste as carbon source. PLIII was able to digest apple pectin and citrus pectins with different degrees of methyl esterification. Interestingly, the PLIII activity was stimulated in the presence of some divalent cations including Pb2+ and was not significantly affected by Hg2+. Like other pectin lyases, PLIII is stimulated by but is not ...

  20. Site-directed mutagenesis of lysine 193 in Escherichia coli isocitrate lyase by use of unique restriction enzyme site elimination.

    OpenAIRE

    Diehl, P; McFadden, B A

    1993-01-01

    By a newly developed double-stranded mutagenesis technique, histidine (H), glutamate (E), arginine (R) and leucine (L) have been substituted for the lysyl 193 residue (K-193) in isocitrate lyase from Escherichia coli. The substitutions for this residue, which is present in a highly conserved, cationic region, significantly affect both the Km for Ds-isocitrate and the apparent kcat of isocitrate lyase. Specifically, the conservative substitutions, K-193-->H (K193H) and K193R, reduce catalytic ...

  1. Crystallization and preliminary X-ray analysis of argininosuccinate lyase from Streptococcus mutans

    International Nuclear Information System (INIS)

    Crystals of argininosuccinate lyase from S. mutans were obtained and X-ray data were collected to 2.5 Å resolution in space group R3. Argininosuccinate lyase (ASL) is an important enzyme in arginine synthesis and the urea cycle, which are highly conserved from bacteria to eukaryotes. The gene encoding Streptococcus mutans ASL (smASL) was amplified and cloned into expression vector pET28a. The recombinant smASL protein was expressed in a soluble form in Escherichia coli strain BL21 (DE3) and purified to homogeneity by two-step column chromatography. Crystals suitable for X-ray analysis were obtained and X-ray diffraction data were collected to a resolution of 2.5 Å. The crystals belonged to space group R3, with unit-cell parameters a = b = 254.5, c = 78.3 Å

  2. Hydroperoxide lyase depletion in transgenic potato plants leads to an increase in aphid performance

    OpenAIRE

    Vancanneyt, Guy; Sanz, Carlos; Farmaki, Theodora; Paneque, Manuel; Ortego, Félix; Castañera, Pedro; Sánchez-Serrano, Jose J.

    2001-01-01

    Hydroperoxide lyases (HPLs) catalyze the cleavage of fatty acid hydroperoxides to aldehydes and oxoacids. These volatile aldehydes play a major role in forming the aroma of many plant fruits and flowers. In addition, they have antimicrobial activity in vitro and thus are thought to be involved in the plant defense response against pest and pathogen attack. An HPL activity present in potato leaves has been characterized and shown to cleave specifically 13-hydroperoxides of both linoleic and li...

  3. Screening and optimization of pectin lyase and polygalacturonase activity from ginseng pathogen Cylindrocarpon Destructans

    OpenAIRE

    Gayathri Sathiyaraj; Sathiyaraj Srinivasan; Ho-Bin Kim; Sathiyamoorthy Subramaniyam; Ok Ran Lee; Yeon-Ju Kim; Deok Chun Yang

    2011-01-01

    Cylindrocarpon destructans isolated from ginseng field was found to produce pectinolytic enzymes. A Taguchi's orthogonal array experimental design was applied to optimize the preliminary production of polygalacturonase (PG) and pectin lyase (PL) using submerged culture condition. This method was applied to evaluate the significant parameters for the production of enzymes. The process variables were pH, pectin concentration, incubation time and temperature. Optimization of process parameters r...

  4. Screening and optimization of pectin lyase and polygalacturonase activity from ginseng pathogen Cylindrocarpon Destructans

    OpenAIRE

    Sathiyaraj, Gayathri; Srinivasan, Sathiyaraj; Kim, Ho-Bin; Subramaniyam, Sathiyamoorthy; Lee, Ok Ran; Kim, Yeon-Ju; Yang, Deok Chun

    2011-01-01

    Cylindrocarpon destructans isolated from ginseng field was found to produce pectinolytic enzymes. A Taguchi’s orthogonal array experimental design was applied to optimize the preliminary production of polygalacturonase (PG) and pectin lyase (PL) using submerged culture condition. This method was applied to evaluate the significant parameters for the production of enzymes. The process variables were pH, pectin concentration, incubation time and temperature. Optimization of process parameters r...

  5. Encapsulated Escherichia coli in alginate beads capable of secreting a heterologous pectin lyase

    OpenAIRE

    Trikka Fotini A; Chaitidou Sotiria A; Papi Rigini M; Kyriakidis Dimitrios A

    2008-01-01

    Abstract Background Production of heterologous proteins in the E. coli periplasm, or into the extracellular fluid has many advantages; therefore naturally occurring signal peptides are selected for proteins translocation. The aim of this study was the production in high yields of a recombinant pectin lyase that is efficiently secreted and the encapsulation of transformed E. coli cells for pectin degradation in a biotechnological process. Results The nucleotide sequence of Bacillus subtilis α-...

  6. Heterologous expression of a Penicillium purpurogenum pectin lyase in Pichia pastoris and its characterization.

    Science.gov (United States)

    Pérez-Fuentes, Claudio; Cristina Ravanal, María; Eyzaguirre, Jaime

    2014-01-01

    Lignocellulose is the major component of plant cell walls and it represents a great source of renewable organic matter. One of lignocellulose constituents is pectin. Pectin is composed of two basic structures: a 'smooth' region and a 'hairy' region. The 'smooth' region (homogalacturonan) is a linear polymer of galacturonic acid residues with α-(1→4) linkages, substituted by methyl and acetyl residues. The 'hairy' region is more complex, containing xylogalacturonan and rhamnogalacturonans I and II. Among the enzymes which degrade pectin (pectinases) is pectin lyase (E.C. 4.2.2.10). This enzyme acts on highly esterified homogalacturonan, catalysing the cleavage of α-(1→4) glycosidic bonds between methoxylated residues of galacturonic acid by means of β-elimination, with the formation of 4,5-unsaturated products. In this work, the gene and cDNA of a pectin lyase from Penicillium purpurogenum have been sequenced, and the cDNA has been expressed in Pichia pastoris. The gene is 1334 pb long, has three introns and codes for a protein of 376 amino acid residues. The recombinant enzyme was purified to homogeneity and characterized. Pectin lyase has a molecular mass of 45 kDa as determined by SDS-PAGE. It is active on highly esterified pectin, and decreases 40% the viscosity of pectin with a degree of esterification ≥85%. The enzyme showed no activity on polygalacturonic acid and pectin from citrus fruit 8% esterified. The optimum pH and temperature for the recombinant enzyme are 6.0 and 50 °C, respectively, and it is stable up to 50 °C when exposed for 3 h. A purified pectin lyase may be useful in biotechnological applications such as the food industry where the liberation of toxic methanol in pectin degradation should be avoided. PMID:24863479

  7. Protein evolution analysis of S-hydroxynitrile lyase by complete sequence design utilizing the INTMSAlign software

    OpenAIRE

    Shogo Nakano; Yasuhisa Asano

    2015-01-01

    Development of software and methods for design of complete sequences of functional proteins could contribute to studies of protein engineering and protein evolution. To this end, we developed the INTMSAlign software, and used it to design functional proteins and evaluate their usefulness. The software could assign both consensus and correlation residues of target proteins. We generated three protein sequences with S-selective hydroxynitrile lyase (S-HNL) activity, which we call designed S-HNL...

  8. Purification and characterization of an extracellular pectate lyase from an Amycolata sp.

    OpenAIRE

    Brühlmann, F

    1995-01-01

    The extracellular pectate lyase (EC 4.2.2.2) of a nonsporulating Amycolata sp. was purified to homogeneity by anion- and cation-exchange chromatographies followed by hydrophobic interaction chromatography. The enzyme cleaved polygalacturonate but not highly esterified pectin in a random endolytic transeliminative mechanism that led to the formation of a wide range of 4,5-unsaturated oligogalacturonates. As shown by high-performance anion-exchange chromatography and pulsed amperometric detecti...

  9. Isocitrate Lyase Activity Is Required for Virulence of the Intracellular Pathogen Rhodococcus equi

    OpenAIRE

    Wall, Daniel M.; Duffy, Pamela S.; DuPont, Chris; Prescott, John F.; Meijer, Wim G.

    2005-01-01

    Rhodococcus equi is an important pathogen of foals, causing severe pyogranulomatous pneumonia. Virulent R. equi strains grow within macrophages, a process which remains poorly characterized. A potential source of carbon for intramacrophage R. equi is membrane lipid-derived fatty acids, which following β oxidation are assimilated via the glyoxylate bypass. To assess the importance of isocitrate lyase, the first enzyme of the glyoxylate bypass, in virulence of a foal isolate of R. equi, a mutan...

  10. Essential histidine pairs indicate conserved haem binding in epsilonproteobacterial cytochrome c haem lyases

    OpenAIRE

    Kern, Melanie; Scheithauer, Juliane; Kranz, Robert G.; Simon, Jörg

    2010-01-01

    Bacterial cytochrome c maturation occurs at the outside of the cytoplasmic membrane, requires transport of haem b across the membrane, and depends on membrane-bound cytochrome c haem lyase (CCHL), an enzyme that catalyses covalent attachment of haem b to apocytochrome c. Epsilonproteobacteria such as Wolinella succinogenes use the cytochrome c biogenesis system II and contain unusually large CCHL proteins of about 900 amino acid residues that appear to be fusions of the CcsB and CcsA proteins...

  11. Engineering broad-spectrum digestion of polyuronides from an exolytic polysaccharide lyase

    OpenAIRE

    MacDonald, Logan C.; Weiler, Elizabeth B.; Berger, Bryan W.

    2016-01-01

    Background Macroalgae represents a promising source of fermentable carbohydrates for use in the production of energy efficient biofuel. The primary carbohydrate in brown algae is the uronic acid-containing alginate, whereas green algae contains a significant amount of glucuronan. A necessary step in the conversion of these polyuronides to bioethanol is saccharification, which can be achieved by enzymatic or chemical degradation. Results Polysaccharide lyases are a class of enzymes which cleav...

  12. Stress-dependent regulation of 13-lipoxygenases and 13-hydroperoxide lyase in olive fruit mesocarp

    OpenAIRE

    Padilla, María Nieves; Hernández, M. Luisa; Sanz, Carlos; Martínez-Rivas, José Manuel

    2014-01-01

    The effect of different environmental stresses on the expression and enzyme activity levels of 13-lipoxygenases (13-LOX) and 13-hydroperoxide lyase (13-HPL) and on the volatile compounds synthesized by their sequential action has been studied in the mesocarp tissue of olive fruit from the Picual and Arbequina cultivars. The results showed that temperature, light, wounding and water regime regulate olive 13-LOXs and 13-HPL genes at transcriptional level. Low temperature and wounding brought ab...

  13. Identification of Bacterial Cell Wall Lyases via Pseudo Amino Acid Composition.

    Science.gov (United States)

    Chen, Xin-Xin; Tang, Hua; Li, Wen-Chao; Wu, Hao; Chen, Wei; Ding, Hui; Lin, Hao

    2016-01-01

    Owing to the abuse of antibiotics, drug resistance of pathogenic bacteria becomes more and more serious. Therefore, it is interesting to develop a more reasonable way to solve this issue. Because they can destroy the bacterial cell structure and then kill the infectious bacterium, the bacterial cell wall lyases are suitable candidates of antibacteria sources. Thus, it is urgent to develop an accurate and efficient computational method to predict the lyases. Based on the consideration, in this paper, a set of objective and rigorous data was collected by searching through the Universal Protein Resource (the UniProt database), whereafter a feature selection technique based on the analysis of variance (ANOVA) was used to acquire optimal feature subset. Finally, the support vector machine (SVM) was used to perform prediction. The jackknife cross-validated results showed that the optimal average accuracy of 84.82% was achieved with the sensitivity of 76.47% and the specificity of 93.16%. For the convenience of other scholars, we built a free online server called Lypred. We believe that Lypred will become a practical tool for the research of cell wall lyases and development of antimicrobial agents. PMID:27437396

  14. Expression and properties of the glyoxysomal and cytosolic forms of isocitrate lyase in Amaranthus caudatus L.

    Science.gov (United States)

    Eprintsev, Alexander T; Fedorin, Dmitry N; Salnikov, Alexei V; Igamberdiev, Abir U

    2015-06-01

    Isocitrate lyase (EC 4.1.3.1) catalyzes the reversible conversion of d-isocitrate to succinate and glyoxylate. It is usually associated with the glyoxylate cycle in glyoxysomes, although the non-glyoxysomal form has been reported and its relation to interconversion of organic acids outside the glyoxylate cycle suggested. We investigated the expression of two isocitrate lyase genes and activities of the glyoxysomal (ICL1) and cytosolic (ICL2) forms of isocitrate lyase in amaranth (Amaranthus caudatus L.) seedlings. Both forms were separated and purified. The cytosolic form had a low optimum pH (6.5) and was activated by Mn(2+) ions, while Mg(2+) was ineffective, and had a lower affinity to d, l-isocitrate (Km 63 μM) as compared to the glyoxysomal form (optimum pH 7.5, K(m) 45 μM), which was activated by Mg(2+). The highest ICL1 activity was observed on the 3rd day of germination; then the activity and expression of the corresponding gene decreased, while the activity of ICL2 and gene expression increased to the 7th day of germination and then remained at the same level. It is concluded that the function of ICL1 is related to the glyoxylate cycle while ICL2 functions independently from the glyoxylate cycle and interconverts organic acids in the cytosol. PMID:25955696

  15. Genetically engineered alginate lyase-PEG conjugates exhibit enhanced catalytic function and reduced immunoreactivity.

    Directory of Open Access Journals (Sweden)

    John W Lamppa

    Full Text Available Alginate lyase enzymes represent prospective biotherapeutic agents for treating bacterial infections, particularly in the cystic fibrosis airway. To effectively deimmunize one therapeutic candidate while maintaining high level catalytic proficiency, a combined genetic engineering-PEGylation strategy was implemented. Rationally designed, site-specific PEGylation variants were constructed by orthogonal maleimide-thiol coupling chemistry. In contrast to random PEGylation of the enzyme by NHS-ester mediated chemistry, controlled mono-PEGylation of A1-III alginate lyase produced a conjugate that maintained wild type levels of activity towards a model substrate. Significantly, the PEGylated variant exhibited enhanced solution phase kinetics with bacterial alginate, the ultimate therapeutic target. The immunoreactivity of the PEGylated enzyme was compared to a wild type control using in vitro binding studies with both enzyme-specific antibodies, from immunized New Zealand white rabbits, and a single chain antibody library, derived from a human volunteer. In both cases, the PEGylated enzyme was found to be substantially less immunoreactive. Underscoring the enzyme's potential for practical utility, >90% of adherent, mucoid, Pseudomonas aeruginosa biofilms were removed from abiotic surfaces following a one hour treatment with the PEGylated variant, whereas the wild type enzyme removed only 75% of biofilms in parallel studies. In aggregate, these results demonstrate that site-specific mono-PEGylation of genetically engineered A1-III alginate lyase yielded an enzyme with enhanced performance relative to therapeutically relevant metrics.

  16. Hydroxynitrile lyase at the diisopropyl ether/water interface: Evidence for interfacial enzyme activity

    Energy Technology Data Exchange (ETDEWEB)

    Hichel, A.; Radke, C.J.; Blanch, H.W. [Univ. of California, Berkeley, CA (United States). Dept. of Chemical Engineering

    1999-11-20

    A novel recycle reactor has been designed to determine the interfacial activity of hydroxynitrile lyase in a diisopropyl ether (DIPE)/water two-phase system. The reactor provides a known interfacial area. Enzyme activity toward mandelonitrile cleavage is continuously measured in the reactor by following benzaldehyde product formation in the DIPE organic phase with an optical flow cell. For the first time, the authors establish that this enzymatic reaction is carried out by the hydroxynitrile lyase residing at the organic solvent/water interface and not in the aqueous bulk phase. Hydroxynitrile lyase adsorbs at the interface and exhibits extraordinary stability. Denaturation does not occur over several hours, although the surface pressure increases under the same conditions over this time span. Increases in surface pressure indicate enzyme penetration through the interface although no loss of enzyme activity is observed. Adsorption of p-Hnl at the interface is fit by the Langmuir equilibrium adsorption model with an adsorption equilibrium constant of 0.032 L mg{sup {minus}1}. For the mandelonitrile cleavage reaction at ambient temperature, p-Hnl follows Michaelis-Menten kinetics at the interface with a Michaelis constant of 14.4 mM and a specific activity close that for the bulk aqueous phase.

  17. Comparative characterization of bovine testicular hyaluronidase and a hyaluronate lyase from Streptococcus agalactiae in pharmaceutical preparations.

    Science.gov (United States)

    Oettl, Martin; Hoechstetter, Julia; Asen, Iris; Bernhardt, Günther; Buschauer, Armin

    2003-03-01

    Although bovine testicular hyaluronidase (BTH) has been used in several medical fields for many years, these drugs are poorly characterized. We compared pharmaceutical BTH preparations (Neopermease, Hylase "Dessau") and a hyaluronate lyase from Streptococcus agalactiae. The BTH preparations were complex mixtures of proteins (SDS-PAGE, gel filtration) with enzymatic activity in different fractions. In the case of Neopermease the highest specific activity was found in the 58 kDa fraction (optimum at pH 3.6), whereas the 77 and 33 kDa fractions showed markedly lower specific activities at an optimal pH of 6.2. Maximum specific activity of the bacterial enzyme (approx. 1000 micromol min(-1) mg(-1)) was found at pH 5.0, being 410- and 5100-times higher compared to Neopermease and Hylase "Dessau", respectively. The hyaluronate lyase preparation was separated into two main proteins [100 kDa (pI=8.9) and 85 kDa (pI=9.2)] which were enzymatically active in SDS substrate-PAGE. Zymography after limited proteolysis of the bacterial enzyme with trypsin revealed active fragments (75-50 kDa). Our results suggest that hyaluronate lyase is an alternative for BTH, of which there has been a shortage, since companies have stopped the production of BTH preparations due to the risk of BSE. PMID:12659938

  18. Phosphoserine Lyase Deoxyribozymes: DNA-Catalyzed Formation of Dehydroalanine Residues in Peptides.

    Science.gov (United States)

    Chandrasekar, Jagadeeswaran; Wylder, Adam C; Silverman, Scott K

    2015-08-01

    Dehydroalanine (Dha) is a nonproteinogenic electrophilic amino acid that is a synthetic intermediate or product in the biosynthesis of several bioactive cyclic peptides such as lantibiotics, thiopeptides, and microcystins. Dha also enables labeling of proteins and synthesis of post-translationally modified proteins and their analogues. However, current chemical approaches to introducing Dha into peptides have substantial limitations. Using in vitro selection, here we show that DNA can catalyze Zn(2+) or Zn(2+)/Mn(2+)-dependent formation of Dha from phosphoserine (pSer), i.e., exhibit pSer lyase activity, a fundamentally new DNA-catalyzed reaction. Two new pSer lyase deoxyribozymes, named Dha-forming deoxyribozymes 1 and 2 (DhaDz1 and DhaDz2), each function with multiple turnover on the model hexapeptide substrate that was used during selection. Using DhaDz1, we generated Dha from pSer within an unrelated linear 13-mer peptide. Subsequent base-promoted intramolecular cyclization of homocysteine into Dha formed a stable cystathionine (thioether) analogue of the complement inhibitor compstatin. These findings establish the fundamental catalytic ability of DNA to eliminate phosphate from pSer to form Dha and suggest that with further development, pSer lyase deoxyribozymes will have broad practical utility for site-specific enzymatic synthesis of Dha from pSer in peptide substrates. PMID:26200899

  19. Identification of Bacterial Cell Wall Lyases via Pseudo Amino Acid Composition

    Science.gov (United States)

    Tang, Hua; Li, Wen-Chao; Wu, Hao; Ding, Hui

    2016-01-01

    Owing to the abuse of antibiotics, drug resistance of pathogenic bacteria becomes more and more serious. Therefore, it is interesting to develop a more reasonable way to solve this issue. Because they can destroy the bacterial cell structure and then kill the infectious bacterium, the bacterial cell wall lyases are suitable candidates of antibacteria sources. Thus, it is urgent to develop an accurate and efficient computational method to predict the lyases. Based on the consideration, in this paper, a set of objective and rigorous data was collected by searching through the Universal Protein Resource (the UniProt database), whereafter a feature selection technique based on the analysis of variance (ANOVA) was used to acquire optimal feature subset. Finally, the support vector machine (SVM) was used to perform prediction. The jackknife cross-validated results showed that the optimal average accuracy of 84.82% was achieved with the sensitivity of 76.47% and the specificity of 93.16%. For the convenience of other scholars, we built a free online server called Lypred. We believe that Lypred will become a practical tool for the research of cell wall lyases and development of antimicrobial agents. PMID:27437396

  20. Characterization of the pelL gene encoding a novel pectate lyase of Erwinia chrysanthemi 3937.

    Science.gov (United States)

    Lojkowska, E; Masclaux, C; Boccara, M; Robert-Baudouy, J; Hugouvieux-Cotte-Pattat, N

    1995-06-01

    Erwinia chrysanthemi 3937 secretes five major isoenzymes of pectate lyases encoded by the pelA, pelB, pelC, pelD and pelE genes. Recently, a new set of pectate lyases was identified in E. chrysanthemi mutants deleted of those pel genes. We cloned the pelL gene, encoding one of these secondary pectate lyases of E. chrysanthemi 3937, from a genomic bank of a strain deleted of the five major pel genes. The nucleotide sequence of the region containing the pelL gene was determined. The pelL reading frame is 1275 bases long, corresponding to a protein of 425 amino acids including a typical amino-terminal signal sequence of 25 amino acids. Comparison of the amino acid sequences of PelL and the exo-pectate lyase PelX of E. chrysanthemi EC16 revealed a low homology, limited to 220 residues of the central part of the proteins. No homology was detected with other bacterial pectinolytic enzymes. Regulation of pelL transcription was analysed using gene fusion. As shown for the other pel genes, the transcription of pelL is dependent on various environmental conditions. It is induced by pectic catabolic products and affected by growth phase, temperature, iron starvation, osmolarity, anaerobiosis, nitrogen starvation and catabolite repression. Regulation of pelL expression appeared to be independent of the KdgR repressor, which controls all the steps of pectin catabolism. In contrast, the pecS gene, which is involved in regulation of the synthesis of the major pectate lyases and of cellulase, also appeared to be involved in pelL expression. The PelL protein is able to macerate plant tissue. This enzyme has a basic isoelectric point, presents an endo-cleaving activity on polygalacturonate or partially methylated pectin, with a basic pH optimum and an absolute requirement for Ca2+. The pelL mutant displayed a reduced virulence on potato tubers and Saintpaulia ionantha plants, demonstrating the important role of this enzyme in soft-rot disease. PMID:8577252

  1. Characterization of the exopolygalacturonate lyase PelX of Erwinia chrysanthemi 3937.

    Science.gov (United States)

    Shevchik, V E; Kester, H C; Benen, J A; Visser, J; Robert-Baudouy, J; Hugouvieux-Cotte-Pattat, N

    1999-03-01

    Erwinia chrysanthemi 3937 secretes several pectinolytic enzymes, among which eight isoenzymes of pectate lyases with an endo-cleaving mode (PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ) have been identified. Two exo-cleaving enzymes, the exopolygalacturonate lyase, PelX, and an exo-poly-alpha-D-galacturonosidase, PehX, have been previously identified in other E. chrysanthemi strains. Using a genomic bank of a 3937 mutant with the major pel genes deleted, we cloned a pectinase gene identified as pelX, encoding the exopolygalacturonate lyase. The deduced amino acid sequence of the 3937 PelX is very similar to the PelX of another E. chrysanthemi strain, EC16, except in the 43 C-terminal amino acids. PelX also has homology to the endo-pectate lyase PelL of E. chrysanthemi but has a N-terminal extension of 324 residues. The transcription of pelX, analyzed by gene fusions, is dependent on several environmental conditions. It is induced by pectic catabolic products and affected by growth phase, oxygen limitation, nitrogen starvation, and catabolite repression. Regulation of pelX expression is dependent on the KdgR repressor, which controls almost all the steps of pectin catabolism, and on the global activator of sugar catabolism, cyclic AMP receptor protein. In contrast, PecS and PecT, two repressors of the transcription of most pectate lyase genes, are not involved in pelX expression. The pelX mutant displayed reduced pathogenicity on chicory leaves, but its virulence on potato tubers or Saintpaulia ionantha plants did not appear to be affected. The purified PelX protein has no maceration activity on plant tissues. Tetragalacturonate is the best substrate of PelX, but PelX also has good activity on longer oligomers. Therefore, the estimated number of binding subsites for PelX is 4, extending from subsites -2 to +2. PelX and PehX were shown to be localized in the periplasm of E. chrysanthemi 3937. PelX catalyzed the formation of unsaturated digalacturonates by

  2. Post-merger evolution of carbon-oxygen + helium white dwarf binaries and the origin of R Coronae Borealis and extreme helium stars

    CERN Document Server

    Zhang, Xianfei; Chen, Xuefei; Han, Zhanwen

    2014-01-01

    Orbital decay by gravitational-wave radiation will cause some close-binary white dwarfs (WDs) to merge within a Hubble time. The results from previous hydrodynamical WD-merger simulations have been used to guide calculations of the post-merger evolution of carbon-oxygen + helium (CO+He) WD binaries. Our models include the formation of a hot corona in addition to a Keplerian disk. We introduce a 'destroyeddisk' model to simulate the effect of direct disk ingestion into the expanding envelope. These calculations indicate significant lifetimes in the domain of the rare R Coronae Borealis (RCB) stars, before a fast evolution through the domain of the hotter extreme helium (EHe) stars. Surface chemistries of the resulting giants are in partial agreement with the observed abundances of RCB and EHe stars. The production of 3He, 18O and 19F are discussed. Evolutionary timescales combined with binary white-dwarf merger rates from binary-star population synthesis are consistent with present-day numbers of RCBs and EHes...

  3. Post-merger evolution of carbon-oxygen + helium white dwarf binaries and the origin of R Coronae Borealis and extreme helium stars

    Science.gov (United States)

    Zhang, Xianfei; Jeffery, C. Simon; Chen, Xuefei; Han, Zhanwen

    2014-11-01

    Orbital decay by gravitational-wave radiation will cause some close-binary white dwarfs (WDs) to merge within a Hubble time. The results from previous hydrodynamical WD-merger simulations have been used to guide calculations of the post-merger evolution of carbon-oxygen + helium (CO+He) WD binaries. Our models include the formation of a hot corona in addition to a Keplerian disc. We introduce a `destroyed-disc' model to simulate the effect of direct disc ingestion into the expanding envelope. These calculations indicate significant lifetimes in the domain of the rare R Coronae Borealis (RCB) stars, before a fast evolution through the domain of the hotter extreme helium (EHe) stars. Surface chemistries of the resulting giants are in partial agreement with the observed abundances of RCB and EHe stars. The production of 3He, 18O and 19F are discussed. Evolutionary time-scales combined with binary WD merger rates from binary-star population synthesis are consistent with present-day numbers of RCBs and EHes, provided that the majority come from relatively recent (carbon-rich abundances of RCB stars, the `destroyed-disc' model yields a high-carbon product with He-WD mass ≥0.30 M⊙, in better agreement with population synthesis results.

  4. CNO abundances and hydrodynamic models of the nova outburst. III - 0.5 solar mass models with enhanced carbon, oxygen, and nitrogen

    Science.gov (United States)

    Starrfield, S.; Sparks, W. M.; Truran, J. W.

    1974-01-01

    Consideration of the evolution of thermonuclear runaways in the hydrogen-rich envelopes of 0.5 solar mass carbon-oxygen white dwarfs. The larger radii of these stars, compared with the 1.00 solar mass white dwarfs, results in a lesser degree of degeneracy at the same depth in the star. Four models of luminosity with .00355 solar luminosity, differing only in the initial abundances of C-12, N-14, and O-16, are presented. The degree of enhancement required to produce mass ejection, and thereby a nova-type outburst, is greater than for the 1.00 solar mass model. Nevertheless, the evolution of the 0.5 solar mass model that ejected material is very similar to that of the 1.00 solar mass models, and it also ejects significant amounts of C-13, N-15, and O-17 into the interstellar medium. The 0.5 solar mass outburst is considerably less intense than the 1.00 solar mass outburst (even under optimum conditions), and this lower mass behavior is interpreted as associated with the observed outburst of the slowest novae.

  5. Cloning and characterization of two thermo- and salt-tolerant oligoalginate lyases from marine bacterium Halomonas sp.

    Science.gov (United States)

    Yang, Xuemei; Li, Shangyong; Wu, Ying; Yu, Wengong; Han, Feng

    2016-05-01

    Two new alginate lyase genes, oalY1 and oalY2, have been cloned from the newly isolated marine bacterium Halomonas sp. QY114 and expressed in Escherichia coli The deduced alginate lyases, OalY1 and OalY2, belonged to polysaccharide lyase (PL) family 17 and showed less than 45% amino acid identity with all of the characterized oligoalginate lyases. OalY1 and OalY2 exhibited the highest activities at 45°C and 50°C, respectively. Both of them showed more than 50% of the highest activity at 60°C, and 20% at 80°C. In addition, they were salt-dependent and salt-tolerant since both of them showed the highest activity in the presence of 0.5 M NaCl and preserved 63% and 68% of activity in the presence of 3 M NaCl. Significantly, OalY1 and OalY2 could degrade both polyM and polyG blocks into alginate monosaccharides in an exo-lytic type, indicating that they are bifunctional alginate lyases. In conclusion, our study indicated that OalY1 and OalY2 are good candidates for alginate saccharification application, and the salt-tolerance may present an exciting new concept for biofuel production from native brown seaweeds. PMID:27030725

  6. Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Jendresen, Christian Bille; Stahlhut, Steen Gustav; Li, Mingji;

    2015-01-01

    Phenylalanine and tyrosine ammonia-lyases form cinnamic acid and p-coumaric acid, which are precursors of a wide range of aromatic compounds of biotechnological interest. Lack of highly active and specific tyrosine ammonia-lyases has previously been a limitation in metabolic engineering approache...

  7. Composite active site of chondroitin lyase ABC accepting both epimers of uronic acid

    Energy Technology Data Exchange (ETDEWEB)

    Shaya, D.; Hahn, Bum-Soo; Bjerkan, Tonje Marita; Kim, Wan Seok; Park, Nam Young; Sim, Joon-Soo; Kim, Yeong-Shik; Cygler, M. (Catholic Univ of Korea); (NUST); (McGill); (Nat); (Natural Products Res Inst, Korea)

    2008-03-19

    Enzymes have evolved as catalysts with high degrees of stereospecificity. When both enantiomers are biologically important, enzymes with two different folds usually catalyze reactions with the individual enantiomers. In rare cases a single enzyme can process both enantiomers efficiently, but no molecular basis for such catalysis has been established. The family of bacterial chondroitin lyases ABC comprises such enzymes. They can degrade both chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans at the nonreducing end of either glucuronic acid (CS) or its epimer iduronic acid (DS) by a {beta}-elimination mechanism, which commences with the removal of the C-5 proton from the uronic acid. Two other structural folds evolved to perform these reactions in an epimer-specific fashion: ({alpha}/{alpha}){sub 5} for CS (chondroitin lyases AC) and {beta}-helix for DS (chondroitin lyases B); their catalytic mechanisms have been established at the molecular level. The structure of chondroitinase ABC from Proteus vulgaris showed surprising similarity to chondroitinase AC, including the presence of a Tyr-His-Glu-Arg catalytic tetrad, which provided a possible mechanism for CS degradation but not for DS degradation. We determined the structure of a distantly related Bacteroides thetaiotaomicron chondroitinase ABC to identify additional structurally conserved residues potentially involved in catalysis. We found a conserved cluster located {approx}12 {angstrom} from the catalytic tetrad. We demonstrate that a histidine in this cluster is essential for catalysis of DS but not CS. The enzyme utilizes a single substrate-binding site while having two partially overlapping active sites catalyzing the respective reactions. The spatial separation of the two sets of residues suggests a substrate-induced conformational change that brings all catalytically essential residues close together.

  8. Structure and Mechanism of the Phycobiliprotein Lyase CpcT*♦

    Science.gov (United States)

    Zhou, Wei; Ding, Wen-Long; Zeng, Xiao-Li; Dong, Liang-Liang; Zhao, Bin; Zhou, Ming; Scheer, Hugo; Zhao, Kai-Hong; Yang, Xiaojing

    2014-01-01

    Pigmentation of light-harvesting phycobiliproteins of cyanobacteria requires covalent attachment of open-chain tetrapyrroles, bilins, to the apoproteins. Thioether formation via addition of a cysteine residue to the 3-ethylidene substituent of bilins is mediated by lyases. T-type lyases are responsible for attachment to Cys-155 of phycobiliprotein β-subunits. We present crystal structures of CpcT (All5339) from Nostoc (Anabaena) sp. PCC 7120 and its complex with phycocyanobilin at 1.95 and 2.50 Å resolution, respectively. CpcT forms a dimer and adopts a calyx-shaped β-barrel fold. Although the overall structure of CpcT is largely retained upon chromophore binding, arginine residues at the opening of the binding pocket undergo major rotameric rearrangements anchoring the propionate groups of phycocyanobilin. Based on the structure and mutational analysis, a reaction mechanism is proposed that accounts for chromophore stabilization and regio- and stereospecificity of the addition reaction. At the dimer interface, a loop extending from one subunit partially shields the opening of the phycocyanobilin binding pocket in the other subunit. Deletion of the loop or disruptions of the dimer interface significantly reduce CpcT lyase activity, suggesting functional relevance of the dimer. Dimerization is further enhanced by chromophore binding. The chromophore is largely buried in the dimer, but in the monomer, the 3-ethylidene group is accessible for the apophycobiliprotein, preferentially from the chromophore α-side. Asp-163 and Tyr-65 at the β- and α-face near the E-configured ethylidene group, respectively, support the acid-catalyzed nucleophilic Michael addition of cysteine 155 of the apoprotein to an N-acylimmonium intermediate proposed by Grubmayr and Wagner (Grubmayr, K., and Wagner, U. G. (1988) Monatsh. Chem. 119, 965–983). PMID:25074932

  9. Pectinolytic bacteria and their secreted pectate lyases: agents for the maceration and solubilization of phytomass for fuels production

    Energy Technology Data Exchange (ETDEWEB)

    Preston, J.F. III; Rice, J.D.; Chow, M.C. (Florida Univ., Gainesville, FL (United States). Dept. of Microbiology and Cell Science)

    1993-01-01

    The objectives of this research have been to identify the pectinolytic enzymes secreted by bacteria and apply these towards the enhanced maceration and solubilization of plant material, focusing on the pectate lyases secreted by the phytopathogenic strains of Erwinia chrysanthemi, the ruminant resident Lachnospira multiparus, and the wood digestor isolate, Clostridium populeti. An HPLC approach has been developed that permits the kinetic analysis of each enzyme with respect to the formation of individual products during the pectate depolymerization process. This approach has demonstrated that each of these organisms secretes a nonrandom trimer-generating pectate lyase with a combination of endolytic and exolytic depolymerizing mechanisms. Two different strains of E. chrysanthemi secrete a battery of pectate lyases that include random endolytic as well as nonrandom dimer - and nonrandom trimer-generating endolytic/exolytic mechanisms. (author)

  10. Strain Improvement of Rhodotorula graminis for Production of a Novel l-Phenylalanine Ammonia-Lyase

    OpenAIRE

    Orndorff, Steve A.; Costantino, Nina; Stewart, David; Durham, Don R.

    1988-01-01

    l-Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) from Rhodotorula rubra has been used in the commercial manufacture of l-phenylalanine from trans-cinnamic acid. In this study, R. graminis PAL was investigated. Mutant strain GX6000 was isolated after ethyl methanesulfonate mutagenesis of wild-type R. graminis GX5007 by selecting for resistance to phenylpropiolic acid, an analog of trans-cinnamic acid. Mutant strain GX6000 produced inducible PAL at levels four- to fivefold higher than had wild-t...

  11. Analysis of pectate lyases produced by soft rot bacteria associated with spoilage of vegetables.

    OpenAIRE

    Liao, C.H.

    1989-01-01

    Isoelectric focusing (IEF) profiles of pectate lyases (PLs) produced by five different groups of soft rot bacteria were analyzed by using the combined techniques of thin-layer polyacrylamide gel IEF and agarose-pectate overlay activity staining. Four strains of soft rot Erwinia spp. produced three or more PL isozymes. All of eight Pseudomonas viridiflava strains examined produced one single PL with a pI of 9.7. All 10 of Pseudomonas fluorescens strains produced two PLs; the major one had a pI...

  12. The use of thioglycolate to distinguish between 3' AP (apurinic/apyrimidinic) endonucleases and AP lyases.

    OpenAIRE

    Bricteux-Grégoire, S; Verly, W G

    1989-01-01

    Addition of thioglycolate and DEAE-Sephadex chromatography were used to analyze the cleavage of the C(3')-O-P bond 3' to AP (apurinic/apyrimidinic) sites in DNA and to distinguish between a mechanism of hydrolysis (which would allow the nicking enzyme to be called 3' AP endonuclease) or beta-elimination (so that the nicking enzyme should be called AP lyase). For this purpose, DNA labelled in the AP sites was first cleaved by rat-liver AP endonuclease, then with the 3' nicking catalyst in the ...

  13. Probing Reversible Chemistry in Coenzyme B12-Dependent Ethanolamine Ammonia Lyase with Kinetic Isotope Effects

    OpenAIRE

    Rentergent, Julius; Scruton, Nigel S; Hay, Sam; Jones, Alex R.

    2015-01-01

    Coenzyme B12-dependent enzymes such as ethanolamine ammonia lyase have remarkable catalytic power and some unique properties that enable detailed analysis of the reaction chemistry and associated dynamics. By selectively deuterating the substrate (ethanolamine) and/or the β-carbon of the 5′-deoxyadenosyl moiety of the intrinsic coenzyme B12, it was possible to experimentally probe both the forward and reverse hydrogen atom transfers between the 5′-deoxyadenosyl radical and substrate duri...

  14. Purification and properties of Bacteroides heparinolyticus heparinase (heparin lyase, EC 4.2.2.7).

    OpenAIRE

    Nakamura, T.(International Center for Elementary Particle Physics and Department of Physics, The University of Tokyo, Tokyo, Japan); Shibata, Y.; Fujimura, S.

    1988-01-01

    Heparinase (heparin lyase, EC 4.2.2.7) was isolated from the cell extract of an oral bacterium, Bacteroides heparinolyticus. It was a basic protein with an isoelectric point of 9.5. Its molecular weight was 63,000. The enzyme was the most active against heparin among the tested mucopolysaccharides. Catalytic properties may be similar to those of heparinase of Flavobacterium heparinum, since the enzymatic degradation products obtained by using the two enzymes were the same on the basis of pape...

  15. Utilization of glyphosate as phosphate source: biochemistry and genetics of bacterial carbon-phosphorous lyase

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Zechel, David L; Jochimsen, Bjarne

    2014-01-01

    of a large group of chemicals, phosphonic acids or phosphonates, which are characterized by a carbon-phosphorus bond. This is in contrast to the general phosphorus compounds utilized and metabolized by microorganisms. Here phosphorus is found as phosphoric acid or phosphate ion, phosphoric acid esters......, or phosphoric acid anhydrides. The latter compounds contain phosphorus that is bound only to oxygen. Hydrolytic, oxidative, and radical-based mechanisms for carbon-phosphorus bond cleavage have been described. This review deals with the radical-based mechanism employed by the carbon-phosphorus lyase...

  16. Identification and Functional Analysis of the Gene Encoding Methionine-γ-Lyase in Brevibacterium linens

    OpenAIRE

    Amarita, Felix; Yvon, Mireille; Nardi, Michele; Chambellon, Emilie; Delettre, Jerôme; Bonnarme, Pascal

    2004-01-01

    The enzymatic degradation of l-methionine and subsequent formation of volatile sulfur compounds (VSCs) is believed to be essential for flavor development in cheese. l-Methionine-γ-lyase (MGL) can convert l-methionine to methanethiol (MTL), α-ketobutyrate, and ammonia. The mgl gene encoding MGL was cloned from the type strain Brevibacterium linens ATCC 9175 known to produce copious amounts of MTL and related VSCs. The disruption of the mgl gene, achieved in strain ATCC 9175, resulted in a 62% ...

  17. Purification and Characterization of l-Methionine γ-Lyase from Brevibacterium linens BL2†

    OpenAIRE

    Dias, Benjamin; Weimer, Bart

    1998-01-01

    l-Methionine γ-lyase (EC 4.4.1.11) was purified to homogeneity from Brevibacterium linens BL2, a coryneform bacterium which has been used successfully as an adjunct bacterium to improve the flavor of Cheddar cheese. The enzyme catalyzes the α,γ elimination of methionine to produce methanethiol, α-ketobutyrate, and ammonia. It is a pyridoxal phosphate-dependent enzyme, with a native molecular mass of approximately 170 kDa, consisting of four identical subunits of 43 kDa each. The purified enzy...

  18. Sugar-cane juice induces pectin lyase and polygalacturonase in Penicillium griseoroseum

    OpenAIRE

    Minussi Rosana Cristina; Soares-Ramos Juliana Rocha Lopes; Coelho Jorge Luiz Cavalcante; Silva Daison Olzany

    1998-01-01

    The use of other inducers as substitutes for pectin was studied aiming to reduce the production costs of pectic enzymes. The effects of sugar-cane juice on the production of pectin lyase (PL) and polygalacturonase (PG) by Penicillium griseoroseum were investigated. The fungus was cultured in a mineral medium (pH 6.3) in a rotary shaker (150 rpm) for 48 h at 25oC. Culture media were supplemented with yeast extract and sucrose or sugar-cane juice. Sugar-cane juice added singly to the medium pro...

  19. Synthesis and Posttranslational Regulation of Pyruvate Formate-Lyase in Lactococcus lactis

    OpenAIRE

    Melchiorsen, Claus Rix; Jokumsen, Kirsten Væver; Villadsen, John; Johnsen, Mads G.; Israelsen, Hans; Arnau, José

    2000-01-01

    The enzyme pyruvate formate-lyase (PFL) from Lactococcus lactis was produced in Escherichia coli and purified to obtain anti-PFL antibodies that were shown to be specific for L. lactis PFL. It was demonstrated that activated L. lactis PFL was sensitive to oxygen, as in E. coli, resulting in the cleavage of the PFL polypeptide. The PFL protein level and its in vivo activity and regulation were shown by Western blotting, enzyme-linked immunosorbent assay, and metabolite measurement to be depend...

  20. Purification and characterization of thermostable pectate-lyases from a newly isolated thermophilic bacterium, Thermoanaerobacter italicus sp. nov.

    Science.gov (United States)

    Kozianowski, G; Canganella, F; Rainey, F A; Hippe, H; Antranikian, G

    1997-11-01

    A novel thermophilic spore-forming anaerobic microorganism (strain Ab9) able to grow on citrus pectin and polygalacturonic acid (pectate) was isolated from a thermal spa in Italy. The newly isolated strain grows optimally at 70 degrees C with a growth rate of 0.23 h(-1) with pectin and 0.12 h(-1) with pectate as substrates. Xylan, starch, and glycogen are also utilized as carbon sources and thermoactive xylanolytic (highest activity at 70 degrees - 75 degrees C), amylolytic as well as pullulolytic enzymes (highest activity at 80 degrees - 85 degrees C) are formed. Two thermoactive pectate lyases were isolated from the supernatant of a 300-l culture of isolate Ab9 after growth on citrus pectin. The two enzymes (lyases a and b) were purified to homogeneity by ammonium sulfate treatment, anion exchange chromatography, hydrophobic chromatography and finally by preparative gel electrophoresis. After sodium dodecylsulfate (SDS) gel electrophoresis, lyase a appeared as a single polypeptide with a molecular mass of 135000 Da whereas lyase b consisted of two subunits with molecular masses of 93000 Da and 158000 Da. Both enzymes displayed similar catalytic properties with optimal activity at pH 9.0 and 80 degrees C. The enzymes were very stable at 70 degrees C and at 80 degrees C with a half-life of more than 60 min. The maximal activity of the purified lyases was observed with orange pectate (100%) and pectate-sodium salt (90%), whereas pectin was attacked to a much lesser extent (50%). The Km values of both lyases for pectate and citrus pectin were 0.5 g(-1) and 5.0 g(-1), respectively. After incubation with polygalacturonic acid, mono-, di-, and trigalacturonate were detected as final products. A 2.5-fold increase of activity was obtained when pectate lyases were incubated in the presence of 1 mM Ca2+. The addition of 1 mM ethylenediaminetetraacetic acid (EDTA) resulted in complete inhibition of the enzymes. These heat-stable enzymes represent the first pectate-lyases

  1. [Preparation and properties of isocitrate lyase isoforms from the cotyledons of Glycine max L].

    Science.gov (United States)

    Eprintsev, A T; Diachenko, E V; Lykova, T V; Kuen, C T H; Popov, V N

    2010-01-01

    A four-stage purification procedure including ammonium sulfate precipitation and ion exchange chromatography on DEAE cellulose has been elaborated for isolation of isocitrate lyase (EC 4.1.3.1) isoforms from the cotyledons of soybean Glycine max L. Electrophoretically homogeneous preparations of two forms of the enzyme with specific activity of 5.28 and 5.81 U/mg protein have been obtained. Comparison of physicochemical, kinetic, and regulation characteristics of the isoforms obtained revealed fundamental differences between them. Thus, the isoform that migrated quickly in PAAG had a much lower affinity to isocitrate (K(M) - 50 microM) than the slowly migrating form (K(M) - 16 microM). It has been shown that the conservation of activity of the isoforms obtained depends on the presence of divalent cations (Mn2+ and Mg2+) in the medium. It is suggested to use the isoforms of isocitrate lyase isolated from soybeans for the development of biosensors for biochemical and kinetic assays. PMID:20198926

  2. Expression, purification and crystallization of l-methionine γ-lyase 2 from Entamoeba histolytica

    International Nuclear Information System (INIS)

    l-Methionine γ-lyase 2 from E. histolytica, a key enzyme in sulfur-containing amino-acid degradation in this protozoan parasite, has been crystallized in a form suitable for X-ray structure analysis. l-Methionine γ-lyase (MGL) is considered to be an attractive target for rational drug development because the enzyme is absent in mammalian hosts. To enable structure-based design of drugs targeting MGL, one of the two MGL isoenzymes (EhMGL2) was crystallized in the orthorhombic space group P212121, with unit-cell parameters a = 88.89, b = 102.68, c = 169.87 Å. The crystal diffracted to a resolution of 2.0 Å. The presence of a tetramer in the asymmetric unit (4 × 43.1 kDa) gives a Matthews coefficient of 2.2 Å3 Da−1. The structure was solved by the molecular-replacement method and structure refinement is now in progress

  3. Creation of a S1P Lyase bacterial surrogate for structure-based drug design.

    Science.gov (United States)

    Argiriadi, Maria A; Banach, David; Radziejewska, Elzbieta; Marchie, Susan; DiMauro, Jennifer; Dinges, Jurgen; Dominguez, Eric; Hutchins, Charles; Judge, Russell A; Queeney, Kara; Wallace, Grier; Harris, Christopher M

    2016-05-01

    S1P Lyase (SPL) has been described as a drug target in the treatment of autoimmune diseases. It plays an important role in maintaining intracellular levels of S1P thereby affecting T cell egress from lymphoid tissues. Several groups have already published approaches to inhibit S1P Lyase with small molecules, which in turn increase endogenous S1P concentrations resulting in immunosuppression. The use of structural biology has previously aided SPL inhibitor design. Novel construct design is at times necessary to provide a reagent for protein crystallography. Here we present a chimeric bacterial protein scaffold used for protein X-ray structures in the presence of early small molecule inhibitors. Mutations were introduced to the bacterial SPL from Symbiobacterium thermophilum which mimic the human enzyme. As a result, two mutant StSPL crystal structures resolved to 2.8Å and 2.2Å resolutions were solved and provide initial structural hypotheses for an isoxazole chemical series, whose optimization is discussed in the accompanying paper. PMID:27013389

  4. Expression, purification and crystallization of l-methionine γ-lyase 2 from Entamoeba histolytica

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Dan [Department of Parasitology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511 (Japan); Yamagata, Wataru; Kamei, Kaeko [Graduate School of Science and Technology, Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Nozaki, Tomoyoshi [Department of Parasitology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511 (Japan); Harada, Shigeharu, E-mail: harada@kit.ac.jp [Graduate School of Science and Technology, Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Department of Parasitology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511 (Japan)

    2006-10-01

    l-Methionine γ-lyase 2 from E. histolytica, a key enzyme in sulfur-containing amino-acid degradation in this protozoan parasite, has been crystallized in a form suitable for X-ray structure analysis. l-Methionine γ-lyase (MGL) is considered to be an attractive target for rational drug development because the enzyme is absent in mammalian hosts. To enable structure-based design of drugs targeting MGL, one of the two MGL isoenzymes (EhMGL2) was crystallized in the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 88.89, b = 102.68, c = 169.87 Å. The crystal diffracted to a resolution of 2.0 Å. The presence of a tetramer in the asymmetric unit (4 × 43.1 kDa) gives a Matthews coefficient of 2.2 Å{sup 3} Da{sup −1}. The structure was solved by the molecular-replacement method and structure refinement is now in progress.

  5. Structural Basis for Streptogramin B Resistance in Staphylococcus aureus by Virginiamycin B Lyase

    Energy Technology Data Exchange (ETDEWEB)

    Korczynska,M.; Mukhtar, T.; Wright, G.; Berghuis, A.

    2007-01-01

    The streptogramin combination therapy of quinupristin-dalfopristin (Synercid) is used to treat infections caused by bacterial pathogens, such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. However, the effectiveness of this therapy is being compromised because of an increased incidence of streptogramin resistance. One of the clinically observed mechanisms of resistance is enzymatic inactivation of the type B streptogramins, such as quinupristin, by a streptogramin B lyase, i.e., virginiamycin B lyase (Vgb). The enzyme catalyzes the linearization of the cyclic antibiotic via a cleavage that requires a divalent metal ion. Here, we present crystal structures of Vgb from S. aureus in its apoenzyme form and in complex with quinupristin and Mg{sup 2+} at 1.65- and 2.8-{angstrom} resolution, respectively. The fold of the enzyme is that of a seven-bladed {beta}-propeller, although the sequence reveals no similarity to other known members of this structural family. Quinupristin binds to a large depression on the surface of the enzyme, where it predominantly forms van der Waals interactions. Validated by site-directed mutagenesis studies, a reaction mechanism is proposed in which the initial abstraction of a proton is facilitated by a Mg{sup 2+}-linked conjugated system. Analysis of the Vgb-quinupristin structure and comparison with the complex between quinupristin and its natural target, the 50S ribosomal subunit, reveals features that can be exploited for developing streptogramins that are impervious to Vgb-mediated resistance.

  6. Characterization and differential expression analysis of artichoke phenylalanine ammonia-lyase-coding sequences.

    Science.gov (United States)

    De Paolis, Angelo; Pignone, Domenico; Morgese, Anita; Sonnante, Gabriella

    2008-01-01

    Sequences encoding phenylalanine ammonia-lyase were isolated from artichoke, by using a sequence homology strategy, by screening a genomic library and by 3'-rapid amplification of cDNA end (RACE) technology. These analyses and Southern blots suggested that, in artichoke, phenylalanine ammonia-lyase (PAL) is encoded by a small gene family. The sequences isolated from genomic DNA possess two exons and one intron at the conserved position as in most plant pal characterized to date. The 3'-RACE analysis also indicated that each member of the artichoke pal gene family was present as a pool of transcripts, different in the length of 3'-untranslated region. The deduced amino acid sequences were highly similar to those of PAL from lettuce and sunflower. One of the artichoke pal genes was completely sequenced, and its 5' upstream region contained TATA, CAAT box and cis regulatory elements identified in other phenylpropanoid pathway genes as playing a role in UV and elicitor induction. The expression of three of the identified artichoke pal sequences was evaluated in different plant parts, in developmental stages and after wounding, using gene-specific primers/probe combinations in real-time polymerase chain reaction assays. The three putative genes were differentially expressed in the plant parts analysed and were developmentally regulated. Moreover, after leaf mechanical injury, all of them were differentially regulated. The possible involvement of the single pal genes in different physiological processes is discussed. PMID:18251868

  7. Structural and biochemical characterization of the bilin lyase CpcS from Thermosynechococcus elongatus.

    Science.gov (United States)

    Kronfel, Christina M; Kuzin, Alexandre P; Forouhar, Farhad; Biswas, Avijit; Su, Min; Lew, Scott; Seetharaman, Jayaraman; Xiao, Rong; Everett, John K; Ma, Li-Chung; Acton, Thomas B; Montelione, Gaetano T; Hunt, John F; Paul, Corry E C; Dragomani, Tierna M; Boutaghou, M Nazim; Cole, Richard B; Riml, Christian; Alvey, Richard M; Bryant, Donald A; Schluchter, Wendy M

    2013-12-01

    Cyanobacterial phycobiliproteins have evolved to capture light energy over most of the visible spectrum due to their bilin chromophores, which are linear tetrapyrroles that have been covalently attached by enzymes called bilin lyases. We report here the crystal structure of a bilin lyase of the CpcS family from Thermosynechococcus elongatus (TeCpcS-III). TeCpcS-III is a 10-stranded β barrel with two alpha helices and belongs to the lipocalin structural family. TeCpcS-III catalyzes both cognate as well as noncognate bilin attachment to a variety of phycobiliprotein subunits. TeCpcS-III ligates phycocyanobilin, phycoerythrobilin, and phytochromobilin to the alpha and beta subunits of allophycocyanin and to the beta subunit of phycocyanin at the Cys82-equivalent position in all cases. The active form of TeCpcS-III is a dimer, which is consistent with the structure observed in the crystal. With the use of the UnaG protein and its association with bilirubin as a guide, a model for the association between the native substrate, phycocyanobilin, and TeCpcS was produced. PMID:24215428

  8. Sugar-cane juice induces pectin lyase and polygalacturonase in Penicillium griseoroseum

    Directory of Open Access Journals (Sweden)

    Minussi Rosana Cristina

    1998-01-01

    Full Text Available The use of other inducers as substitutes for pectin was studied aiming to reduce the production costs of pectic enzymes. The effects of sugar-cane juice on the production of pectin lyase (PL and polygalacturonase (PG by Penicillium griseoroseum were investigated. The fungus was cultured in a mineral medium (pH 6.3 in a rotary shaker (150 rpm for 48 h at 25oC. Culture media were supplemented with yeast extract and sucrose or sugar-cane juice. Sugar-cane juice added singly to the medium promoted higher PL activity and mycelial dry weight when compared to pectin and the use of sugar-cane juice and yeast extract yielded levels of PG activity that were similar to those obtained with sucrose-yeast extract or pectin. The results indicated that, even at low concentrations, sugar-cane juice was capable of inducing pectin lyase and polygalacturonase with no cellulase activity in P. griseoroseum.

  9. Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy.

    Science.gov (United States)

    Rolando, Monica; Escoll, Pedro; Nora, Tamara; Botti, Joëlle; Boitez, Valérie; Bedia, Carmen; Daniels, Craig; Abraham, Gilu; Stogios, Peter J; Skarina, Tatiana; Christophe, Charlotte; Dervins-Ravault, Delphine; Cazalet, Christel; Hilbi, Hubert; Rupasinghe, Thusitha W T; Tull, Dedreia; McConville, Malcolm J; Ong, Sze Ying; Hartland, Elizabeth L; Codogno, Patrice; Levade, Thierry; Naderer, Thomas; Savchenko, Alexei; Buchrieser, Carmen

    2016-02-16

    Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen's Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis. PMID:26831115

  10. Molecular and Functional Analyses of the metC Gene of Lactococcus lactis, Encoding Cystathionine β-Lyase

    NARCIS (Netherlands)

    Fernández, María; Doesburg, Wim van; Rutten, Ger A.M.; Marugg, Joey D.; Alting, Arno C.; Kranenburg, Richard van; Kuipers, Oscar P.

    2000-01-01

    The enzymatic degradation of amino acids in cheese is believed to generate aroma compounds and therefore to be essential for flavor development. Cystathionine β-lyase (CBL) can convert cystathionine to homocysteine but is also able to catalyze an α,γ elimination. With methionine as a substrate, it p

  11. Purification and characterization of a novel UV lesion-specific DNA glycosylase/AP lyase from Bacillus sphaericus.

    Science.gov (United States)

    Vasquez, D A; Nyaga, S G; Lloyd, R S

    2000-05-31

    The purification and characterization of a pyrimidine dimer-specific glycosylase/AP lyase from Bacillus sphaericus (Bsp-pdg) are reported. Bsp-pdg is highly specific for DNA containing the cis-syn cyclobutane pyrimidine dimer, displaying no detectable activity on oligonucleotides with trans-syn I, trans-syn II, (6-4), or Dewar photoproducts. Like other glycosylase/AP lyases that sequentially cleave the N--glycosyl bond of the 5' pyrimidine of a cyclobutane pyrimidine dimer, and the phosphodiester backbone, this enzyme appears to utilize a primary amine as the attacking nucleophile. The formation of a covalent enzyme-DNA imino intermediate is evidenced by the ability to trap this protein-DNA complex by reduction with sodium borohydride. Also consistent with its AP lyase activity, Bsp-pdg was shown to incise an AP site-containing oligonucleotide, yielding beta- and delta-elimination products. N-terminal amino acid sequence analysis of this 26 kDa protein revealed little amino acid homology to any previously reported protein. This is the first report of a glycosylase/AP lyase enzyme from Bacillus sphaericus that is specific for cis-syn pyrimidine dimers. PMID:10844244

  12. Vertical and temporal variability of DMSP lyase activity in a coccolithophorid bloom in the northern North Sea

    Science.gov (United States)

    Steinke, Michael; Malin, Gill; Gibb, Stuart W.; Burkill, Peter H.

    The climatically relevant trace gas dimethyl sulphide (DMS) is produced within the microbial food-web from the algal metabolite dimethylsulphoniopropionate (DMSP). The presence of DMSP lyase isozymes is necessary for this process. Measurements of in vitro DMSP lyase activity (DLA) were conducted in the northern North Sea in June 1999 in order to investigate the vertical and temporal variability of activity in a Lagrangian time-series process study. DLA ranged from 4 to 207 nM h -1, with maximum values close to the surface and between 30 and 50 m depth. DLA increased towards the surface relative to chlorophyll a, as did the non-photosynthetic but photoprotective pigment diadinoxanthin, DMS and dissolved dimethylsulphoxide, a likely oxidation product of DMS. These observations support the hypothesis that DMSP lyases can be affected by irradiance levels, and that DMSP and its cleavage products could be involved in scavenging oxygen radicals; hence, they may function as antioxidants in marine algae. Linear regression analysis of our field data showed reduced biomass of some oligotrich and non-oligotrich ciliates at higher levels of DLA, a finding that could be supportive of a role for phytoplankton DMSP lyases in chemical defence.

  13. Purification and Characterization of a Unique Pectin Lyase from Aspergillus giganteus Able to Release Unsaturated Monogalacturonate during Pectin Degradation.

    Science.gov (United States)

    Pedrolli, Danielle Biscaro; Carmona, Eleonora Cano

    2014-01-01

    A pectin lyase, named PLIII, was purified to homogeneity from the culture filtrate of Aspergillus giganteus grown in submerged culture containing orange peel waste as carbon source. PLIII was able to digest apple pectin and citrus pectins with different degrees of methyl esterification. Interestingly, the PLIII activity was stimulated in the presence of some divalent cations including Pb(2+) and was not significantly affected by Hg(2+). Like other pectin lyases, PLIII is stimulated by but is not dependent on Ca(2+). The main soluble product released during the degradation of pectic substances promoted by the PLIII is compatible with an unsaturated monogalacturonate. PLIII is a unique enzyme able to release unsaturated monogalacturonate as the only soluble product during the degradation of pectic substances; therefore, PLIII was classified as an exo-pectin lyase. To our knowledge, this is the first characterization of an exo-pectin lyase. The PLIII described in this work is potentially useful for ethanol production from pectin-rich biomass, besides other common applications for alkaline pectinases like preparation of textile fibers, coffee and tea fermentation, vegetable oil extraction, and the treatment of pulp in papermaking. PMID:25610636

  14. Identification, expression, and characterization of a novel bacterial RGI Lyase enzyme for the production of bio-functional fibers

    DEFF Research Database (Denmark)

    da Silva, Ines Isabel Cardoso Rodrigues; Larsen, Dorte Møller; Meyer, Anne S.;

    2011-01-01

    A gene encoding a putative rhamnogalacturonan I (RGI) Lyase (EC 4.2.2.-) from Bacillus licheniformis (DSM13) was selected after a homology search and phylogenetic analysis and optimized with respect to codon usage. The designed gene was transformed into Pichia pastoris and the enzyme was produced...... in the eukaryotic host with a high titer in a 5l bioreactor. The RGI Lyase was purified by Cu2+ affinity chromatography and 1.1g pure enzyme was achieved pr. L. When the denatured protein was deglycosylated with EndoH, the molecular weight of the protein decreased to 65kDa, which correlated with the...... predicted molecular weight of the mature RGI Lyase of 596 amino acids. By use of a statistical design approach, with potato rhamnogalacturonan as the substrate, the optimal reaction conditions for the RGI Lyase were established to be: 61°C, pH 8.1, and 2mM of both Ca2+ and Mn2+ (specific activity 18.4U...

  15. Improvement of enantioselectivity of the B-type halohydrin hydrogen-halide-lyase from Corynebacterium sp. N-1074.

    Science.gov (United States)

    Watanabe, Fumiaki; Yu, Fujio; Ohtaki, Akashi; Yamanaka, Yasuaki; Noguchi, Keiichi; Odaka, Masafumi; Yohda, Masafumi

    2016-09-01

    Halohydrin hydrogen-halide-lyase (H-Lyase) is a bacterial enzyme involved in the degradation of halohydrins. This enzyme catalyzes the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins, producing the corresponding epoxides. The H-Lyases have been classified into A, B and C subtypes based on amino acid sequence similarities. These enzymes have attracted much attention as industrial catalysts in the synthesis of chiral chemicals from prochiral halohydrins. In the present study, we constructed mutants of B-type H-Lyase from Corynebacterium sp. N-1074 (HheB) displaying higher enantioselectivity by structure-based site-directed mutagenesis and random mutagenesis. A triple mutant of HheB exhibited 98.5% enantioselectivity, the highest ever reported, toward (R)-4-chloro-3-hydroxy-butyronitrile production, with the yield reaching approximately two-fold that of the wild-type enzyme. We discuss the structural basis of the high enantioselectivity and productivity of the mutant by comparing the crystal structures of the mutant HheB and the wild-type enzyme in complex with or without the substrate analogue. PMID:27215832

  16. C12 derivatives of the hydroperoxide lyase pathway are produced by product recycling through lipoxygenase-2 in Nicotiana attenuata leaves

    NARCIS (Netherlands)

    M. Kallenbach; P.A. Gilardoni; S. Allmann; I.T. Baldwin; G. Bonaventure

    2011-01-01

    In response to diverse stresses, the hydroperoxide lyase (HPL) pathway produces C(6) aldehydes and 12-oxo-(9Z )-dodecenoic acid ((9Z )-traumatin). Since the original characterization of (10E )-traumatin and traumatic acid, little has been added to our knowledge of the metabolism and fluxes associate

  17. Structure of PhnP: a phosphodiesterase of the carbon-phosphorous lyase pathway for phosphonate degradation

    DEFF Research Database (Denmark)

    Podzelinska, Kateryna; He, Shu-Mei; Wathier, Matthew;

    2009-01-01

    Carbon-phosphorus lyase is a multienzyme system encoded by the phn operon that enables bacteria to metabolize organophosphonates when the preferred nutrient, inorganic phosphate, is scarce. One of the enzymes encoded by this operon, PhnP, is predicted by sequence homology to be a metal-dependent ...

  18. Design of thermostable rhamnogalacturonan lyase mutants from Bacillus licheniformis by combination of targeted single point mutations

    DEFF Research Database (Denmark)

    da Silva, Ines Isabel Cardoso Rodrigues; Jers, Carsten; Otten, Harm; Nyffenegger, Christian; Larsen, Dorte Møller; Derkx, Patrick M. F.; Meyer, Anne S.; Mikkelsen, Jørn Dalgaard; Larsen, Sine

    2014-01-01

    the wild-type RGI lyase in Bacillus subtilis as opposed to in Pichia pastoris; this effect is suggested to be a negative result of glycosylation of the P. pastoris expressed enzyme. A ~ twofold improvement in thermal stability at 60 °C, accompanied by less significant increases in Tm of the enzyme...

  19. A 5-methylcytosine DNA glycosylase/lyase demethylates the retrotransposon Tos17 and promotes its transposition in rice

    KAUST Repository

    La, Honggui

    2011-09-06

    DNA 5-methylcytosine (5-meC) is an important epigenetic mark for transcriptional gene silencing in many eukaryotes. In Arabidopsis, 5-meC DNA glycosylase/lyases actively remove 5-meC to counter-act transcriptional gene silencing in a locus-specific manner, and have been suggested to maintain the expression of transposons. However, it is unclear whether plant DNA demethylases can promote the transposition of transposons. Here we report the functional characterization of the DNA glycosylase/lyase DNG701 in rice. DNG701 encodes a large (1,812 amino acid residues) DNA glycosylase domain protein. Recombinant DNG701 protein showed 5-meC DNA glycosylase and lyase activities in vitro. Knockout or knockdown of DNG701 in rice plants led to DNA hypermethylation and reduced expression of the retrotransposon Tos17. Tos17 showed less transposition in calli derived from dng701 knockout mutant seeds compared with that in wild-type calli. Overexpression of DNG701 in both rice calli and transgenic plants substantially reduced DNA methylation levels of Tos17 and enhanced its expression. The overexpression also led to more frequent transposition of Tos17 in calli. Our results demonstrate that rice DNG701 is a 5-meC DNA glycosylase/lyase responsible for the demethylation of Tos17 and this DNA demethylase plays a critical role in promoting Tos17 transposition in rice calli.

  20. Five phosphonate operon gene products as components of a multi-subunit complex of the carbon-phosphorus lyase pathway

    DEFF Research Database (Denmark)

    Jochimsen, Bjarne; Lolle, Signe; McSorley, Fern R.;

    2011-01-01

    Organophosphonate utilization by Escherichia coli requires the 14 cistrons of the phnCDEFGHIJKLMNOP operon, of which the carbon-phosphorus lyase has been postulated to consist of the seven polypeptides specified by phnG to phnM. A 5,660-bp DNA fragment encompassing phnGHIJKLM is cloned, followed by...

  1. Possible regulatory role of phenylalanine ammonia-lyase in the production of anthocyanins in asparagus (Asparagus officinalis L)

    NARCIS (Netherlands)

    Flores, F.B.; Oosterhaven, J.; Martinez-Madrid, M.C.; Romojaro, F.

    2005-01-01

    The regulatory role of phenylalanine ammonia-lyase (PAL) in the light-induced accumulation of anthocyanins in the epidermis of asparagus spears has been analysed. A correlation between the stimulation of PAL activity and the rise in total anthocyanin content has been observed. Light radiation induce

  2. Activities of methionine-γ-lyase in the acidophilic archaeon “Ferroplasma acidarmanus” strain fer1

    Directory of Open Access Journals (Sweden)

    Khan MA

    2013-04-01

    Full Text Available M A Khan,1 Madeline M López-Muñoz,2 Charles W Kaspar,3 Kai F Hung1 1Department of Biological Sciences, Eastern Illinois University, Charleston, IL, USA; 2Department of Biology, Universidad de Puerto Rico, Mayaguez, Puerto Rico; 3Bacteriology Department, University of Wisconsin, Madison, WI, USA Abstract: Biogeochemical processes on exposed pyrite ores result in extremely high levels of sulfuric acid at these locations. Acidophiles that thrive in these conditions must overcome significant challenges, including an environment with proton concentrations at pH 3 or below. The role of sulfur metabolism in the archaeon “Ferroplasma acidarmanus” strain fer1's ability to thrive in this environment was investigated due to its growth-dependent production of methanethiol, a volatile organic sulfur compound. Two putative sequences for methionine-γ-lyase (EC 4.4.1.11, an enzyme known to carry out α, γ-elimination on L-methionine to produce methanethiol, were identified in fer1. Bioinformatic analyses identified a conserved pyridoxal-5'-phosphate (PLP binding domain and a partially conserved catalytic domain in both putative sequences. Detection of PLP-dependent and L-methionine-dependent production of α-keto compounds and thiol groups in fer1 confirmed the presence of methionine-γ-lyase activity. Further, fer1 lysate was capable of processing related substrates, including D-methionine, L-cysteine, L-cystathionine, and L/D-homocysteine. When the two putative fer1 methionine-γ-lyase gene-coded proteins were expressed in Escherichia coli cells, one sequence demonstrated an ability to carry out α, γ-elimination activity, while the other exhibited γ-replacement activity. These fer1 methionine-γ-lyases also exhibited optimum pH, substrate specificity, and catalytic preferences that are different from methionine-γ-lyases from other organisms. These differences are discussed in the context of molecular phylogeny constructed using a maximum

  3. Inhibition of the cystathionine-γ-lyase/hydrogen sulfide pathway in rat vascular smooth muscle cells by cobalt-60 gamma radiation

    Institute of Scientific and Technical Information of China (English)

    ZHONG Guang-zhen; YANG Xin-chun; JIA Li-ping; CHEN Feng-rong; CUI Ming

    2009-01-01

    Background Radiation is a promising treatment for in stent restenosis and restenosis following percutaneous transluminal coronary angioplasty, which has troubled interventional cardiologists for a long time. It inhibits neointima hyperplasia, vascular remodeling, and increases the mean luminal diameter. The mechanism of intracoronary brachytherapy for restenosis is not well understood. Endogenous gaseous transmitters including nitric oxide and carbon monoxide are closely related to restenosis. Hydrogen sulfide, a new endogenous gaseous transmitter, is able to inhibit the proliferation of vascular smooth muscle cells and vascular remodeling. This study aimed to clarify the effect of radiation on cystathionine-y-lyase/hydrogen sulfide pathway in rat smooth muscle cells.Methods We studied the effect of radiation on the cystathionine-γ-lyase/hydrogen sulfide pathway. Rat vascular smooth muscle cells were radiated with 60Co y at doses of 14 Gy and 25 Gy respectively. Then the mRNA level of cystathionine-γ-lyase was studied by quantitative reverse-transcription competitive polymerase chain reaction. Hydrogen sulfide concentration in culture medium was determined by methylene blue spectrophotometry. Cystathionine-γ-lyase activity in vascular smooth muscle cells was also studied.Results 60Co y radiation at a dose of 1 Gy did not affect the cystathionine-γ-lyase/hydrogen sulfide pathway significantly. However, 60Co y radiation at doses of 14 Gy and 25 Gy decreased the hydrogen sulfide synthesis by 21.9% (P <0.05) and 26.8% (P <0.01 ) respectively. At the same time, they decreased the cystathionine-γ-lyase activity by 15.1% (P <0.05) and 20.5% (P <0.01) respectively, and cystathionine-γ-lyase mRNA expression by 29.3% (P <0.01 ) and 38.2% (P <0.01) respectively.Conclusion Appropriate 60Co γ radiation inhibits the H2S synthesis by inhibiting the gene expression of cystathionine-γ-lyase and the cystathionine-y-lyase activity.

  4. Biochemical, Kinetic, and Spectroscopic Characterization of Ruegeria pomeroyi DddW--A Mononuclear Iron-Dependent DMSP Lyase.

    Directory of Open Access Journals (Sweden)

    Adam E Brummett

    Full Text Available The osmolyte dimethylsulfoniopropionate (DMSP is a key nutrient in marine environments and its catabolism by bacteria through enzymes known as DMSP lyases generates dimethylsulfide (DMS, a gas of importance in climate regulation, the sulfur cycle, and signaling to higher organisms. Despite the environmental significance of DMSP lyases, little is known about how they function at the mechanistic level. In this study we biochemically characterize DddW, a DMSP lyase from the model roseobacter Ruegeria pomeroyi DSS-3. DddW is a 16.9 kDa enzyme that contains a C-terminal cupin domain and liberates acrylate, a proton, and DMS from the DMSP substrate. Our studies show that as-purified DddW is a metalloenzyme, like the DddQ and DddP DMSP lyases, but contains an iron cofactor. The metal cofactor is essential for DddW DMSP lyase activity since addition of the metal chelator EDTA abolishes its enzymatic activity, as do substitution mutations of key metal-binding residues in the cupin motif (His81, His83, Glu87, and His121. Measurements of metal binding affinity and catalytic activity indicate that Fe(II is most likely the preferred catalytic metal ion with a nanomolar binding affinity. Stoichiometry studies suggest DddW requires one Fe(II per monomer. Electronic absorption and electron paramagnetic resonance (EPR studies show an interaction between NO and Fe(II-DddW, with NO binding to the EPR silent Fe(II site giving rise to an EPR active species (g = 4.29, 3.95, 2.00. The change in the rhombicity of the EPR signal is observed in the presence of DMSP, indicating that substrate binds to the iron site without displacing bound NO. This work provides insight into the mechanism of DMSP cleavage catalyzed by DddW.

  5. Active site proton delivery and the lyase activity of human CYP17A1

    Energy Technology Data Exchange (ETDEWEB)

    Khatri, Yogan; Gregory, Michael C.; Grinkova, Yelena V.; Denisov, Ilia G.; Sligar, Stephen G., E-mail: s-sligar@illinois.edu

    2014-01-03

    Highlights: •The disruption of PREG/PROG hydroxylation activity by T306A showed the participation of Cpd I. •T306A supports the involvement of a nucleophilic peroxo-anion during lyase activity. •The presence of cytochrome b{sub 5} augments C–C lyase activity. •Δ5-Steroids are preferred substrates for CYP17 catalysis. -- Abstract: Cytochrome P450 CYP17A1 catalyzes a series of reactions that lie at the intersection of corticoid and androgen biosynthesis and thus occupies an essential role in steroid hormone metabolism. This multifunctional enzyme catalyzes the 17α-hydroxylation of Δ4- and Δ5-steroids progesterone and pregnenolone to form the corresponding 17α-hydroxy products through its hydroxylase activity, and a subsequent 17,20-carbon–carbon scission of pregnene-side chain produce the androgens androstenedione (AD) and dehydroepiandrosterone (DHEA). While the former hydroxylation reaction is believed to proceed through a conventional “Compound I” rebound mechanism, it has been suggested that the latter carbon cleavage is initiated by an iron-peroxy intermediate. We report on the role of Thr306 in CYP17 catalysis. Thr306 is a member of the conserved acid/alcohol pair thought to be essential for the efficient delivery of protons required for hydroperoxoanion heterolysis and formation of Compound I in the cytochromes P450. Wild type and T306A CYP17A1 self-assembled in Nanodiscs were used to quantitate turnover and coupling efficiencies of CYP17’s physiological Δ4- and Δ5-substrates. We observed that T306A co-incorporated in Nanodiscs with its redox partner cytochrome P450 oxidoreductase, coupled NADPH only by 0.9% and 0.7% compared to the wild type (97% and 22%) during the conversion of pregnenolone and progesterone, respectively, to the corresponding 17-OH products. Despite increased oxidation of pyridine nucleotide, hydroxylase activity was drastically diminished in the T306A mutant, suggesting a high degree of uncoupling in which reducing

  6. Active site proton delivery and the lyase activity of human CYP17A1

    International Nuclear Information System (INIS)

    Highlights: •The disruption of PREG/PROG hydroxylation activity by T306A showed the participation of Cpd I. •T306A supports the involvement of a nucleophilic peroxo-anion during lyase activity. •The presence of cytochrome b5 augments C–C lyase activity. •Δ5-Steroids are preferred substrates for CYP17 catalysis. -- Abstract: Cytochrome P450 CYP17A1 catalyzes a series of reactions that lie at the intersection of corticoid and androgen biosynthesis and thus occupies an essential role in steroid hormone metabolism. This multifunctional enzyme catalyzes the 17α-hydroxylation of Δ4- and Δ5-steroids progesterone and pregnenolone to form the corresponding 17α-hydroxy products through its hydroxylase activity, and a subsequent 17,20-carbon–carbon scission of pregnene-side chain produce the androgens androstenedione (AD) and dehydroepiandrosterone (DHEA). While the former hydroxylation reaction is believed to proceed through a conventional “Compound I” rebound mechanism, it has been suggested that the latter carbon cleavage is initiated by an iron-peroxy intermediate. We report on the role of Thr306 in CYP17 catalysis. Thr306 is a member of the conserved acid/alcohol pair thought to be essential for the efficient delivery of protons required for hydroperoxoanion heterolysis and formation of Compound I in the cytochromes P450. Wild type and T306A CYP17A1 self-assembled in Nanodiscs were used to quantitate turnover and coupling efficiencies of CYP17’s physiological Δ4- and Δ5-substrates. We observed that T306A co-incorporated in Nanodiscs with its redox partner cytochrome P450 oxidoreductase, coupled NADPH only by 0.9% and 0.7% compared to the wild type (97% and 22%) during the conversion of pregnenolone and progesterone, respectively, to the corresponding 17-OH products. Despite increased oxidation of pyridine nucleotide, hydroxylase activity was drastically diminished in the T306A mutant, suggesting a high degree of uncoupling in which reducing

  7. 莱钢顶底复吹转炉碳氧积稳定控制技术研究%Research of Carbon Oxygen Product Stability Control Technology on the Long Life Converter in Laiwu Steel

    Institute of Scientific and Technical Information of China (English)

    高志滨; 温福新; 于亮涛

    2015-01-01

    Through improving the design of bottom blowing air brick process, developing new type of converter slag splashing technology, optimization intelligent of converter steelmaking and bottom blowing model, to realize the combined-blowing converter low carbon oxygen deposition and stability control at a lower level. Using a laser thickness gauge to monitor the furnace type to guarantee the stability of furnace type, prevent the carbon oxygen volume fluctuations and using the static decarburization technology to further reduce carbon and oxygen. After the process implementation, the carbon oxygen volumes of the No.1, No.2 or No.3 converters were controlled in 0.002 5 and the carbon oxygen product of No.4 converter for dephosphorization was controlled in 0.002 3;The average total iron content of the final slag of 4 converters was reduced to 12.83%from 14.38%and the average oxygen content in molten steel at the end of blowing was decreased to 450 × 10-6 from 520 × 10-6. The direct economic benefit of oxygen carbon product stability control technology for long life of converter is 23.9 million Yuan.%通过改进底吹透气砖工艺设计、开发新型转炉溅渣工艺、优化转炉智能炼钢及底吹模型等,实现复吹转炉碳氧积的降低并稳定控制在较低水平;利用激光测厚仪对炉型进行监控,确保炉型稳定,防止碳氧积波动;利用静止脱碳技术进一步降低碳氧积。工艺实施后,1#~3#120 t转炉碳氧积控制在0.0025,4#脱磷炉碳氧积控制在0.0023;4座转炉终渣平均全铁含量由14.38%降为12.83%,吹炼终点钢水氧含量平均由520×10-6降为450×10-6,长寿转炉碳氧积稳定控制技术年直接经济效益2390.04万元。

  8. Sphingosine 1-phosphate lyase enzyme assay using a BODIPY-labeled substrate

    International Nuclear Information System (INIS)

    Sphingosine 1-phosphate lyase (SPL) is responsible for the irreversible catabolism of sphingosine 1-phosphate, which signals through five membrane receptors to mediate cell stress responses, angiogenesis, and lymphocyte trafficking. The standard assay for SPL activity utilizes a radioactive dihydrosphingosine 1-phosphate substrate and is expensive and cumbersome. In this study, we describe an SPL assay that employs an ω-labeled BODIPY-sphingosine 1-phosphate substrate, allowing fluorescent product detection by HPLC and incorporating advantages of the BODIPY fluorophore. The major aldehyde product is confirmed by reaction with 2,4-dinitrophenylhydrazine. The SPL-catalyzed reaction is linear over a 30 min time period and yields a Km of 35 μM for BODIPY-sphingosine 1-phosphate.

  9. Determining the extent of heparan sulfate depolymerisation following heparin lyase treatment.

    Science.gov (United States)

    Carnachan, Susan M; Bell, Tracey J; Sims, Ian M; Smith, Raymond A A; Nurcombe, Victor; Cool, Simon M; Hinkley, Simon F R

    2016-11-01

    The depolymerisation of porcine mucosal heparan sulfate under the action of heparin lyases and analysis by size-exclusion chromatography (SEC) is described. Heparan sulfate treated to enzymic bond scission producing a Δ4,5 double-bond and quantified by SEC with ultraviolet-visible (UV) spectroscopic detection (230nm) indicated that the majority of the biopolymer (>85%) was reduced to disaccharides (degree of polymerisation (DP)=2). However, analysis of the SEC eluant using refractive index (RI), which reflects the mass contribution of the oligosaccharides rather than the molar response of a UV chromophore, indicated that a considerable proportion of the digested HS, up to 43%, was present with DP >2. This was supported by a mass balance analysis. These results contradict the accepted literature where "complete digestion" is routinely reported. Herein we report on the composition and methodology utilised to ascertain the extent of depolymerization and disaccharide composition of this important biopolymer. PMID:27516308

  10. An acidic pectin lyase from Aspergillus niger with favourable efficiency in fruit juice clarification.

    Science.gov (United States)

    Xu, S X; Qin, X; Liu, B; Zhang, D Q; Zhang, W; Wu, K; Zhang, Y H

    2015-02-01

    The pectin lyase gene pnl-zj5a from Aspergillus niger ZJ5 was identified and expressed in Pichia pastoris. PNL-ZJ5A was purified by ultrafiltration, anion exchange and gel chromatography. The Km and Vmax values determined using citrus pectin were 0.66 mg ml(-1) and 32.6 μmol min(-1) mg(-1) , respectively. PNL-ZJ5A exhibited optimal activity at 43°C and retained activity over 25-50°C. PNL-ZJ5A was optimally active at pH 5 and effective in apple juice clarification. Compared with controls, PNL-ZJ5A increased the fruit juice yield significantly. Furthermore, PNL-ZJ5A reduced the viscosity of apple juice by 38.8% and increased its transmittance by 86.3%. PNL-ZJ5A combined with a commercial pectin esterase resulted in higher juice volume. PMID:25382689

  11. Intermediate binding of phycocyanobilin to the lyase, CpeS1, and transfer to apoprotein.

    Science.gov (United States)

    Tu, Jun-Ming; Kupka, Michaela; Böhm, Stephan; Plöscher, Matthias; Eichacker, Lutz; Zhao, Kai-Hong; Scheer, Hugo

    2008-01-01

    The phycobilin: Cysteine-84-phycobiliprotein lyase, CpeS1, catalyzes phycocyanobilin (PCB) and phycoerythrobilin attachment to nearly all cysteine-84 (consensus sequence) binding sites of phycoerythrin, phycoerythrocyanin, phycocyanin and allophycocyanin (Zhao et al. (2007) Proc Natl Acad Sci 104:14300-14305). We now show that CpeS1 can bind PCB, as assayed by Ni(2+) chelating affinity chromatography. Binding is rapid, and the chromophore is bound in an extended conformation similar to that in phycobiliproteins but only poorly fluorescent. Upon addition of apo-biliproteins, the chromophore is transferred to the latter much slower ( approximately 1 h), indicating that chromophorylated CpeS1 is an intermediate in the enzymatic reaction. In addition, imidazole is bound to PCB, as shown by mass spectroscopy of tryptic digests of the intermediate CpeS1-PCB complex. PMID:17912606

  12. Crystallization and preliminary X-ray analysis of argininosuccinate lyase from Streptococcus mutans

    Science.gov (United States)

    Cao, Yan-Li; Li, Gui-Lan; Wang, Kai-Tuo; Zhang, Hong-Yin; Li, Lan-Fen

    2011-01-01

    Argininosuccinate lyase (ASL) is an important enzyme in arginine synthesis and the urea cycle, which are highly conserved from bacteria to eukaryotes. The gene encoding Streptococcus mutans ASL (smASL) was amplified and cloned into expression vector pET28a. The recombinant smASL protein was expressed in a soluble form in Escherichia coli strain BL21 (DE3) and purified to homogeneity by two-step column chromatography. Crystals suitable for X-ray analysis were obtained and X-ray diffraction data were collected to a resolution of 2.5 Å. The crystals belonged to space group R3, with unit-cell parameters a = b = 254.5, c = 78.3 Å. PMID:21636911

  13. Stress-dependent regulation of 13-lipoxygenases and 13-hydroperoxide lyase in olive fruit mesocarp.

    Science.gov (United States)

    Padilla, María N; Hernández, M Luisa; Sanz, Carlos; Martínez-Rivas, José M

    2014-06-01

    The effect of different environmental stresses on the expression and enzyme activity levels of 13-lipoxygenases (13-LOX) and 13-hydroperoxide lyase (13-HPL) and on the volatile compounds synthesized by their sequential action has been studied in the mesocarp tissue of olive fruit from the Picual and Arbequina cultivars. The results showed that temperature, light, wounding and water regime regulate olive 13-LOXs and 13-HPL genes at transcriptional level. Low temperature and wounding brought about an increase in LOX and HPL enzyme activities. A very slight increase in the total content of six straight-chain carbons (C6) volatile compounds was also observed in the case of low temperature and wounding treatments. The physiological roles of 13-LOXs and 13-HPL in the olive fruit stress response are discussed. PMID:24629805

  14. Mechanistic deductions from kinetic isotope effects and pH studies of pyridoxal phosphate dependent carbon-carbon lyases: Erwinia herbicola and Citrobacter freundii tyrosine phenol-lyase

    International Nuclear Information System (INIS)

    The pH dependence of the kinetic parameters and primary deuterium isotope effects have been determined for tyrosine phenol-lyase from both Erwinia herbicola and Citrobacter freundii. The primary deuterium isotope effects indicate that proton abstraction from the 2-position of the substrate is partially rate-limiting for both enzymes. The C. freundii enzyme primary deuterium isotope effects [DV = 3.5 and D(V/Ktyr) = 2.5] are pH independent, indicating that tyrosine is not sticky (i.e., does not dissociate slower than it reacts to give products). Since Vmax for both tyrosine and the alternate substrate S-methyl-L-cysteine is also pH independent, substrate binds only to the correctly protonated form of the enzyme. For the E. herbicola enzyme, both Vmax and V/K for tyrosine or S-methyl-L-cysteine are pH dependent, as well as both DV and D(V/Ktyr). Thus, while both the protonated and unprotonated enzyme can bind substrate, and may be interconverted directly, only the unprotonated Michaelis complex is catalytically competent. At pH 9.5, DV = 2.5 and D(V/Ktyr) = 1.5. However, at pH 6.4 the isotope effect on both parameters is equal to 4.1. From these data, the forward commitment factor (cf = 5.2) and catalytic ratio (cvf = 1.1) for tyrosine and S-methyl-L-cysteine (cf = 2.2, cvf = 24) are calculated. Also, the Michaelis complex partition ratio (cf/cvf) for substrate and products is calculated to be 4.7 for tyrosine and 0.1 for S-methyl-L-cysteine

  15. Mechanistic deductions from kinetic isotope effects and pH studies of pyridoxal phosphate dependent carbon-carbon lyases: Erwinia herbicola and Citrobacter freundii tyrosine phenol-lyase

    Energy Technology Data Exchange (ETDEWEB)

    Kiick, D.M.; Phillips, R.S.

    1988-09-20

    The pH dependence of the kinetic parameters and primary deuterium isotope effects have been determined for tyrosine phenol-lyase from both Erwinia herbicola and Citrobacter freundii. The primary deuterium isotope effects indicate that proton abstraction from the 2-position of the substrate is partially rate-limiting for both enzymes. The C. freundii enzyme primary deuterium isotope effects (DV = 3.5 and D(V/Ktyr) = 2.5) are pH independent, indicating that tyrosine is not sticky (i.e., does not dissociate slower than it reacts to give products). Since Vmax for both tyrosine and the alternate substrate S-methyl-L-cysteine is also pH independent, substrate binds only to the correctly protonated form of the enzyme. For the E. herbicola enzyme, both Vmax and V/K for tyrosine or S-methyl-L-cysteine are pH dependent, as well as both DV and D(V/Ktyr). Thus, while both the protonated and unprotonated enzyme can bind substrate, and may be interconverted directly, only the unprotonated Michaelis complex is catalytically competent. At pH 9.5, DV = 2.5 and D(V/Ktyr) = 1.5. However, at pH 6.4 the isotope effect on both parameters is equal to 4.1. From these data, the forward commitment factor (cf = 5.2) and catalytic ratio (cvf = 1.1) for tyrosine and S-methyl-L-cysteine (cf = 2.2, cvf = 24) are calculated. Also, the Michaelis complex partition ratio (cf/cvf) for substrate and products is calculated to be 4.7 for tyrosine and 0.1 for S-methyl-L-cysteine.

  16. Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of N-acetylneuraminate lyase from methicillin-resistant Staphylococcus aureus

    International Nuclear Information System (INIS)

    N-Acetylneuraminate lyase, an enzyme involved in the bacterial uptake and metabolism of sialic acid, is a promising target for antibiotic development against pathogenic bacteria. Here, the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of N-acetylneuraminate lyase from methicillin-resistant S. aureus to 1.70 Å resolution are reported. The enzyme N-acetylneuraminate lyase (EC 4.1.3.3) is involved in the metabolism of sialic acids. Specifically, the enzyme catalyzes the retro-aldol cleavage of N-acetylneuraminic acid to form N-acetyl-d-mannosamine and pyruvate. Sialic acids comprise a large family of nine-carbon amino sugars, all of which are derived from the parent compound N-acetylneuraminic acid. In recent years, N-acetylneuraminate lyase has received considerable attention from both mechanistic and structural viewpoints and has been recognized as a potential antimicrobial drug target. The N-acetylneuraminate lyase gene was cloned from methicillin-resistant Staphylococcus aureus genomic DNA, and recombinant protein was expressed and purified from Escherichia coli BL21 (DE3). The enzyme crystallized in a number of crystal forms, predominantly from PEG precipitants, with the best crystal diffracting to beyond 1.70 Å resolution in space group P21. Molecular replacement indicates the presence of eight monomers per asymmetric unit. Understanding the structural biology of N-acetylneuraminate lyase in pathogenic bacteria, such as methicillin-resistant S. aureus, will provide insights for the development of future antimicrobials

  17. Expression, crystallization and preliminary X-ray crystallographic study of ethanolamine ammonia-lyase from Escherichia coli

    International Nuclear Information System (INIS)

    Ethanolamine ammonia-lyase from E. coli has been overexpressed, purified and crystallized. The crystals diffracted to 2.2 Å resolution using synchrotron radiation. Ethanolamine ammonia-lyase (EAL) catalyzes the adenosylcobalamin-dependent conversion of ethanolamine to acetaldehyde and ammonia. The wild-type enzyme shows a very low solubility. N-terminal truncation of the Escherichia coli EAL β-subunit dramatically increases the solubility of the enzyme without altering its catalytic properties. Two deletion mutants of the enzyme [EAL(βΔ4–30) and EAL(βΔ4–43)] have been overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method. Crystals of EAL(βΔ4–30) and EAL(βΔ4–43) diffracted to approximately 8.0 and 2.1 Å resolution, respectively

  18. Degumming of ramie fiber and the production of reducing sugars from waste peels using nanoparticle supplemented pectate lyase.

    Science.gov (United States)

    Mukhopadhyay, Arka; Dutta, Nalok; Chattopadhyay, Dhrubajyoti; Chakrabarti, Krishanu

    2013-06-01

    Banana, citrus and potato peels were subjected to treatment with hydroxyapatite nanoparticle (NP) supplemented purified pectate lyase (NP-PL), isolated from Bacillus megaterium AK2 to produce reducing sugar (RS). At both 50 and 90°C production of RS by NP-PL was almost twofold greater than that by untreated pectate lyase (PL) from each of the three peels. The optimal production of RS from banana and citrus peels were after 24 and 6h of incubation while it was 24 and 4h for potato peels at 50 and 90°C, respectively, on NP-PL treatment. NP-PL could degum raw, decorticated ramie fibers as well as enhance fiber tenacity and fineness. The weight loss of the fibers were 24% and 31% better (compared to PL treatment) after 24 and 48 h of processing. These findings have potential implications for the bio-ethanol, bio-fuel and textile industries. PMID:23587821

  19. The Structure of Chondroitin B Lyase Complexed with Glycosaminoglycan Oligosaccharides Unravels a Calcium-dependent Catalytic Machinery*

    OpenAIRE

    Michel, Gurvan; Pojasek, Kevin; Li, Yunge; Sulea, Traian; Linhardt, Robert J.; Raman, Rahul; Prabhakar, Vikas; Sasisekharan, Ram; Cygler, Miroslaw

    2004-01-01

    Chondroitinase B from Pedobacter heparinus is the only known enzyme strictly specific for dermatan sulfate and is a widely used enzymatic tool for the structural characterization of glycosaminoglycans. This β-helical polysaccharide lyase belongs to family PL-6 and cleaves the β(1,4) linkage of dermatan sulfate in a random manner, yielding 4,5-unsaturated dermatan sulfate disaccharides as the product. The previously reported structure of its complex with a dermatan sulfate disaccharide product...

  20. Molecular and Functional Analyses of the metC Gene of Lactococcus lactis, Encoding Cystathionine β-Lyase

    OpenAIRE

    Fernández, María; Doesburg, Wim van; Rutten, Ger A.M.; Marugg, Joey D.; Alting, Arno C.; van Kranenburg, Richard; Oscar P. Kuipers

    2000-01-01

    The enzymatic degradation of amino acids in cheese is believed to generate aroma compounds and therefore to be essential for flavor development. Cystathionine β-lyase (CBL) can convert cystathionine to homocysteine but is also able to catalyze an α,γ elimination. With methionine as a substrate, it produces volatile sulfur compounds which are important for flavor formation in Gouda cheese. The metC gene, which encodes CBL, was cloned from the Lactococcus lactis model strain MG1363 and from str...

  1. Developmental and environmental regulation of a phenylalanine ammonia-lyase-beta-glucuronidase gene fusion in transgenic tobacco plants.

    OpenAIRE

    Liang, X W; Dron, M; J. Schmid; Dixon, R. A.; Lamb, C J

    1989-01-01

    A 1.1-kilobase promoter fragment of the bean (Phaseolus vulgaris L.) phenylalanine ammonia-lyase (EC 4.3.1.5) gene PAL2 was translationally fused to the beta-glucuronidase reporter gene and transferred to tobacco by Agrobacterium tumefaciens-mediated leaf disk transformation. The distribution of beta-glucuronidase activity in these transgenic plants is very similar to that of endogenous PAL2 transcripts in bean, with very high levels in petals; marked accumulation in anthers, stigmas, roots, ...

  2. Sunlight-stimulated phenylalanine ammonia-lyase (PAL) activity and anthocyanin accumulation in exocarp of ‘Mahajanaka’ mango

    OpenAIRE

    Kobkiat Saengnil

    2011-01-01

    The activity of phenylalanine ammonia-lyase (PAL) required for anthocyanin synthesis was stimulated by sunlight exposure resulting in the development of red colour in ‘Mahajanaka’ mango exocarp, which occurred only on the sunlight-exposed side of the fruit. The accumulation of anthocyanin was concurrent with the increase in PAL activity in the mature stage of the fruit. The exposed side of the fruit had higher PAL activity, endogenous sugar content, and anthocyanin accumulation than the unexp...

  3. Phenolics and Flavonoids Compounds, Phenylanine Ammonia Lyase and Antioxidant Activity Responses to Elevated CO2 in Labisia pumila (Myrisinaceae)

    OpenAIRE

    Jaafar, Hawa Z. E.; Ehsan Karimi; Mohd Hafiz Ibrahim

    2012-01-01

    A split plot 3 × 3 experiment was designed to examine the impact of three concentrations of CO2 (400, 800 and 1,200 µmol·mol−1) on the phenolic and flavonoid compound profiles, phenylalanine ammonia lyase (PAL) and antioxidant activity in three varieties of Labisia pumila Benth. (var. alata, pumila and lanceolata) after 15 weeks of exposure. HPLC analysis revealed ...

  4. ATP-citrate lyase is required for production of cytosolic acetyl coenzyme A and development in Aspergillus nidulans.

    Science.gov (United States)

    Hynes, Michael J; Murray, Sandra L

    2010-07-01

    Acetyl coenzyme A (CoA) is a central metabolite in carbon and energy metabolism and in the biosynthesis of cellular molecules. A source of cytoplasmic acetyl-CoA is essential for the production of fatty acids and sterols and for protein acetylation, including histone acetylation in the nucleus. In Saccharomyces cerevisiae and Candida albicans acetyl-CoA is produced from acetate by cytoplasmic acetyl-CoA synthetase, while in plants and animals acetyl-CoA is derived from citrate via ATP-citrate lyase. In the filamentous ascomycete Aspergillus nidulans, tandem divergently transcribed genes (aclA and aclB) encode the subunits of ATP-citrate lyase, and we have deleted these genes. Growth is greatly diminished on carbon sources that do not result in cytoplasmic acetyl-CoA, such as glucose and proline, while growth is not affected on carbon sources that result in the production of cytoplasmic acetyl-CoA, such as acetate and ethanol. Addition of acetate restores growth on glucose or proline, and this is dependent on facA, which encodes cytoplasmic acetyl-CoA synthetase, but not on the regulatory gene facB. Transcription of aclA and aclB is repressed by growth on acetate or ethanol. Loss of ATP-citrate lyase results in severe developmental effects, with the production of asexual spores (conidia) being greatly reduced and a complete absence of sexual development. This is in contrast to Sordaria macrospora, in which fruiting body formation is initiated but maturation is defective in an ATP-citrate lyase mutant. Addition of acetate does not repair these defects, indicating a specific requirement for high levels of cytoplasmic acetyl-CoA during differentiation. Complementation in heterokaryons between aclA and aclB deletions for all phenotypes indicates that the tandem gene arrangement is not essential. PMID:20495057

  5. Catalytic mechanism of S-type phycobiliprotein lyase: chaperone-like action and functional amino acid residues.

    Science.gov (United States)

    Kupka, Michaela; Zhang, Juan; Fu, Wei-Lei; Tu, Jun-Ming; Böhm, Stephan; Su, Ping; Chen, Yu; Zhou, Ming; Scheer, Hugo; Zhao, Kai-Hong

    2009-12-25

    The phycobilin:cysteine 84-phycobiliprotein lyase, CpcS1, catalyzes phycocyanobilin (PCB) and phycoerythrobilin (PEB) attachment at nearly all cysteine 82 binding sites (consensus numbering) of phycoerythrin, phycoerythrocyanin, phycocyanin, and allophycocyanin (Zhao, K. H., Su, P., Tu, J. M., Wang, X., Liu, H., Plöscher, M., Eichacker, L., Yang, B., Zhou, M., and Scheer, H. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 14300-14305). We now show that CpcS1 binds PCB and PEB rapidly with bi-exponential kinetics (38/119 and 12/8300 ms, respectively). Chromophore binding to the lyase is reversible and much faster than the spontaneous, but low fidelity chromophore addition to the apo-protein in the absence of the lyase. This indicates kinetic control by the enzyme, which then transfers the chromophore to the apo-protein in a slow (tens of minutes) but stereo- and regioselectively corrects the reaction. This mode of action is reminiscent of chaperones but does not require ATP. The amino acid residues Arg-18 and Arg-149 of the lyase are essential for chromophore attachment in vitro and in Escherichia coli, mutations of His-21, His-22, Trp-75, Trp-140, and Arg-147 result in reduced activity (<30% of wild type in vitro). Mutants R147Q and W69M were active but had reduced capacity for PCB binding; additionally, with W69M there was loss of fidelity in chromophore attachment. Imidazole is a non-competitive inhibitor, supporting a bilin-binding function of histidine. Evidence was obtained that CpcS1 also catalyzes exchange of C-beta84-bound PCB in biliproteins by PEB. PMID:19864423

  6. Production of L-dihydroxyphenylalanine in Escherichia coli with the tyrosine phenol-lyase gene cloned from Erwinia herbicola.

    OpenAIRE

    Foor, F; Morin, N.; Bostian, K A

    1993-01-01

    The gene (tutA) encoding tyrosine phenol-lyase from Erwinia herbicola was cloned into Escherichia coli, and fusions to the lac and tac promoters were constructed. The enzyme was expressed at high levels in E. coli in the presence of isopropyl-beta-D-thiogalactopyranoside or lactose as an inducer. L-Dihydroxyphenylalanine was synthesized in high yield from catechol, pyruvate, and ammonia by induced cells.

  7. Heterologous production of methionine-γ-lyase from brevibacterium linens in lactococcus lactis and formation of volatile sulfur compounds

    OpenAIRE

    Hanniffy, Sean; Philo, Mark; Peláez, Carmen; Gasson, M. J.; Requena, Teresa; Martínez-Cuesta, M. Carmen

    2009-01-01

    The conversion of methionine to volatile sulfur compounds (VSCs) is of great importance in flavor formation during cheese ripening and is the focus of biotechnological approaches toward flavor improvement. A synthetic mgl gene encoding methionine-γ-lyase (MGL) from Brevibacterium linens BL2 was cloned into a Lactococcus lactis expression plasmid under the control of the nisin-inducible promoter PnisA. When expressed in L. lactis and purified as a recombinant protein, MGL was shown to degrade ...

  8. Cloning and characterization of a pectin lyase gene from Colletotrichum lindemuthianum and comparative phylogenetic/structural analyses with genes from phytopathogenic and saprophytic/opportunistic microorganisms

    Directory of Open Access Journals (Sweden)

    Lara-Márquez Alicia

    2011-12-01

    Full Text Available Abstract Background Microorganisms produce cell-wall-degrading enzymes as part of their strategies for plant invasion/nutrition. Among these, pectin lyases (PNLs catalyze the depolymerization of esterified pectin by a β-elimination mechanism. PNLs are grouped together with pectate lyases (PL in Family 1 of the polysaccharide lyases, as they share a conserved structure in a parallel β-helix. The best-characterized fungal pectin lyases are obtained from saprophytic/opportunistic fungi in the genera Aspergillus and Penicillium and from some pathogens such as Colletotrichum gloeosporioides. The organism used in the present study, Colletotrichum lindemuthianum, is a phytopathogenic fungus that can be subdivided into different physiological races with different capacities to infect its host, Phaseolus vulgaris. These include the non-pathogenic and pathogenic strains known as races 0 and 1472, respectively. Results Here we report the isolation and sequence analysis of the Clpnl2 gene, which encodes the pectin lyase 2 of C. lindemuthianum, and its expression in pathogenic and non-pathogenic races of C. lindemuthianum grown on different carbon sources. In addition, we performed a phylogenetic analysis of the deduced amino acid sequence of Clpnl2 based on reported sequences of PNLs from other sources and compared the three-dimensional structure of Clpnl2, as predicted by homology modeling, with those of other organisms. Both analyses revealed an early separation of bacterial pectin lyases from those found in fungi and oomycetes. Furthermore, two groups could be distinguished among the enzymes from fungi and oomycetes: one comprising enzymes from mostly saprophytic/opportunistic fungi and the other formed mainly by enzymes from pathogenic fungi and oomycetes. Clpnl2 was found in the latter group and was grouped together with the pectin lyase from C. gloeosporioides. Conclusions The Clpnl2 gene of C. lindemuthianum shares the characteristic elements of

  9. Molecular and Functional Characterization of Sphingosine-1-Phosphate Lyase Homolog from Higher Plants

    Institute of Scientific and Technical Information of China (English)

    Yan Niu; Kunling Chen; Jizhou Wang; Xin Liu; Huanju Qin; Aimin Zhang; Daowen Wang

    2007-01-01

    Sphingosine-1-phosphate lyase (SPL) is involved in degrading the conserved sphingolipid signaling molecule sphingoaine-1-phosphate. However, molecular studies on plant SPL have not been reported to date. Here, we present bloinformatic, molecular and functional analyses of putative SPL proteins from Arabldopsis thaliana and rice (designated as AtSPL and OsSPL, respectively). Amino acid sequence comparison revealed that plant SPL contained the pyridoxal-dependent decarboxylase domain and the conserved residue that may be involved in substrate catalysis. When expressed in Saccharomyces cerevisiae, AtSPL and OsSPL corrected the hypersensitive phenotype of the yeast dpl1 deletion strain, which is deficient in endogenous SPL activity, to exogenous supplied sphingolipid long chain bases (LCBs), suggesting that plant SPL protein is functional in vivo in degrading phosphorylated LCBs. In Arabidopsis, AtSPL transcripts were detected in roots, stems, leaves, flowers and siliques. In pAtSPL-AtSPL::GUS transgenlc lines, the AtSPL::GUS fusion protein was found in a variety of vegetative and reproductive tissues. AtSPL expression level was dynamically regulated during leaf development and senescence, and was steadily and significantly increased in Arabidopsis seedlings treated with the cell death-inducing fungal toxin fumonisin B1. The potential function of SPL in Arabidopsis is discussed.

  10. Screening and optimization of pectin lyase and polygalacturonase activity from ginseng pathogen Cylindrocarpon Destructans

    Directory of Open Access Journals (Sweden)

    Gayathri Sathiyaraj

    2011-06-01

    Full Text Available Cylindrocarpon destructans isolated from ginseng field was found to produce pectinolytic enzymes. A Taguchi's orthogonal array experimental design was applied to optimize the preliminary production of polygalacturonase (PG and pectin lyase (PL using submerged culture condition. This method was applied to evaluate the significant parameters for the production of enzymes. The process variables were pH, pectin concentration, incubation time and temperature. Optimization of process parameters resulted in high levels of enzyme (PG and PL production after ten days of incubation at a pH of 5.0 at 25°C in the presence of 1.5% pectin. Among different nitrogen sources, urea and peptone showed high production of PG and PL, respectively. The enzyme production and mycelial growth seems to have direct influence on the culture conditions; therefore, at stationary state high enzyme production and mycelial growth were obtained than agitation state. Along with this, optimization of enzyme activity was also determined using various physiological parameters like, temperature, incubation time and pH. Taguchi's data was also analyzed using one step ANOVA statistical method.

  11. Screening and optimization of pectin lyase and polygalacturonase activity from ginseng pathogen Cylindrocarpon Destructans.

    Science.gov (United States)

    Sathiyaraj, Gayathri; Srinivasan, Sathiyaraj; Kim, Ho-Bin; Subramaniyam, Sathiyamoorthy; Lee, Ok Ran; Kim, Yeon-Ju; Yang, Deok Chun

    2011-04-01

    Cylindrocarpon destructans isolated from ginseng field was found to produce pectinolytic enzymes. A Taguchi's orthogonal array experimental design was applied to optimize the preliminary production of polygalacturonase (PG) and pectin lyase (PL) using submerged culture condition. This method was applied to evaluate the significant parameters for the production of enzymes. The process variables were pH, pectin concentration, incubation time and temperature. Optimization of process parameters resulted in high levels of enzyme (PG and PL) production after ten days of incubation at a pH of 5.0 at 25°C in the presence of 1.5% pectin. Among different nitrogen sources, urea and peptone showed high production of PG and PL, respectively. The enzyme production and mycelial growth seems to have direct influence on the culture conditions; therefore, at stationary state high enzyme production and mycelial growth were obtained than agitation state. Along with this, optimization of enzyme activity was also determined using various physiological parameters like, temperature, incubation time and pH. Taguchi's data was also analyzed using one step ANOVA statistical method. PMID:24031695

  12. Potential Inhibitors for Isocitrate Lyase of Mycobacterium tuberculosis and Non-M. tuberculosis: A Summary

    Directory of Open Access Journals (Sweden)

    Yie-Vern Lee

    2015-01-01

    Full Text Available Isocitrate lyase (ICL is the first enzyme involved in glyoxylate cycle. Many plants and microorganisms are relying on glyoxylate cycle enzymes to survive upon downregulation of tricarboxylic acid cycle (TCA cycle, especially Mycobacterium tuberculosis (MTB. In fact, ICL is a potential drug target for MTB in dormancy. With the urge for new antitubercular drug to overcome tuberculosis treat such as multidrug resistant strain and HIV-coinfection, the pace of drug discovery has to be increased. There are many approaches to discovering potential inhibitor for MTB ICL and we hereby review the updated list of them. The potential inhibitors can be either a natural compound or synthetic compound. Moreover, these compounds are not necessary to be discovered only from MTB ICL, as it can also be discovered by a non-MTB ICL. Our review is categorized into four sections, namely, (a MTB ICL with natural compounds; (b MTB ICL with synthetic compounds; (c non-MTB ICL with natural compounds; and (d non-MTB ICL with synthetic compounds. Each of the approaches is capable of overcoming different challenges of inhibitor discovery. We hope that this paper will benefit the discovery of better inhibitor for ICL.

  13. The Salmonella effector protein SpvC, a phosphothreonine lyase is functional in plant cells

    KAUST Repository

    Neumann, Christina

    2014-10-17

    Salmonella is one of the most prominent causes of food poisoning and growing evidence indicates that contaminated fruits and vegetables are an increasing concern for human health. Successful infection demands the suppression of the host immune system, which is often achieved via injection of bacterial effector proteins into host cells. In this report we present the function of Salmonella effector protein in plant cell, supporting the new concept of trans-kingdom competence of this bacterium. We screened a range of Salmonella Typhimurium effector proteins for interference with plant immunity. Among these, the phosphothreonine lyase SpvC attenuated the induction of immunity-related genes when present in plant cells. Using in vitro and in vivo systems we show that this effector protein interacts with and dephosphorylates activated Arabidopsis Mitogen-activated Protein Kinase 6 (MPK6), thereby inhibiting defense signaling. Moreover, the requirement of Salmonella SpvC was shown by the decreased proliferation of the ΔspvC mutant in Arabidopsis plants. These results suggest that some Salmonella effector proteins could have a conserved function during proliferation in different hosts. The fact that Salmonella and other Enterobacteriaceae use plants as hosts strongly suggests that plants represent a much larger reservoir for animal pathogens than so far estimated.

  14. Screening Peptide Inhibitors Using Phage Peptide Library with Isocitrate Lyase in Mycobacterium tuberculosis as Target

    Institute of Scientific and Technical Information of China (English)

    YIN Yu-he; NIU Xue; SUN Bo; TENG Guo-sheng; ZHAO Yun-hui; WU Cong-mei

    2011-01-01

    When devoured by macrophages,Mycobacterium tuberculosis remains persistent in macrophages and gains energy through the glyoxylate bypass to maintain its long-term existence in host cells.Therefore it is possible to stop persistent infections by interdicting the glyoxylate bypass in which the isocitrate lyase(ICL) is the key rate-limiting enzyme and a persistence factor.ICL is the target of anti-TB(TB:tubercular) drugs,which could screen ICL out and effectively inhibit the activity of ICL in Mycobacterium tuberculosis,and because of this,anti-TB drugs can be used to kill persistent Mycobacterium tuberculosis.In this study,the ICL gene of the Mycobacterium tuberculosis H37Rv was cloned successfully and recombinant protein with bioactivity was obtained through the enzyme characteristic appraisal.The specific activity of the recombined ICL is 24 μmol·mg-1 -min-1.The recombined ICL protein was used as the target,and phages which can specifically combine to ICL were screened in the phage 7 peptide library.According to the results of the ELISA and DNA sequence detection,eventually three 7-peptide chains were synthesized.Then the peptide chains were reacted with ICL,respectively,to detect their inhibitory effects on ICL.The results show that all the three 7-peptide chains possessed varying inhibitory effects on the activity of ICL.This study provided lead compounds for the research and development of new peptide anti-TB drugs.

  15. Crystal structure and mechanism of the Staphylococcus cohnii virginiamycin B lyase (Vgb).

    Science.gov (United States)

    Lipka, Magdalena; Filipek, Renata; Bochtler, Matthias

    2008-04-01

    The semisynthetic streptogramin antibiotic quinupristin/dalfopristin (trade name Synercid, Aventis Pharma) is a mixture of the A-type streptogramin dalfopristin and the B-type streptogramin quinupristin, a capped hexapeptide macrolactone. Quinupristin/dalfopristin was developed to combat multidrug resistant pathogens, but suffers from its own problems with drug resistance. Virginiamycin B lyase (Vgb) inactivates the quinupristin component of Synercid by lactone ring opening. Remarkably, the enzyme promotes this reaction by intramolecular beta-elimination without the involvement of a water molecule. Recently, structures of S. aureus Vgb in the presence and absence of substrate were reported and used together with detailed mutagenesis data to suggest a catalytic mechanism. Here, we report an independent determination of the S. cohnii Vgb crystal structure and a biochemical characterization of the enzyme. As expected, the S. cohnii and S. aureus Vgb structures and active sites are very similar. Moreover, both enzymes catalyze quinupristin lactone ring opening with similar rate constants, albeit perhaps with different dependencies on divalent metal ions. Replacement of the conserved active site residues His228, Glu268, or His270 with alanine reduces or abolishes S. cohnii Vgb activity. Residue Lys285 in S. cohnii Vgb is spatially equivalent to the S. aureus Vgb active site residue Glu284. A glutamate but not an alanine residue can substitute for the lysine without significant loss of activity. PMID:18341294

  16. Adenylosuccinate lyase (ADSL) and infantile autism: Absence of previously reported point mutation

    Energy Technology Data Exchange (ETDEWEB)

    Fon, E.A.; Sarrazin, J.; Rouleau, G.A. [Montreal General Hospital (Canada)] [and others

    1995-12-18

    Autism is a heterogeneous neuropsychiatric syndrome of unknown etiology. There is evidence that a deficiency in the enzyme adenylosuccinate lyase (ADSL), essential for de novo purine biosynthesis, could be involved in the pathogenesis of certain cases. A point mutation in the ADSL gene, resulting in a predicted serine-to-proline substitution and conferring structural instability to the mutant enzyme, has been reported previously in 3 affected siblings. In order to determine the prevalence of the mutation, we PCR-amplified the exon spanning the site of this mutation from the genomic DNA of patients fulfilling DSM-III-R criteria for autistic disorder. None of the 119 patients tested were found to have this mutation. Furthermore, on preliminary screening using single-strand conformation polymorphism (SSCP), no novel mutations were detected in the coding sequence of four ADSL exons, spanning approximately 50% of the cDNA. In light of these findings, it appears that mutations in the ADSL gene represent a distinctly uncommon cause of autism. 12 refs., 2 figs.

  17. Mitochondrial Sulfide Detoxification Requires a Functional Isoform O-Acetylserine(thiol)lyase C in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Consolación (A)lvarez; Irene García; Luis C.Romero; Cecilia Gotor

    2012-01-01

    In non-cyanogenic species,the main source of cyanide derives from ethylene and camalexin biosyntheses.In mitochondria,cyanide is a potent inhibitor of the cytochrome c oxidase and is metabolized bythe β-cyanoalanine synthase CYS-C1,catalyzing the conversion of cysteine and cyanide to hydrogen sulfide and β-cyanoalanine.The hydrogen sulfide released also inhibits the cytochrome c oxidase and needs to be detoxified by the O-acetylserine(thiol)lyase mitochondrial isoform,OAS-C,which catalyzes the incorporation of sulfide to O-acetylserine to produce cysteine,thus generating a cyclic pathway in the mitochondria.The loss of functional OAS-C isoforms causes phenotypic characteristics very similar to the loss of the CYS-C1 enzyme,showing defects in root hair formation.Genetic complementation with the OAS-C gene rescues the impairment of root hair elongation,restoring the wild-type phenotype.The mitochondria compromise their capacity to properly detoxify cyanide and the resulting sulfide because the latter cannot re-assimilate into cysteine in the oas-c null mutant.Consequently,we observe an accumulation of sulfide and cyanide and of the alternative oxidase,which is unable to prevent the production of reactive oxygen species probably due to the accumulation of both toxic molecules.Our results allow us to suggest that the significance of OAS-C is related to its role in the proper sulfide and cyanide detoxification in mitochondria.

  18. Recombinant l-phenylalanine ammonia lyase from Rhodosporidium toruloides as a potential anticancer agent.

    Science.gov (United States)

    Babich, Olga O; Pokrovsky, Vadim S; Anisimova, Natalia Yu; Sokolov, Nikolai N; Prosekov, Alexander Yu

    2013-01-01

    The recombinant producer strain expressing Rhodosporidium toruloides l-phenylalanine ammonia lyase (PAL) has been obtained, and a purification procedure of PAL has been developed. The purified enzyme, PAL, has the following biochemical and catalytic characteristics: Km for l-Phe of 0.49 mM, pH optimum at 8.5, and temperature optimum at 50°C. PAL exhibited a significant cytotoxic effect toward the following cell lines: MCF7 (IC50 = 1.97 U/mL), DU145 (IC50 = 7.3 U/mL), which are comparable with E. coli l-asparaginase type-II cytotoxicity in vitro. Administration of PAL (200-400 U/kg) to L5178y-bearing mice for five times (a total dose of 1000-2000 U/kg) was well tolerated and showed the increase of life span (ILS) = 12-16%, P < 0.05. Data obtained suggest that PAL from R. toruloides has a potential for cancer treatment. PMID:23718781

  19. Molecular cloning and sequence analysis of a phenylalanine ammonia-lyase gene from dendrobium.

    Directory of Open Access Journals (Sweden)

    Qing Jin

    Full Text Available In this study, a phenylalanine ammonia-lyase (PAL gene was cloned from Dendrobium candidum using homology cloning and RACE. The full-length sequence and catalytic active sites that appear in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum are also found: PAL cDNA of D. candidum (designated Dc-PAL1, GenBank No. JQ765748 has 2,458 bps and contains a complete open reading frame (ORF of 2,142 bps, which encodes 713 amino acid residues. The amino acid sequence of DcPAL1 has more than 80% sequence identity with the PAL genes of other plants, as indicated by multiple alignments. The dominant sites and catalytic active sites, which are similar to that showing in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum, are also found in DcPAL1. Phylogenetic tree analysis revealed that DcPAL is more closely related to PALs from orchidaceae plants than to those of other plants. The differential expression patterns of PAL in protocorm-like body, leaf, stem, and root, suggest that the PAL gene performs multiple physiological functions in Dendrobium candidum.

  20. Control of phenylalanine ammonia-lyase gene promoters from pea by UV radiation

    International Nuclear Information System (INIS)

    The gene fusion system was used to study UV light-control of PS PAL1 and PS PAL2 genes encoding phenylalanine ammonia-lyase of pea. The induction of pea PAL promoters was analysed in transgenic tobacco plants. Binary plasmids (derivatives of pBI101.2 vector) containing 5' regulatory fragments of PS PAL1 and PS PAL2 linked to reporter genes (GUS, LUC) were constructed. The analyses were performed with the use of single constructs (containing one variant of PS PAL promoter and one reporter gene) and dual constructs (containing both PS PAL1 and PS PAL2 promoters connected with different reporter genes). The use of dual constructs enabled the evaluation of both PS PAL promoters activity in the same plant. The analyses of in vitro grown plants have shown that both PAL promoters are strongly induced in leaves subjected to UV radiation. In some cases, the UV-stimulated expression exceeded the exposed areas. This phenomenon was observed more often in the leaves of plants containing the PS PAL1::GUS than PS PAL2::GUS construct. Removal of boxes 2, 4, 5 from PS PAL1 promoter and deletion of its 5' end region (-339 to -1394) decreases the level of gene expression but does not eliminate its responsiveness to UV

  1. Molecular Cloning and Characterization of Hydroperoxide Lyase Gene in the Leaves of Tea Plant (Camellia sinensis).

    Science.gov (United States)

    Deng, Wei-Wei; Wu, Yi-Lin; Li, Ye-Yun; Tan, Zhen; Wei, Chao-Ling

    2016-03-01

    Hydroperoxide lyase (HPL, E.C. 4.1.2.) is the major enzyme in the biosynthesis of natural volatile aldehydes and alcohols in plants, however, little was known about HPL in tea plants (Camellia sinensis). A unique cDNA fragment was isolated by suppressive subtractive hybridization (SSH) from a tea plant subjected to herbivory by tea geometrid Ectropis obliqua. This full length cDNA acquired by RACE was 1476 bp and encoded 491 amino acids. DNA and protein BLAST searches showed high homology to HPL sequences from other plants. The His-tag expression vector pET-32a(+)/CsHPL was constructed and transferred into Escherichia coli Rosetta (DE3). The expression product of recombinant CsHPL in E. coli was about 60 kDa. The enzyme activity of CsHPL was 0.20 μmol·min(-1)·mg(-1). Quantitative RT-PCR analysis indicated CsHPL was strongly up-regulated in tea plants after Ectropis obliqua attack, suggesting that it may be an important candidate for defense against insects in tea plants. PMID:26886573

  2. Phylogenetic analysis of the genus Plasmodium based on the gene encoding adenylosuccinate lyase.

    Science.gov (United States)

    Kedzierski, Lukasz; Escalante, Ananias A; Isea, Raul; Black, Casilda G; Barnwell, John W; Coppel, Ross L

    2002-07-01

    Phylogenetic studies of the genus Plasmodium have been performed using sequences of the nuclear, mitochondrial and plastid genes. Here we have analyzed the adenylosuccinate lyase (ASL) gene, which encodes an enzyme involved in the salvage of host purines needed by malaria parasites for DNA synthesis. The ASL gene is present in several eukaryotic as well as prokaryotic organisms and does not have repeat regions, which facilitates the accuracy of the alignment. Furthermore, it has been shown that ASL is not subject to positive natural selection. We have sequenced the ASL gene of several different Plasmodium species infecting humans, rodents, monkeys and birds and used the obtained sequences along with the previously known P. falciparum ASL sequence, for structural and phylogenetic analysis of the genus Plasmodium. The genetic divergence of ASL is comparable with that observed in other nuclear genes such as cysteine proteinase, although ASL cannot be considered conserved when compared to aldolase or superoxide dismutase, which exhibit a slower rate of evolution. Nevertheless, a protein like ASL has a rate of evolution that provides enough information for elucidating evolutionary relationships. We modeled 3D structures of the ASL protein based on sequences used in the phylogenetic analysis and obtained a consistent structure for four different species despite the divergence observed. Such models would facilitate alignment in further studies with a greater number of plasmodial species or other Apicomplexa. PMID:12798008

  3. Purification and characterization of selenocysteine beta-lyase from Citrobacter freundii

    International Nuclear Information System (INIS)

    The purification and characterization of bacterial selenocysteine beta-lyase, an enzyme which specifically catalyzes the cleavage of L-selenocysteine to L-alanine and Se0, are presented. The enzyme, purified to near homogeneity from Citrobacter freundii, is monomeric with a molecular weight of ca. 64,000 and contains 1 mol of pyridoxal 5'-phosphate as a cofactor per mol of enzyme. L-Selenocysteine is the sole substrate. L-Cysteine is a competitive inhibitor of the enzyme. The enzyme also catalyzes the alpha, beta elimination of beta-chloro-L-alanine to form NH3, pyruvate, and Cl- and is irreversibly inactivated during the reaction. The physicochemical properties, e.g., amino acid composition and subunit structure, of the bacterial enzyme are fairly different from those of the pig liver enzyme. However, the catalytic properties of both enzymes, e.g., substrate specificity and inactivation by the substrate or a mechanism-based inactivator, beta-chloro-L-alanine, are very similar

  4. Purification and characterization of selenocysteine beta-lyase from Citrobacter freundii

    Energy Technology Data Exchange (ETDEWEB)

    Chocat, P.; Esaki, N.; Tanizawa, K.; Nakamura, K.; Tanaka, H.; Soda, K.

    1985-08-01

    The purification and characterization of bacterial selenocysteine beta-lyase, an enzyme which specifically catalyzes the cleavage of L-selenocysteine to L-alanine and Se0, are presented. The enzyme, purified to near homogeneity from Citrobacter freundii, is monomeric with a molecular weight of ca. 64,000 and contains 1 mol of pyridoxal 5'-phosphate as a cofactor per mol of enzyme. L-Selenocysteine is the sole substrate. L-Cysteine is a competitive inhibitor of the enzyme. The enzyme also catalyzes the alpha, beta elimination of beta-chloro-L-alanine to form NH3, pyruvate, and Cl- and is irreversibly inactivated during the reaction. The physicochemical properties, e.g., amino acid composition and subunit structure, of the bacterial enzyme are fairly different from those of the pig liver enzyme. However, the catalytic properties of both enzymes, e.g., substrate specificity and inactivation by the substrate or a mechanism-based inactivator, beta-chloro-L-alanine, are very similar.

  5. Studies on the chemical mechanism of E. coli pyruvate-formate lyase

    International Nuclear Information System (INIS)

    Pyruvate-formate lyase (PFL) catalyzes the CoA dependent dismutation of pyruvate into acetyl-CoA and formate. The activated enzyme contains a radical moiety essential for this reaction. They have initiated studies to elucidate the mechanism by which this enzymic process occurs. Their studies have centered on the inactivation of PFL by a variety of pyruvate and formate analogs, including the known inhibitors O2, hypophosphite, and 3-fluoropyruvate, and by some newly discovered compounds, propargylic and acrylic acids. Inactivation is first order, and readily observable at inhibitor concentrations of 5 mM or less. Propargylic acid is especially reactive; at 1 mM the enzyme half-life is 2 minutes. Furthermore, a variety of other acetylenic analogs such as acetylene dicarboxylate, 2-butynoate, and propargyl alcohol do not inhibit PFL, suggesting some specificity for propargylic acid. Of particular significance is their finding of isotope effects on the rate of inactivation by 2H2-hypophosphite and 3-2H-propargylic acid (3.2 at 1 mM). Such data suggest to us that inactivation may involve rate-determining hydrogen abstraction or hydrogen shift from the inhibitor molecule. Anaerobic gel filtration of chemically inactivated enzyme does not lead to recovery of activity, although subsequent treatment with a PFL activating system does lead to partial activity restoration

  6. Diagnosis of adenylosuccinate lyase deficiency by metabolomic profiling in plasma reveals a phenotypic spectrum.

    Science.gov (United States)

    Donti, Taraka R; Cappuccio, Gerarda; Hubert, Leroy; Neira, Juanita; Atwal, Paldeep S; Miller, Marcus J; Cardon, Aaron L; Sutton, V Reid; Porter, Brenda E; Baumer, Fiona M; Wangler, Michael F; Sun, Qin; Emrick, Lisa T; Elsea, Sarah H

    2016-09-01

    Adenylosuccinate lyase (ADSL) deficiency is a rare autosomal recessive neurometabolic disorder that presents with a broad-spectrum of neurological and physiological symptoms. The ADSL gene produces an enzyme with binary molecular roles in de novo purine synthesis and purine nucleotide recycling. The biochemical phenotype of ADSL deficiency, accumulation of SAICAr and succinyladenosine (S-Ado) in biofluids of affected individuals, serves as the traditional target for diagnosis with targeted quantitative urine purine analysis employed as the predominate method of detection. In this study, we report the diagnosis of ADSL deficiency using an alternative method, untargeted metabolomic profiling, an analytical scheme capable of generating semi-quantitative z-score values for over 1000 unique compounds in a single analysis of a specimen. Using this method to analyze plasma, we diagnosed ADSL deficiency in four patients and confirmed these findings with targeted quantitative biochemical analysis and molecular genetic testing. ADSL deficiency is part of a large a group of neurometabolic disorders, with a wide range of severity and sharing a broad differential diagnosis. This phenotypic similarity among these many inborn errors of metabolism (IEMs) has classically stood as a hurdle in their initial diagnosis and subsequent treatment. The findings presented here demonstrate the clinical utility of metabolomic profiling in the diagnosis of ADSL deficiency and highlights the potential of this technology in the diagnostic evaluation of individuals with neurologic phenotypes. PMID:27504266

  7. Discovery and molecular and biocatalytic properties of hydroxynitrile lyase from an invasive millipede, Chamberlinius hualienensis.

    Science.gov (United States)

    Dadashipour, Mohammad; Ishida, Yuko; Yamamoto, Kazunori; Asano, Yasuhisa

    2015-08-25

    Hydroxynitrile lyase (HNL) catalyzes the degradation of cyanohydrins and causes the release of hydrogen cyanide (cyanogenesis). HNL can enantioselectively produce cyanohydrins, which are valuable building blocks for the synthesis of fine chemicals and pharmaceuticals, and is used as an important biocatalyst in industrial biotechnology. Currently, HNLs are isolated from plants and bacteria. Because industrial biotechnology requires more efficient and stable enzymes for sustainable development, we must continuously explore other potential enzyme sources for the desired HNLs. Despite the abundance of cyanogenic millipedes in the world, there has been no precise study of the HNLs from these arthropods. Here we report the isolation of HNL from the cyanide-emitting invasive millipede Chamberlinius hualienensis, along with its molecular properties and application in biocatalysis. The purified enzyme displays a very high specific activity in the synthesis of mandelonitrile. It is a glycosylated homodimer protein and shows no apparent sequence identity or homology with proteins in the known databases. It shows biocatalytic activity for the condensation of various aromatic aldehydes with potassium cyanide to produce cyanohydrins and has high stability over a wide range of temperatures and pH values. It catalyzes the synthesis of (R)-mandelonitrile from benzaldehyde with a 99% enantiomeric excess, without using any organic solvents. Arthropod fauna comprise 80% of terrestrial animals. We propose that these animals can be valuable resources for exploring not only HNLs but also diverse, efficient, and stable biocatalysts in industrial biotechnology. PMID:26261304

  8. Enzymatic Hydrolysis of Alginate to Produce Oligosaccharides by a New Purified Endo-Type Alginate Lyase

    Science.gov (United States)

    Zhu, Benwei; Chen, Meijuan; Yin, Heng; Du, Yuguang; Ning, Limin

    2016-01-01

    Enzymatic hydrolysis of sodium alginate to produce alginate oligosaccharides has drawn increasing attention due to its advantages of containing a wild reaction condition, excellent gel properties and specific products easy for purification. However, the efficient commercial enzyme tools are rarely available. A new alginate lyase with high activity (24,038 U/mg) has been purified from a newly isolated marine strain, Cellulophaga sp. NJ-1. The enzyme was most active at 50 °C and pH 8.0 and maintained stability at a broad pH range (6.0–10.0) and temperature below 40 °C. It had broad substrate specificity toward sodium alginate, heteropolymeric MG blocks (polyMG), homopolymeric M blocks (polyM) and homopolymeric G blocks (polyG), and possessed higher affinity toward polyG (15.63 mM) as well as polyMG (23.90 mM) than polyM (53.61 mM) and sodium alginate (27.21 mM). The TLC and MS spectroscopy analysis of degradation products suggested that it completely hydrolyzed sodium alginate into oligosaccharides of low degrees of polymerization (DPs). The excellent properties would make it a promising tool for full use of sodium alginate to produce oligosaccharides. PMID:27275826

  9. Purification and properties of S-alkyl-L-cysteine lyase from seedlings of Acacia farnesiana Willd.

    Science.gov (United States)

    Mazelis, M; Creveling, R K

    1975-06-01

    1. An S-alkyl-L-cysteine lyase (EC 4.4.1.6) was purified to apparent homogeneity from extracts of acetone-dried powders of the hypocotyls of etiolated 5-day-old seedlings of Acacia farnesiana Willd. 2. The enzyme catalyses a beta-elimination reaction and will utilize both the thioether and sulphoxide form of the substrate. 3. There is a braod specificity with regard to the alkyl substituent, but cystathionine is utilized very poorly. 4. The pH optimum is 7.8 and the Km value for the probable natural substrate L-djenkolate is 0.3 mM. 5. Both sodium dodecyl sulphate-polyacrylamide-gel electrophoresis and ultracentirfugal analysis give a molecular weight of about 144000. 6. One mol of pyridoxal phosphate is bound/mol of enzyme. 7. The energy of activation with L-djenkolate as the substrate is 53.1 kJ/mol. 8. The enzyme has a partial specific volume of 0.56 and S20,w 7.26S. PMID:241329

  10. Crystallization and preliminary X-ray analysis of alginate lyases A1-II and A1-II′ from Sphingomonas sp. A1

    International Nuclear Information System (INIS)

    The crystallization and preliminary characterization of the family PL-7 alginate lyases A1-II and A1-II′ from Sphingomonas sp. A1 are presented. Alginate lyases depolymerize alginate, a heteropolysaccharide consisting of α-l-guluronate and β-d-mannuronate, through a β-elimination reaction. The alginate lyases A1-II (25 kDa) and A1-II′ (25 kDa) from Sphingomonas sp. A1, which belong to polysaccharide lyase family PL-7, exhibit 68% homology in primary structure but have different substrate specificities. To determine clearly the structural basis for substrate recognition in the depolymerization mechanism by alginate lyases, both proteins were crystallized at 293 K using the vapour-diffusion method. A crystal of A1-II belonged to space group P21 and diffracted to 2.2 Å resolution, with unit-cell parameters a = 51.3, b = 30.1, c = 101.6 Å, β = 100.2°, while a crystal of A1-II′ belonged to space group P212121 and diffracted to 1.0 Å resolution, with unit-cell parameters a = 34.6, b = 68.5, c = 80.3 Å

  11. A continuous spectrophotometric assay and nonlinear kinetic analysis of methionine γ-lyase catalysis.

    Science.gov (United States)

    Foo, Timothy C; Terentis, Andrew C; Venkatachalam, Kallidaikurichi V

    2016-08-15

    In this article, we present a new, easy-to-implement assay for methionine γ-lyase (MGL)-catalyzed γ-elimination reactions of l-methionine and its analogues that produce α-ketobutyrate (α-KB) as product. The assay employs ultraviolet-visible (UV-Vis) spectrophotometry to continuously monitor the rate of formation of α-KB by its absorbance at 315 nm. We also employ a nonlinear data analysis method that obviates the need for an "initial slope" determination, which can introduce errors when the progress curves are nonlinear. The spectrophotometric assay is validated through product analysis by (1)H NMR (nuclear magnetic resonance), which showed that under the conditions of study l-methionine (l-met) and l-methionine sulfone (l-met sulfone) substrates were converted to α-KB product with greater than 99% yield. Using this assay method, we determined for the first time the Michaelis-Menten parameters for a recombinant form of MGL from Porphyromonas gingivalis, obtaining respective kcat and Km values of 328 ± 8 min(-1) and 1.2 ± 0.1 mM for l-met γ-elimination and 2048 ± 59 min(-1) and 38 ± 2 mM for l-met sulfone γ-elimination reactions. We envisage that this assay method will be useful for determining the activity of MGL γ-elimination reactions that produce α-KB as the end product. PMID:27235171

  12. Genome-wide characterization of phenylalanine ammonia-lyase gene family in watermelon (Citrullus lanatus).

    Science.gov (United States)

    Dong, Chun-Juan; Shang, Qing-Mao

    2013-07-01

    Phenylalanine ammonia-lyase (PAL), the first enzyme in the phenylpropanoid pathway, plays a critical role in plant growth, development, and adaptation. PAL enzymes are encoded by a gene family in plants. Here, we report a genome-wide search for PAL genes in watermelon. A total of 12 PAL genes, designated ClPAL1-12, are identified . Nine are arranged in tandem in two duplication blocks located on chromosomes 4 and 7, and the other three ClPAL genes are distributed as single copies on chromosomes 2, 3, and 8. Both the cDNA and protein sequences of ClPALs share an overall high identity with each other. A phylogenetic analysis places 11 of the ClPALs into a separate cucurbit subclade, whereas ClPAL2, which belongs to neither monocots nor dicots, may serve as an ancestral PAL in plants. In the cucurbit subclade, seven ClPALs form homologous pairs with their counterparts from cucumber. Expression profiling reveals that 11 of the ClPAL genes are expressed and show preferential expression in the stems and male and female flowers. Six of the 12 ClPALs are moderately or strongly expressed in the fruits, particularly in the pulp, suggesting the potential roles of PAL in the development of fruit color and flavor. A promoter motif analysis of the ClPAL genes implies redundant but distinctive cis-regulatory structures for stress responsiveness. Finally, duplication events during the evolution and expansion of the ClPAL gene family are discussed, and the relationships between the ClPAL genes and their cucumber orthologs are estimated. PMID:23546528

  13. Simultaneous determination of the lipoxygenase and hydroperxide lyase specificity in olive fruit pulp

    Directory of Open Access Journals (Sweden)

    Salas, Joaquín J.

    2000-06-01

    Full Text Available Olive pulp lipoxygenase regiospecificity and hydroperoxide lyase substrate specificity are important parameters in order to justify the volatile composition of olive oil. A new radiolabelling method to determine simultaneously these properties using only thin layer chromatography steps is described in the present work. The method involves incubation of an enzyme preparation from olive pulp with radiolabelled linoleate, followed by the fractionation of the resulting lipid products, previously treated with 2,4-dinitrophenyl hydrazine, on thin layer chromatography plates coated with polyethylenglycol 400. The results obtained are in agreement with previous studies carried out by other methods.La regioespecificidad de la lipoxigenasa y la especificidad del sustrato hidroperóxido liasa de pulpa de aceituna son parámetros importantes en la justificación de la composición en volátiles del aceite de oliva. En este trabajo se describe un nuevo método de marcaje radioactivo para determinar simultáneamente estas propiedades, usando solo etapas de cromatografía en capa fina. El método implica la incubación de una preparación enzimática de pulpa de aceituna con linoleato marcado, seguido del fraccionamiento de los productos lipídicos resultantes, previamente tratados con 2,4-dinitrofenil hidrazina, sobre placas de cromatografía en capa fina soportadas con polietilenglicol 400. Los resultados obtenidos están de acuerdo con estudios previos llevados a cabo con otros métodos.

  14. The variability in DMSP content and DMSP lyase activity in marine dinoflagellates

    Science.gov (United States)

    Caruana, Amandine M. N.; Malin, Gill

    2014-01-01

    More than 20 years ago Maureen Keller and co-workers published a study that identified dinoflagellates as an important marine phytoplankton group with respect to the production of dimethylsulphoniopropionate (DMSP). Here, we present a synthesis and analysis of all the DMSP and DMSP lyase activity (DLA) measurements currently available for dinoflagellates. The data cover 110 species and strains and reveal over 6 orders of magnitude variability in intracellular DMSP concentrations and substantial variations in DLA in 23 strains. Inter-specific variability was explored with reference to a range of biological characteristics. The presence of a theca did not appear to be related to DMSP concentration but there was a potential relationship with toxicity (P = 0.06) and bioluminescent species produced significantly lower concentrations (P plastid types (P plastids contained lower amounts of DMSP than those with peridinin plastids (P plastids tended to have higher DMSP concentrations. Heterotrophic dinoflagellates were also considered given their importance in the natural environment. They are the only heterotrophs known to synthesise DMSP and this ability may support the theory that they are of photosynthetic origin. However, the heterotrophic species investigated so far suggest wide variability in DMSP content and the species Oxyrrhis marina had no detectable DMSP. The oceanic province of origin significantly affected the DMSP concentrations (P < 0.05) with higher DMSP content observed in dinoflagellates from the Mediterranean province, the Kuroshio Current province and the East Coastal Australian province. Overall this study supports the concept that DMSP-containing dinoflagellates are an important potential source of DMS to the global atmosphere and highlights current gaps in knowledge.

  15. Partial deficiency of sphingosine-1-phosphate lyase confers protection in experimental autoimmune encephalomyelitis.

    Directory of Open Access Journals (Sweden)

    Andreas Billich

    Full Text Available BACKGROUND: Sphingosine-1-phosphate (S1P regulates the egress of T cells from lymphoid organs; levels of S1P in the tissues are controlled by S1P lyase (Sgpl1. Hence, Sgpl1 offers a target to block T cell-dependent inflammatory processes. However, the involvement of Sgpl1 in models of disease has not been fully elucidated yet, since Sgpl1 KO mice have a short life-span. METHODOLOGY: We generated inducible Sgpl1 KO mice featuring partial reduction of Sgpl1 activity and analyzed them with respect to sphingolipid levels, T-cell distribution, and response in models of inflammation. PRINCIPAL FINDINGS: The partially Sgpl1 deficient mice are viable but feature profound reduction of peripheral T cells, similar to the constitutive KO mice. While thymic T cell development in these mice appears normal, mature T cells are retained in thymus and lymph nodes, leading to reduced T cell numbers in spleen and blood, with a skewing towards increased proportions of memory T cells and T regulatory cells. The therapeutic relevance of Sgpl1 is demonstrated by the fact that the inducible KO mice are protected in experimental autoimmune encephalomyelitis (EAE. T cell immigration into the CNS was found to be profoundly reduced. Since S1P levels in the brain of the animals are unchanged, we conclude that protection in EAE is due to the peripheral effect on T cells, leading to reduced CNS immigration, rather than on local effects in the CNS. SIGNIFICANCE: The data suggest Sgpl1 as a novel therapeutic target for the treatment of multiple sclerosis.

  16. Molecular analysis of human argininosuccinate lyase: Mutant characterization and alternative splicing of the coding region

    International Nuclear Information System (INIS)

    Argininosuccinic acid lyase (ASAL) deficiency is a clinically heterogeneous autosomal recessive urea cycle disorder. The authors previously established by complementation analysis that 29 ASAL-deficient patients have heterogeneous mutations in a single gene. To prove that the ASAL structural gene is the affected locus, they sequenced polymerase chain reaction-amplified ASAL cDNA of a representative mutant from the single complementation group. Fibroblast strain 944 from a late-onset patient who was the product of a consanguineous mating, had only a single base-pair change in the coding region, a C-283→ T transition at a CpG dinucleotide in exon 3. This substitution converts Arg-95 to Cys (R95C), occurs in a stretch of 13 residues that is identical in yeast and human ASAL, and was present in both of the patient's alleles but not in 14 other mutant or 10 normal alleles. They observed that amplified cDNA from mutant 944 and normal cells (liver, keratinocytes, lymphoblasts, and fibroblasts) contained, in addition to the expected 5' 513-base-pair band, a prominent 318-base-pair ASAL band formed by the splicing of exon 2 from the transcript. The short transcript maintains the ASAL reading frame but removes Lys-51, a residue that may be essential for catalysis, since it binds the argininosuccinate substrate. They conclude (i) that the identification of the R95C mutation in strain 944 demonstrates that virtually all ASAL deficiency results from defects in the ASAL structural gene and (ii) that minor alternative splicing of the coding region occurs at the ASAL locus

  17. Production of protocatechuic acid by Corynebacterium glutamicum expressing chorismate-pyruvate lyase from Escherichia coli.

    Science.gov (United States)

    Okai, Naoko; Miyoshi, Takanori; Takeshima, Yasunobu; Kuwahara, Hiroaki; Ogino, Chiaki; Kondo, Akihiko

    2016-01-01

    Protocatechuic acid (3,4-dihydroxybenzoic acid; PCA) serves as a building block for polymers and pharmaceuticals. In this study, the biosynthetic pathway for PCA from glucose was engineered in Corynebacterium glutamicum. The pathway to PCA-employed elements of the chorismate pathway by using chorismate-pyruvate lyase (CPL) and 4-hydroxybenzoate hydroxylase (4-HBA hydroxylase). As C. glutamicum has the potential to synthesize the aromatic amino acid intermediate chorismate and possesses 4-HBA hydroxylase, we focused on expressing Escherichia coli CPL in a phenylalanine-producing strain of C. glutamicum ATCC21420. To secrete PCA, the gene (ubiC) encoding CPL from E. coli was expressed in C. glutamicum ATCC 21420 (strain F(UbiC)). The formation of 28.8 mg/L of extracellular 4-HBA (36 h) and 213 ± 29 mg/L of extracellular PCA (80 h) was obtained by the C. glutamicum strain F(UbiC) from glucose. The strain ATCC21420 was also found to produce extracellular PCA. PCA fermentation was performed using C. glutamicum strain F(UbiC) in a bioreactor at the optimized pH of 7.5. C. glutamicum F(UbiC) produced 615 ± 2.1 mg/L of PCA from 50 g/L of glucose after 72 h. Further, fed-batch fermentation of PCA by C. glutamicum F(UbiC) was performed with feedings of glucose every 24 h. The maximum production of PCA (1140.0 ± 11.6 mg/L) was achieved when 117.0 g/L of glucose was added over 96 h of fed-batch fermentation. PMID:26392137

  18. Sphingosine-1-phosphate lyase downregulation promotes colon carcinogenesis through STAT3-activated microRNAs.

    Science.gov (United States)

    Degagné, Emilie; Pandurangan, Ashok; Bandhuvula, Padmavathi; Kumar, Ashok; Eltanawy, Abeer; Zhang, Meng; Yoshinaga, Yuko; Nefedov, Mikhail; de Jong, Pieter J; Fong, Loren G; Young, Stephen G; Bittman, Robert; Ahmedi, Yasmin; Saba, Julie D

    2014-12-01

    Growing evidence supports a link between inflammation and cancer; however, mediators of the transition between inflammation and carcinogenesis remain incompletely understood. Sphingosine-1-phosphate (S1P) lyase (SPL) irreversibly degrades the bioactive sphingolipid S1P and is highly expressed in enterocytes but downregulated in colon cancer. Here, we investigated the role of SPL in colitis-associated cancer (CAC). We generated mice with intestinal epithelium-specific Sgpl1 deletion and chemically induced colitis and tumor formation in these animals. Compared with control animals, mice lacking intestinal SPL exhibited greater disease activity, colon shortening, cytokine levels, S1P accumulation, tumors, STAT3 activation, STAT3-activated microRNAs (miRNAs), and suppression of miR-targeted anti-oncogene products. This phenotype was attenuated by STAT3 inhibition. In fibroblasts, silencing SPL promoted tumorigenic transformation through a pathway involving extracellular transport of S1P through S1P transporter spinster homolog 2 (SPNS2), S1P receptor activation, JAK2/STAT3-dependent miR-181b-1 induction, and silencing of miR-181b-1 target cylindromatosis (CYLD). Colon biopsies from patients with inflammatory bowel disease revealed enhanced S1P and STAT3 signaling. In mice with chemical-induced CAC, oral administration of plant-type sphingolipids called sphingadienes increased colonic SPL levels and reduced S1P levels, STAT3 signaling, cytokine levels, and tumorigenesis, indicating that SPL prevents transformation and carcinogenesis. Together, our results suggest that dietary sphingolipids can augment or prevent colon cancer, depending upon whether they are metabolized to S1P or promote S1P metabolism through the actions of SPL. PMID:25347472

  19. 3-Hydroxy-3-methylglutaryl-CoA lyase deficiency studied using 2-dimensional proton nuclear magnetic resonance spectroscopy

    International Nuclear Information System (INIS)

    1H-NMR spectroscopy has been applied to identify components in the urine of subjects with a deficiency of the enzyme 3-hydroxy-3-methylglutaryl-CoA lyase. One-dimensional spectra of samples from a pair of non-identical twins with this disorder were very similar and are probably diagnostic. The most intense signals were from singlets. Complete assignment of these major components was made possible by the use of 2-dimensional chemical shift correlated spectroscopy since several long-range couplings were detected. 2-dimensional spectroscopic techniques may therefore be of value in the identification of singlets in multicomponent systems. (Auth.)

  20. Purification and properties of alpha-pinene oxide lyase from Nocardia sp. strain P18.3.

    OpenAIRE

    Griffiths, E T; Harries, P C; Jeffcoat, R; Trudgill, P W

    1987-01-01

    alpha-Pinene oxide is an intermediate in the degradation of alpha-pinene by Nocardia sp. strain P18.3 and some Pseudomonas strains. The epoxide is cleaved by a lyase which catalyzes a concerted reaction in which both rings of the bicyclic structure are cleaved with the formation of cis-2-methyl-5-isopropylhexa-2,5-dienal. The enzyme has been purified to homogeneity from Nocardia sp. strain P18.3. It was induced by growth with alpha-pinene and constituted 6 to 7% of the soluble protein of cell...

  1. pH Regulation of Pectate Lyase Secretion Modulates the Attack of Colletotrichum gloeosporioides on Avocado Fruits†

    OpenAIRE

    Yakoby, Nir; Kobiler, Ilana; Dinoor, Amos; Prusky, Dov

    2000-01-01

    Growth of Colletotrichum gloeosporioides in pectolytic enzyme-inducing medium (PEIM) increased the pH of the medium from 3.8 to 6.5. Pectate lyase (PL) secretion was detected when the pH reached 5.8, and the level of secretion increased up to pH 6.5. PL gene (pel) transcript production began at pH 5.0 and increased up to pH 5.7. PL secretion was never detected when the pH of the inducing medium was lower than 5.8 or when C. gloeosporioides hyphae were transferred from PL-secreting conditions ...

  2. Role of cystathionine gamma-lyase in immediate renal impairment and inflammatory response in acute ischemic kidney injury

    OpenAIRE

    Lajos Markó; Szijártó, István A.; Filipovic, Milos R.; Mario Kaßmann; András Balogh; Joon-Keun Park; Lukasz Przybyl; Gabriele N’diaye; Stephanie Krämer; Juliane Anders; Isao Ishii; Müller, Dominik N.; Maik Gollasch

    2016-01-01

    Hydrogen sulfide (H2S) is known to act protectively during renal ischemia/reperfusion injury (IRI). However, the role of the endogenous H2S in acute kidney injury (AKI) is largely unclear. Here, we analyzed the role of cystathionine gamma-lyase (CTH) in acute renal IRI using CTH-deficient (Cth(-/-)) mice whose renal H2S levels were approximately 50% of control (wild-type) mice. Although levels of serum creatinine and renal expression of AKI marker proteins were equivalent between Cth(-/-) and...

  3. Complementation of a phycocyanin-bilin lyase from Synechocystis sp. PCC 6803 with a nucleomorph-encoded open reading frame from the cryptophyte Guillardia theta

    Directory of Open Access Journals (Sweden)

    Nyalwidhe Julius

    2008-05-01

    Full Text Available Abstract Background Cryptophytes are highly compartmentalized organisms, expressing a secondary minimized eukaryotic genome in the nucleomorph and its surrounding remnant cytoplasm, in addition to the cell nucleus, the mitochondrion and the plastid. Because the members of the nucleomorph-encoded proteome may contribute to essential cellular pathways, elucidating nucleomorph-encoded functions is of utmost interest. Unfortunately, cryptophytes are inaccessible for genetic transformations thus far. Therefore the functions of nucleomorph-encoded proteins must be elucidated indirectly by application of methods in genetically accessible organisms. Results Orf222, one of the uncharacterized nucleomorph-specific open reading frames of the cryptophyte Guillardia theta, shows homology to slr1649 of Synechocystis sp. PCC 6803. Recently a further homolog from Synechococcus sp. PCC 7002 was characterized to encode a phycocyanin-β155-bilin lyase. Here we show by insertion mutagenesis that the Synechocystis sp. PCC 6803 slr1649-encoded protein also acts as a bilin lyase, and additionally contributes to linker attachment and/or stability of phycobilisomes. Finally, our results indicate that the phycocyanin-β155-bilin lyase of Synechocystis sp. PCC 6803 can be complemented in vivo by the nucleomorph-encoded open reading frame orf222. Conclusion Our data show that the loss of phycocyanin-lyase function causes pleiotropic effects in Synechocystis sp. PCC 6803 and indicate that after separating from a common ancestor protein, the phycoerythrin lyase from Guillardia theta has retained its capacity to couple a bilin group to other phycobiliproteins. This is a further, unexpected example of the universality of phycobiliprotein lyases.

  4. Two Novel Alliin Lyase (Alliinase Genes from Twisted-Leaf Garlic (Allium obliquum and Mountain Garlic (Allium senescens var. montanum

    Directory of Open Access Journals (Sweden)

    Bogdan DRUGĂ

    2011-11-01

    Full Text Available Alliinase (Alliin lyase EC 4.4.1.4, a pyridoxal phosphate-dependent lyase, represents one of the major protein components of Allium species. The enzyme is a homodimeric glycoprotein and catalyzes the synthesis of allicin (diallyl thiosulfinate, a biologically active compound, pyruvate, and ammonia starting from the specific non-protein sulfur-containing amino acid alliin ((+S-allyl-L-cysteine sulfoxide. Using newly developed specific primers two new alliinase genes from Allium obliquum and Allium senescens ssp. montanum were amplified and sequenced, as well as their homologs, from Allium fistulosum and Allium schoeonoprasum. The G+C content of the alliinase region ranges between that of other dicot plants and that reported in monocot cereal plants, in all four species. Investigations of gene expression revealed a significantly higher enzyme expression level in bulbs than in leaves in all four taxa. The deduced alliinase sequences displayed a high variability among different species, since the lowest sequence similarity was found to be 55.5% between Allium senescens var. montanum and Allium cepa, while the highest similarity is 77.5%, between Allium senescens var. montanum and Allium fistulosum. Leucine is the most common amino acid in all four alliinases, while cysteine is also more frequent that in other enzymes, suggesting a high stability of the molecules due to the possible disulfide bonds.

  5. Crystallization and preliminary X-ray analysis of l-methionine γ-lyase 1 from Entamoeba histolytica

    International Nuclear Information System (INIS)

    l-Methionine γ-lyase 1, a key enzyme in sulfur-containing amino-acid degradation, from the protozoan parasite E. histolytica was crystallized in a form suitable for X-ray structure analysis. l-Methionine γ-lyase (MGL) is a pyridoxal phosphate-dependent enzyme that is involved in the degradation of sulfur-containing amino acids. MGL is an attractive drug target against amoebiasis because the mammalian host of its causative agent Entamoeba histolytica lacks MGL. For the development of anti-amoebic agents based on the structure of MGL, one of two MGL isoenzymes (EhMGL1) was crystallized in the monoclinic space group P21, with unit-cell parameters a = 99.12, b = 85.38, c = 115.37 Å, β = 101.82°. The crystals diffract to beyond 2.0 Å resolution. The presence of a tetramer in the asymmetric unit (4 × 42.4 kDa) gives a Matthews coefficient of 2.8 Å3 Da−1 and a solvent content of 56%. The structure was solved by the molecular-replacement method and structure refinement is now in progress

  6. Crystallization and preliminary X-ray analysis of l-methionine γ-lyase 1 from Entamoeba histolytica

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Dan [Institute for Advanced Biosciences, Keio University, 246-2 Mizukami, Kakuganji, Tsuruoka, Yamagata 997-0052 (Japan); Center for Integrated Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Department of Parasitology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511 (Japan); Karaki, Tsuyoshi; Shimizu, Akira; Kamei, Kaeko; Harada, Shigeharu, E-mail: harada@kit.ac.jp [Graduate School of Science and Technology, Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Nozaki, Tomoyoshi [Department of Parasitology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511 (Japan); Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640 (Japan); Institute for Advanced Biosciences, Keio University, 246-2 Mizukami, Kakuganji, Tsuruoka, Yamagata 997-0052 (Japan)

    2008-08-01

    l-Methionine γ-lyase 1, a key enzyme in sulfur-containing amino-acid degradation, from the protozoan parasite E. histolytica was crystallized in a form suitable for X-ray structure analysis. l-Methionine γ-lyase (MGL) is a pyridoxal phosphate-dependent enzyme that is involved in the degradation of sulfur-containing amino acids. MGL is an attractive drug target against amoebiasis because the mammalian host of its causative agent Entamoeba histolytica lacks MGL. For the development of anti-amoebic agents based on the structure of MGL, one of two MGL isoenzymes (EhMGL1) was crystallized in the monoclinic space group P2{sub 1}, with unit-cell parameters a = 99.12, b = 85.38, c = 115.37 Å, β = 101.82°. The crystals diffract to beyond 2.0 Å resolution. The presence of a tetramer in the asymmetric unit (4 × 42.4 kDa) gives a Matthews coefficient of 2.8 Å{sup 3} Da{sup −1} and a solvent content of 56%. The structure was solved by the molecular-replacement method and structure refinement is now in progress.

  7. The nanostructural characterization of strawberry pectins in pectate lyase or polygalacturonase silenced fruits elucidates their role in softening.

    Science.gov (United States)

    Posé, Sara; Kirby, Andrew R; Paniagua, Candelas; Waldron, Keith W; Morris, Victor J; Quesada, Miguel A; Mercado, José A

    2015-11-01

    To ascertain the role of pectin disassembly in fruit softening, chelated- (CSP) and sodium carbonate-soluble (SSP) pectins from plants with a pectate lyase, FaplC, or a polygalacturonase, FaPG1, downregulated by antisense transformation were characterized at the nanostructural level. Fruits from transgenic plants were firmer than the control, although FaPG1 suppression had a greater effect on firmness. Size exclusion chromatography showed that the average molecular masses of both transgenic pectins were higher than that of the control. Atomic force microscopy analysis of pectins confirmed the higher degree of polymerization as result of pectinase silencing. The mean length values for CSP chains increased from 84 nm in the control to 95.5 and 101 nm, in antisense FaplC and antisense FaPG1 samples, respectively. Similarly, SSP polyuronides were longer in transgenic fruits (61, 67.5 and 71 nm, in the control, antisense FaplC and antisense FaPG1 samples, respectively). Transgenic pectins showed a more complex structure, with a higher percentage of branched chains than the control, especially in the case of FaPG1 silenced fruits. Supramolecular pectin aggregates, supposedly formed by homogalacturonan and rhamnogalacturonan I, were more frequently observed in antisense FaPG1 samples. The larger modifications in the nanostructure of pectins in FaPG1 silenced fruits when compared with antisense pectate lyase plants correlate with the higher impact of polygalacturonase silencing on reducing strawberry fruit softening. PMID:26256334

  8. Comparison of expression, purification and characterization of a new pectate lyase from Phytophthora capsici using two different methods

    Directory of Open Access Journals (Sweden)

    Zhang Xiuguo

    2011-04-01

    Full Text Available Abstract Background Pectate lyases (PELs play an important role in the infection process of plant pathogens and also have a commercial significance in industrial applications. Most of the PELs were expressed as soluble recombinant proteins, while a few recombinant proteins were insoluble. The production of a large-scale soluble recombinant PEL would allow not only a more detailed structural and functional characterization of this enzyme but also may have important applications in the food industry. Results We cloned a new pectate lyase gene (Pcpel2 from Phytophthora capsici. Pcpel2 was constructed by pET system and pMAL system, and both constructs were used to express the PCPEL2 in Escherichia coli BL21 (DE3 pLysS. The expressed products were purified using affinity chromatography and gel filtration chromatography. The purity, specific activity and pathogenicity of the purified PCPEL2 expressed by the pMAL system were higher than the purified PCPEL2 expressed by the pET system. In addition, some other characteristics of the purified PCPEL2 differed from the two systems, such as crystallographic features. Purified PCPEL2 expressed by the pMAL system was crystallized by the hanging-drop vapour-diffusion method at 289 K, and initial crystals were grown. Conclusion The two different methods and comparison presented here would be highly valuable in obtaining an ideal enzyme for the downstream experiments, and supply an useful alternative to purify some insoluble recombinant proteins.

  9. Structural and Functional Studies on Salmonella Typhimurium Ethanolamine Ammonia-Lyase

    Science.gov (United States)

    Bovell, Adonis

    Ethanolamine ammonia-lyase (EAL), a coenzyme-B12 (AdoCbl) dependent bacterial enzyme, catalyzes the deamination of select amino-alcohols by using a radical mechanism. Extensive high-resolution spectroscopic determinations of reactant intermediate-state structures and detailed kinetic and thermodynamic studies have been conducted for the Salmonella typhimurium enzyme. A statistically robust homology model for the full [(EutB-EutC) 2]3 oligomer of S. typhimurium EAL is constructed from the Escherichia coli crystal structure. This structure establishes a platform for detailed, microscopic interpretation of the molecular mechanism of EAL catalysis. The model is used to describe the hierarchy of EutB and EutC subunit interactions in the native oligomer and to guide a genetic and biochemical approach to the long-standing challenge of functional oligomer reconstitution from isolated subunits. The model is used to direct site-directed mutagenesis of EAL, leading to the creation of the EutB-F258W mutant, whose fluorescence is sensitive to the binding of AdoCbl. The AdoCbl-EAL dissociation constant is determined to be 1.2 microM, which places limits on the timescale of cofactor exchange kinetics. A series of cysteine-replaced mutants of EAL was created, and progress was made towards the goal of a mutant EAL for site-directed spin labeling studies. The primary cysteine attachment site in wild-type EAL for the 4-maleimido-TEMPO spin label was identified as EutC-C37. The localization of spin labels on EAL enables the interpretation of electron paramagnetic resonance (EPR) studies that probe distal effects on protein structure caused by cofactor binding. Previously determined rate constants for decay of the cryotrapped substrate radical, and kcat values at ambient temperature, for 1H- and 2H-labelled substrate, are united in a single model that describes the sequential radical rearrangement and hydrogen atom transfer steps, from 190 to 295 K. The model indicates that hydrogen

  10. Mechanistic studies of the spore photoproduct lyase via a single cysteine mutation.

    Science.gov (United States)

    Yang, Linlin; Lin, Gengjie; Nelson, Renae S; Jian, Yajun; Telser, Joshua; Li, Lei

    2012-09-11

    5-Thyminyl-5,6-dihydrothymine (also called spore photoproduct or SP) is the exclusive DNA photodamage product in bacterial endospores. It is repaired by a radical SAM (S-adenosylmethionine) enzyme, the spore photoproduct lyase (SPL), at the bacterial early germination phase. Our previous studies proved that SPL utilizes the 5'-dA• generated by the SAM cleavage reaction to abstract the H(6proR) atom to initiate the SP repair process. The resulting thymine allylic radical was suggested to take an H atom from an unknown protein source, most likely cysteine 141. Here we show that C141 can be readily alkylated in the native SPL by an iodoacetamide treatment, suggesting that it is accessible to the TpT radical. SP repair by the SPL C141A mutant yields TpTSO(2)(-) and TpT simultaneously from the very beginning of the reaction; no lag phase is observed for TpTSO(2)(-) formation. Should any other protein residue serve as the H donor, its presence would result in TpT being the major product at least for the first enzyme turnover. These observations provide strong evidence to support C141 as the direct H atom donor. Moreover, because of the lack of this intrinsic H donor, the C141A mutant produces TpT via an unprecedented thymine cation radical reduction (proton-coupled electron transfer) process, contrasting to the H atom transfer mechanism in the wild-type (WT) SPL reaction. The C141A mutant repairs SP at a rate that is ~3-fold slower than that of the WT enzyme. Formation of TpTSO(2)(-) and TpT exhibits a V(max) deuterium kinetic isotope effect (KIE) of 1.7 ± 0.2, which is smaller than the (D)V(max) KIE of 2.8 ± 0.3 determined for the WT SPL reaction. These findings suggest that removing the intrinsic H atom donor disturbs the rate-limiting process during enzyme catalysis. As expected, the prereduced C141A mutant supports only ~0.4 turnover, which is in sharp contrast to the >5 turnovers exhibited by the WT SPL reaction, suggesting that the enzyme catalytic cycle (SAM

  11. Essential histidine pairs indicate conserved haem binding in epsilonproteobacterial cytochrome c haem lyases

    Science.gov (United States)

    Kern, Melanie; Scheithauer, Juliane; Kranz, Robert G.; Simon, Jörg

    2010-01-01

    Bacterial cytochrome c maturation occurs at the outside of the cytoplasmic membrane, requires transport of haem b across the membrane, and depends on membrane-bound cytochrome c haem lyase (CCHL), an enzyme that catalyses covalent attachment of haem b to apocytochrome c. Epsilonproteobacteria such as Wolinella succinogenes use the cytochrome c biogenesis system II and contain unusually large CCHL proteins of about 900 amino acid residues that appear to be fusions of the CcsB and CcsA proteins found in other bacteria. CcsBA-type CCHLs have been proposed to act as haem transporters that contain two haem b coordination sites located at different sides of the membrane and formed by histidine pairs. W. succinogenes cells contain three CcsBA-type CCHL isoenzymes (NrfI, CcsA1 and CcsA2) that are known to differ in their specificity for apocytochromes and apparently recognize different haem c binding motifs such as CX2CH (by CcsA2), CX2CK (by NrfI) and CX15CH (by CcsA1). In this study, conserved histidine residues were individually replaced by alanine in each of the W. succinogenes CCHLs. Characterization of NrfI and CcsA1 variants in W. succinogenes demonstrated that a set of four histidines is essential for maturing the dedicated multihaem cytochromes c NrfA and MccA, respectively. The function of W. succinogenes CcsA2 variants produced in Escherichia coli was also found to depend on each of these four conserved histidine residues. The presence of imidazole in the growth medium of both W. succinogenes and E. coli rescued the cytochrome c biogenesis activity of most histidine variants, albeit to different extents, thereby implying the presence of two functionally distinct histidine pairs in each CCHL. The data support a model in which two conserved haem b binding sites are involved in haem transport catalysed by CcsBA-type CCHLs. PMID:20705660

  12. Correlation of Rutin Accumulation with 3-O-Glucosyl Transferase and Phenylalanine Ammonia-lyase Activities During the Ripening of Tomato Fruit

    NARCIS (Netherlands)

    Capanoglu, E.; Beekwilder, J.; Matros, A.; Boyacioglu, D.; Hall, R.D.; Mock, H.P.

    2012-01-01

    In tomato, the predominant flavonoid is quercetin-3-rutinoside (rutin). In this study, we aim to investigate the phenylalanine ammonia-lyase (PAL) and the quercetin-3-O-glucosyl transferase (3-GT) reactions in the formation of rutin during tomato fruit ripening. Tomatoes of the Moneymaker variety at

  13. The structure of RdDddP from Roseobacter denitrificans reveals that DMSP lyases in the DddP-family are metalloenzymes.

    Directory of Open Access Journals (Sweden)

    Jan-Hendrik Hehemann

    Full Text Available Marine microbes degrade dimethylsulfoniopropionate (DMSP, which is produced in large quantities by marine algae and plants, with DMSP lyases into acrylate and the gas dimethyl sulfide (DMS. Approximately 10% of the DMS vents from the sea into the atmosphere and this emission returns sulfur, which arrives in the sea through rivers and runoff, back to terrestrial systems via clouds and rain. Despite their key role in this sulfur cycle DMSP lyases are poorly understood at the molecular level. Here we report the first X-ray crystal structure of the putative DMSP lyase RdDddP from Roseobacter denitrificans, which belongs to the abundant DddP family. This structure, determined to 2.15 Å resolution, shows that RdDddP is a homodimeric metalloprotein with a binuclear center of two metal ions located 2.7 Å apart in the active site of the enzyme. Consistent with the crystallographic data, inductively coupled plasma mass spectrometry (ICP-MS and total reflection X-ray fluorescence (TRXF revealed the bound metal species to be primarily iron. A 3D structure guided analysis of environmental DddP lyase sequences elucidated the critical residues for metal binding are invariant, suggesting all proteins in the DddP family are metalloenzymes.

  14. Establishment of chondroitin B lyase-based analytical methods for sensitive and quantitative detection of dermatan sulfate in heparin.

    Science.gov (United States)

    Wu, Jingjun; Ji, Yang; Su, Nan; Li, Ye; Liu, Xinxin; Mei, Xiang; Zhou, Qianqian; Zhang, Chong; Xing, Xin-Hui

    2016-06-25

    Dermatan sulfate (DS) is one of the hardest impurities to remove from heparin products due to their high structural similarity. The development of a sensitive and feasible method for quantitative detection of DS in heparin is essential to ensure the clinical safety of heparin pharmaceuticals. In the current study, based on the substrate specificity of chondroitin B lyase, ultraviolet spectrophotometric and strong anion-exchange high-performance liquid chromatographic methods were established for detection of DS in heparin. The former method facilitated analysis in heparin with DS concentrations greater than 0.1mgmL(-1) at 232nm, with good linearity, precision and recovery. The latter method allowed sensitive and accurate detection of DS at concentrations lower than 0.1mgmL(-1), exhibiting good linearity, precision and recovery. The linear range of DS detection using the latter method was between 0.01 and 0.5mgmL(-1). PMID:27083825

  15. Polyphenol Oxidase, Peroxidase and Phenylalanine Ammonium Lyase Induced in Postharvest Peach Fruits by Inoculation with Pichia membranefaciens or Rhizopus stolonifer

    Institute of Scientific and Technical Information of China (English)

    QIN Guo-zheng; TIAN Shi-ping; LIU Hai-bo; XU Yong

    2002-01-01

    Rhizopus rot of peach fruits could be significantly suppressed by Pichia membranefaciens.Polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonium-lyase (PAL) activities inducedby inoculation with P. membranefaciens or R. stolonifer were studied in postharvest peach fruits. The activ-ities of PPO and PAL in peaches increased significantly after being inoculated with P. membranefaciens + R.stolonifer by 24 h, the activities maintained at a high level throughout the experiment. Under the condition ofinfected with R. stolonifer alone, activity of PPO and PAL could also increased, but the levels were lowerthan those treated with P. membranefaciens+ R. stolonifer. However, fruits inoculaed with P. membrane-faciens+ R. stolonifer or R. stolonifer alone did not stimulated POD activity. The results suggest that theactivation of these defense enzymes is involved in the action of P. membranefaciens against R. stolonifer.

  16. Preliminary structural investigations of the Eut-L shell protein of the ethanolamine ammonia-lyase metabolosome of Escherichia coli

    International Nuclear Information System (INIS)

    Preliminary X-ray analysis of crystals of the bacterial microcompartment shell protein Eut-L from Escherichia coli is reported. The ethanolamine ammonia-lyase microcompartment is composed of five different shell proteins that have been proposed to assemble into symmetrically shaped polyhedral particles of varying sizes. Here, preliminary X-ray analysis of crystals of the bacterial microcompartment shell protein Eut-L from Escherichia coli is reported. Cloning, overexpression and purification resulted in highly pure protein that crystallized readily under many different conditions. In all cases the protein forms thin hexagonal plate-shaped crystals belonging to space group P3 that are of unusually high stability against different solvent conditions. The crystals diffracted to a resolution of 2.0 Å using synchrotron radiation but proved to be radiation-sensitive. Preparations of heavy-atom-derivatized crystals for use in determining the three-dimensional structure are under way

  17. Structural dynamics of a methionine γ-lyase for calicheamicin biosynthesis: Rotation of the conserved tyrosine stacking with pyridoxal phosphate.

    Science.gov (United States)

    Cao, Hongnan; Tan, Kemin; Wang, Fengbin; Bigelow, Lance; Yennamalli, Ragothaman M; Jedrzejczak, Robert; Babnigg, Gyorgy; Bingman, Craig A; Joachimiak, Andrzej; Kharel, Madan K; Singh, Shanteri; Thorson, Jon S; Phillips, George N

    2016-05-01

    CalE6 from Micromonospora echinospora is a (pyridoxal 5' phosphate) PLP-dependent methionine γ-lyase involved in the biosynthesis of calicheamicins. We report the crystal structure of a CalE6 2-(N-morpholino)ethanesulfonic acid complex showing ligand-induced rotation of Tyr100, which stacks with PLP, resembling the corresponding tyrosine rotation of true catalytic intermediates of CalE6 homologs. Elastic network modeling and crystallographic ensemble refinement reveal mobility of the N-terminal loop, which involves both tetrameric assembly and PLP binding. Modeling and comparative structural analysis of PLP-dependent enzymes involved in Cys/Met metabolism shine light on the functional implications of the intrinsic dynamic properties of CalE6 in catalysis and holoenzyme maturation. PMID:27191010

  18. Analysis of promoter activity of members of the PECTATE LYASE-LIKE (PLL gene family in cell separation in Arabidopsis

    Directory of Open Access Journals (Sweden)

    van Nocker Steven

    2010-07-01

    Full Text Available Abstract Background Pectate lyases depolymerize pectins by catalyzing the eliminative cleavage of α-1,4-linked galacturonic acid. Pectate lyase-like (PLL genes make up among the largest and most complex families in plants, but their cellular and organismal roles have not been well characterized, and the activity of these genes has been assessed only at the level of entire organs or plant parts, potentially obscuring important sub-organ or cell-type-specific activities. As a first step to understand the potential functional diversity of PLL genes in plants and specificity of individual genes, we utilized a reporter gene approach to document the spatial and temporal promoter activity for 23 of the 26 members of the Arabidopsis thaliana (Arabidopsis PLL gene family throughout development, focusing on processes involving cell separation. Results Numerous PLL promoters directed activity in localized domains programmed for cell separation, such as the abscission zones of the sepal, petal, stamen, and seed, as well as the fruit dehiscence zone. Several drove activity in cell types expected to facilitate separation, including the style and root endodermal and cortical layers during lateral root emergence. However, PLL promoters were active in domains not obviously programmed for separation, including the stipule, hydathode and root axis. Nearly all PLL promoters showed extensive overlap of activity in most of the regions analyzed. Conclusions Our results document potential for involvement of PLL genes in numerous aspects of growth and development both dependent and independent of cell separation. Although the complexity of the PLL gene family allows for enormous potential for gene specialization through spatial or temporal regulation, the high degree of overlap of activity among the PLL promoters suggests extensive redundancy. Alternatively, functional specialization might be determined at the post-transcriptional or protein level.

  19. The Sphingosine-1-Phosphate Lyase (LegS2) Contributes to the Restriction of Legionella pneumophila in Murine Macrophages.

    Science.gov (United States)

    Abu Khweek, Arwa; Kanneganti, Apurva; Guttridge D, Denis C; Amer, Amal O

    2016-01-01

    L. pneumophila is the causative agent of Legionnaires' disease, a human illness characterized by severe pneumonia. In contrast to those derived from humans, macrophages derived from most mouse strains restrict L. pneumophila replication. The restriction of L. pneumophila replication has been shown to require bacterial flagellin, a component of the type IV secretion system as well as the cytosolic NOD-like receptor (NLR) Nlrc4/ Ipaf. These events lead to caspase-1 activation which, in turn, activates caspase-7. Following caspase-7 activation, the phagosome-containing L. pneumophila fuses with the lysosome, resulting in the restriction of L. pneumophila growth. The LegS2 effector is injected by the type IV secretion system and functions as a sphingosine 1-phosphate lyase. It is homologous to the eukaryotic sphingosine lyase (SPL), an enzyme required in the terminal steps of sphingolipid metabolism. Herein, we show that mice Bone Marrow-Derived Macrophages (BMDMs) and human Monocyte-Derived Macrophages (hMDMs) are more permissive to L. pneumophila legS2 mutants than wild-type (WT) strains. This permissiveness to L. pneumophila legS2 is neither attributed to abolished caspase-1, caspase-7 or caspase-3 activation, nor due to the impairment of phagosome-lysosome fusion. Instead, an infection with the legS2 mutant resulted in the reduction of some inflammatory cytokines and their corresponding mRNA; this effect is mediated by the inhibition of the nuclear transcription factor kappa-B (NF-κB). Moreover, BMDMs infected with L. pneumophila legS2 mutant showed elongated mitochondria that resembles mitochondrial fusion. Therefore, the absence of LegS2 effector is associated with reduced NF-κB activation and atypical morphology of mitochondria. PMID:26741365

  20. Structural Insights into an Oxalate-producing Serine Hydrolase with an Unusual Oxyanion Hole and Additional Lyase Activity.

    Science.gov (United States)

    Oh, Juntaek; Hwang, Ingyu; Rhee, Sangkee

    2016-07-15

    In Burkholderia species, the production of oxalate, an acidic molecule, is a key event for bacterial growth in the stationary phase. Oxalate plays a central role in maintaining environmental pH, which counteracts inevitable population-collapsing alkaline toxicity in amino acid-based culture medium. In the phytopathogen Burkholderia glumae, two enzymes are responsible for oxalate production. First, the enzyme oxalate biosynthetic component A (ObcA) catalyzes the formation of a tetrahedral C6-CoA adduct from the substrates acetyl-CoA and oxaloacetate. Then the ObcB enzyme liberates three products from the C6-CoA adduct: oxalate, acetoacetate, and CoA. Interestingly, these two stepwise reactions are catalyzed by a single bifunctional enzyme, Obc1, from Burkholderia thailandensis and Burkholderia pseudomallei Obc1 has an ObcA-like N-terminal domain and shows ObcB activity in its C-terminal domain despite no sequence homology with ObcB. We report the crystal structure of Obc1 in its apo and glycerol-bound form at 2.5 Å and 2.8 Å resolution, respectively. The Obc1 N-terminal domain is essentially identical both in structure and function to that of ObcA. Its C-terminal domain has an α/β hydrolase fold that has a catalytic triad for oxalate production and a novel oxyanion hole distinct from the canonical HGGG motif in other α/β hydrolases. Functional analyses through mutagenesis studies suggested that His-934 is an additional catalytic acid/base for its lyase activity and liberates two additional products, acetoacetate and CoA. These results provide structural and functional insights into bacterial oxalogenesis and an example of divergent evolution of the α/β hydrolase fold, which has both hydrolase and lyase activity. PMID:27226606

  1. The Sphingosine-1-Phosphate Lyase (LegS2) Contributes to the Restriction of Legionella pneumophila in Murine Macrophages

    Science.gov (United States)

    Abu Khweek, Arwa; Kanneganti, Apurva; C. Guttridge D, Denis; Amer, Amal O.

    2016-01-01

    L. pneumophila is the causative agent of Legionnaires’ disease, a human illness characterized by severe pneumonia. In contrast to those derived from humans, macrophages derived from most mouse strains restrict L. pneumophila replication. The restriction of L. pneumophila replication has been shown to require bacterial flagellin, a component of the type IV secretion system as well as the cytosolic NOD-like receptor (NLR) Nlrc4/ Ipaf. These events lead to caspase-1 activation which, in turn, activates caspase-7. Following caspase-7 activation, the phagosome-containing L. pneumophila fuses with the lysosome, resulting in the restriction of L. pneumophila growth. The LegS2 effector is injected by the type IV secretion system and functions as a sphingosine 1-phosphate lyase. It is homologous to the eukaryotic sphingosine lyase (SPL), an enzyme required in the terminal steps of sphingolipid metabolism. Herein, we show that mice Bone Marrow-Derived Macrophages (BMDMs) and human Monocyte-Derived Macrophages (hMDMs) are more permissive to L. pneumophila legS2 mutants than wild-type (WT) strains. This permissiveness to L. pneumophila legS2 is neither attributed to abolished caspase-1, caspase-7 or caspase-3 activation, nor due to the impairment of phagosome-lysosome fusion. Instead, an infection with the legS2 mutant resulted in the reduction of some inflammatory cytokines and their corresponding mRNA; this effect is mediated by the inhibition of the nuclear transcription factor kappa-B (NF-κB). Moreover, BMDMs infected with L. pneumophila legS2 mutant showed elongated mitochondria that resembles mitochondrial fusion. Therefore, the absence of LegS2 effector is associated with reduced NF-κB activation and atypical morphology of mitochondria. PMID:26741365

  2. Purification, molecular cloning, and expression of 2-hydroxyphytanoyl- CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during à-oxidation of 3- methyl-branched fatty acids

    CERN Document Server

    Foulon, V; Croes, K; Waelkens, E

    1999-01-01

    Purification, molecular cloning, and expression of 2-hydroxyphytanoyl- CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during à-oxidation of 3- methyl-branched fatty acids

  3. Residues C123 and D58 of the 2-methylisocitrate lyase (PrpB) enzyme of Salmonella enterica are essential for catalysis.

    Science.gov (United States)

    Grimek, T L; Holden, H; Rayment, I; Escalante-Semerena, J C

    2003-08-01

    The prpB gene of Salmonella enterica serovar Typhimurium LT2 encodes a protein with 2-methylisocitrate (2-MIC) lyase activity, which cleaves 2-MIC into pyruvate and succinate during the conversion of propionate to pyruvate via the 2-methylcitric acid cycle. This paper reports the isolation and kinetic characterization of wild-type and five mutant PrpB proteins. Wild-type PrpB protein had a molecular mass of approximately 32 kDa per subunit, and the biologically active enzyme was comprised of four subunits. Optimal 2-MIC lyase activity was measured at pH 7.5 and 50 degrees C, and the reaction required Mg(2+) ions; equimolar concentrations of Mn(2+) ions were a poor substitute for Mg(2+) (28% specific activity). Dithiothreitol (DTT) or reduced glutathione (GSH) was required for optimal activity; the role of DTT or GSH was apparently not to reduce disulfide bonds, since the disulfide-specific reducing agent Tris(2-carboxyethyl)phosphine hydrochloride failed to substitute for DTT or GSH. The K(m) of PrpB for 2-MIC was measured at 19 micro M, with a k(cat) of 105 s(-1). Mutations in the prpB gene were introduced by site-directed mutagenesis based on the active-site residues deemed important for catalysis in the closely related phosphoenolpyruvate mutase and isocitrate lyase enzymes. Residues D58, K121, C123, and H125 of PrpB were changed to alanine, and residue R122 was changed to lysine. Nondenaturing polyacrylamide gel electrophoresis indicated that all mutant PrpB proteins retained the same oligomeric state of the wild-type enzyme, which is known to form tetramers. The PrpB(K121A), PrpB(H125A), and PrpB(R122K) mutant proteins formed enzymes that had 1,050-, 750-, and 2-fold decreases in k(cat) for 2-MIC lyase activity, respectively. The PrpB(D58A) and PrpB(C123A) proteins formed tetramers that displayed no detectable 2-MIC lyase activity indicating that both of these residues are essential for catalysis. Based on the proposed mechanism of the closely related

  4. A rechargeable carbon-oxygen battery

    DEFF Research Database (Denmark)

    2014-01-01

    The invention relates to a rechargeable battery and a method to operate a rechargeable battery having high efficiency and high energy density for storing energy. The battery stores electrical energy in the bonds of carbon and oxygen atoms by converting carbon dioxide into solid carbon and oxygen....

  5. Solidification of carbon-oxygen white dwarfs

    Science.gov (United States)

    Schatzman, E.

    1982-01-01

    The internal structure of white dwarfs is discussed. Highly correlated plasmas are reviewed. Implications for phase separation in the core of cooling white dwarfs are considered. The consequences for evolution of white dwarfs are addressed.

  6. Integration of Apo-α-Phycocyanin into Phycobilisomes and Its Association with FNRL in the Absence of the Phycocyanin α-Subunit Lyase (CpcF) in Synechocystis sp. PCC 6803

    OpenAIRE

    Zhang, Pengpeng; Frankel, Laurie K.; Terry M Bricker

    2014-01-01

    Phycocyanin is an important component of the phycobilisome, which is the principal light-harvesting complex in cyanobacteria. The covalent attachment of the phycocyanobilin chromophore to phycocyanin is catalyzed by the enzyme phycocyanin lyase. The photosynthetic properties and phycobilisome assembly state were characterized in wild type and two mutants which lack holo-α-phycocyanin. Insertional inactivation of the phycocyanin α-subunit lyase (ΔcpcF mutant) prevents the ligation of phycocyan...

  7. Trifluoromethionine, a Prodrug Designed against Methionine γ-Lyase-Containing Pathogens, Has Efficacy In Vitro and In Vivo against Trichomonas vaginalis

    OpenAIRE

    Coombs, Graham H.; Mottram, Jeremy C.

    2001-01-01

    Methionine γ-lyase, the enzyme which catalyzes the single-step conversion of methionine to α-ketobutyrate, ammonia, and methanethiol, is highly active in many anaerobic pathogenic microorganisms but has no counterpart in mammals. This study tested the hypothesis that this pathogen-specific enzyme can be exploited as a drug target by prodrugs that are exclusively activated by it. Trifluoromethionine was confirmed as such a prodrug and shown to be highly toxic in vitro to the anaerobic protozoa...

  8. Hydroxynitrile Lyases with α/β-Hydrolase Fold: Two Enzymes with Almost Identical 3D Structures but Opposite Enantioselectivities and Different Reaction Mechanisms

    OpenAIRE

    Andexer, Jennifer N; Staunig, Nicole; Eggert, Thorsten; Kratky, Christoph; Pohl, Martina; Gruber, Karl

    2012-01-01

    Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins to yield hydrocyanic acid (HCN) and the respective carbonyl compound and are key enzymes in the process of cyanogenesis in plants. In organic syntheses, HNLs are used as biocatalysts for the formation of enantiopure cyanohydrins. We determined the structure of the recently identified, R-selective HNL from Arabidopsis thaliana (AtHNL) at a crystallographic resolution of 2.5 Å. The structure exhibits an α/β-hydrolase fold, very ...

  9. Reduced Photoinhibition under Low Irradiance Enhanced Kacip Fatimah (Labisia pumila Benth) Secondary Metabolites, Phenyl Alanine Lyase and Antioxidant Activity

    OpenAIRE

    Jaafar, Hawa Z. E.; Mohd Hafiz Ibrahim

    2012-01-01

    A randomized complete block design experiment was designed to characterize the relationship between production of total flavonoids and phenolics, anthocyanin, photosynthesis, maximum efficiency of photosystem II (Fv/Fm), electron transfer rate (Fm/Fo), phenyl alanine lyase activity (PAL) and antioxidant (DPPH) in Labisia pumila var. alata, under four levels of irradiance (225, 500, 625 and 900 µmol/m2/s) for...

  10. Prognostic Value of Malic Enzyme and ATP-Citrate Lyase in Non-Small Cell Lung Cancer of the Young and the Elderly

    OpenAIRE

    Csanadi, Agnes; Kayser, Claudia; Donauer, Marcel; Gumpp, Vera; Aumann, Konrad; Rawluk, Justyna; Prasse, Antje; zur Hausen, Axel; Wiesemann, Sebastian; Werner, Martin; Kayser, Gian

    2015-01-01

    Background Lung cancer is the leading cause of death among malignancies worldwide. Understanding its biology is therefore of pivotal importance to improve patient’s prognosis. In contrast to non-neoplastic tissues, cancer cells utilize glucose mainly for production of basic cellular modules ‘(i.e. nucleotides, aminoacids, fatty acids). In cancer, Malic enzyme (ME) and ATP-citrate lyase (ACLY) are key enzymes linking aerobic glycolysis and fatty acid synthesis and may therefore be of biologica...

  11. Uncovering divergent evolution of α/β-hydrolases: a surprising residue substitution needed to convert Hevea brasiliensis hydroxynitrile lyase into an esterase

    OpenAIRE

    Nedrud, David M.; Lin, Hui; Lopez, Gilsinia; Padhi, Santosh K.; Legatt, Graig A.; Kaz-lauskas, Romas J.

    2014-01-01

    Hevea brasiliensis hydroxynitrile lyase (HbHNL) and salicylic acid binding protein 2 (SABP2, an esterase) share 45% amino acid sequence identity, the same protein fold, and even the same catalytic triad of Ser-His-Asp. However, they catalyze different reactions: cleavage of hydroxynitriles and hydrolysis of esters, respectively. To understand how other active site differences in the two enzymes enable the same catalytic triad to catalyze different reactions, we substituted amino acid residues...

  12. ¹³C metabolic flux analysis identifies an unusual route for pyruvate dissimilation in mycobacteria which requires isocitrate lyase and carbon dioxide fixation.

    Directory of Open Access Journals (Sweden)

    Dany J V Beste

    2011-07-01

    Full Text Available Mycobacterium tuberculosis requires the enzyme isocitrate lyase (ICL for growth and virulence in vivo. The demonstration that M. tuberculosis also requires ICL for survival during nutrient starvation and has a role during steady state growth in a glycerol limited chemostat indicates a function for this enzyme which extends beyond fat metabolism. As isocitrate lyase is a potential drug target elucidating the role of this enzyme is of importance; however, the role of isocitrate lyase has never been investigated at the level of in vivo fluxes. Here we show that deletion of one of the two icl genes impairs the replication of Mycobacterium bovis BCG at slow growth rate in a carbon limited chemostat. In order to further understand the role of isocitrate lyase in the central metabolism of mycobacteria the effect of growth rate on the in vivo fluxes was studied for the first time using ¹³C-metabolic flux analysis (MFA. Tracer experiments were performed with steady state chemostat cultures of BCG or M. tuberculosis supplied with ¹³C labeled glycerol or sodium bicarbonate. Through measurements of the ¹³C isotopomer labeling patterns in protein-derived amino acids and enzymatic activity assays we have identified the activity of a novel pathway for pyruvate dissimilation. We named this the GAS pathway because it utilizes the Glyoxylate shunt and Anapleurotic reactions for oxidation of pyruvate, and Succinyl CoA synthetase for the generation of succinyl CoA combined with a very low flux through the succinate--oxaloacetate segment of the tricarboxylic acid cycle. We confirm that M. tuberculosis can fix carbon from CO₂ into biomass. As the human host is abundant in CO₂ this finding requires further investigation in vivo as CO₂ fixation may provide a point of vulnerability that could be targeted with novel drugs. This study also provides a platform for further studies into the metabolism of M. tuberculosis using ¹³C-MFA.

  13. Production of Salicylic Acid Precursors Is a Major Function of Phenylalanine Ammonia-Lyase in the Resistance of Arabidopsis to Peronospora parasitica

    OpenAIRE

    Slusarenko, A. J.; Mauch-Mani, Brigitte

    2011-01-01

    Arabidopsis ecotype Columbia (Col-0) seedlings, transformed with a phenylalanine ammonia-lyase 1 promoter (PAL1)--glucuronidase (GUS) reporter construct, were inoculated with virulent and avirulent isolates of Peronospora parasitica. The PAL1 promoter was constitutively active in the light in vascular tissue but was induced only in the vicinity of fungal structures in the incompatible interaction. A double-staining procedure was developed to distinguish between GUS activity and fungal struct...

  14. Biochemical Properties of Pectate Lyases Produced by Three Different Bacillus Strains Isolated from Fermenting Cocoa Beans and Characterization of Their Cloned Genes▿ †

    OpenAIRE

    Ouattara, Honoré G.; Reverchon, Sylvie; Niamke, Sébastien L.; Nasser, William

    2010-01-01

    Pectinolytic enzymes play an important role in cocoa fermentation. In this study, we characterized three extracellular pectate lyases (Pels) produced by bacilli isolated from fermenting cocoa beans. These enzymes, named Pel-22, Pel-66, and Pel-90, were synthesized by Bacillus pumilus BS22, Bacillus subtilis BS66, and Bacillus fusiformis BS90, respectively. The three Pels were produced under their natural conditions and purified from the supernatants using a one-step chromatography method. The...

  15. Expression, purification, crystallization and preliminary X-ray analysis of the polysaccharide lyase RB5312 from the marine planctomycete Rhodopirellula baltica

    International Nuclear Information System (INIS)

    This study describes the crystallization and preliminary X-ray analysis of the family PL1 polysaccharide lyase RB5312 from the marine bacterium R. baltica. Purified recombinant protein was crystallized; the crystals belonged to space group P212121 and diffracted X-rays to a resolution of 1.8 Å. Polysaccharide lyases belonging to family PL1 act on pectins. These anionic polymers are usually produced by terrestrial plants and therefore pectinolytic enzymes are not frequently observed in marine microorganisms. The protein RB5312 from the marine bacterium Rhodopirellula baltica is distantly related to family PL1 pectate lyases, but its exact function is unclear. In this study, the expression and purification of a recombinant form of RB5312 are described. This protein was crystallized using the hanging-drop vapour-diffusion method. The crystals belongs to space group P212121, with unit-cell parameters a = 39.05, b = 144.05, c = 153.97 Å, α = β = γ = 90°. A complete data set was collected to 1.8 Å resolution from a native crystal

  16. Uncovering divergent evolution of α/β-hydrolases: a surprising residue substitution needed to convert Hevea brasiliensis hydroxynitrile lyase into an esterase.

    Science.gov (United States)

    Nedrud, David M; Lin, Hui; Lopez, Gilsinia; Padhi, Santosh K; Legatt, Graig A; Kaz-Lauskas, Romas J

    2014-11-01

    Hevea brasiliensis hydroxynitrile lyase (HbHNL) and salicylic acid binding protein 2 (SABP2, an esterase) share 45% amino acid sequence identity, the same protein fold, and even the same catalytic triad of Ser-His-Asp. However, they catalyze different reactions: cleavage of hydroxynitriles and hydrolysis of esters, respectively. To understand how other active site differences in the two enzymes enable the same catalytic triad to catalyze different reactions, we substituted amino acid residues in HbHNL with the corresponding residues from SABP2, expecting hydroxynitrile lyase activity to decrease and esterase activity to increase. Previous mechanistic studies and x-ray crystallography suggested that esterase activity requires removal of an active site lysine and threonine from the hydroxynitrile lyase. The Thr11Gly Lys236Gly substitutions in HbHNL reduced hydroxynitrile lyase activity for cleavage of mandelonitrile 100-fold, but increased esterase activity only threefold to kcat ~ 0.1 min(-1) for hydrolysis of p-nitrophenyl acetate. Adding a third substitution - Glu79His - increased esterase activity more than tenfold to kcat ~ 1.6 min(-1). The specificity constant (kcat/KM) for this triple substitution variant versus wild type HbHNL shifted more than one million-fold from hydroxynitrile lyase activity (acetone cyanohydrin substrate) to esterase activity (p-nitrophenyl acetate substrate). The contribution of Glu79His to esterase activity was surprising since esterases and lipases contain many different amino acids at this position, including glutamate. Saturation mutagenesis at position 79 showed that 13 of 19 possible amino acid substitutions increased esterase activity, suggesting that removal of glutamate, not addition of histidine, increased esterase activity. Molecular modeling indicates that Glu79 disrupts esterase activity in HbHNL when its negatively charged side chain distorts the orientation of the catalytic histidine. Naturally occurring glutamate at the

  17. Phenylalanine ammonia-lyase (pal) and peroxidase activity in brown rust infected tissues of pakistani wheat cultivars

    International Nuclear Information System (INIS)

    Besides other factors resistance and susceptibility is the outcome of biochemical processes such as activities of defense-related enzymes. So in this study, Phenylalanine ammonia-lyase (PAL) and Peroxidase activity of resistant (Inqilab-91) and susceptible (Kirin-95) wheat cultivars were determined through spectrophotometer to address the biochemical aspect related to the disease after 8 hours, 24 hours, 48 hours and 72 hours of leaf rust inoculation. The results have shown that these enzymes were present in both the resistant and susceptible cultivars but the activity was more pronounced in the resistant one. The effect of PAL and peroxidase activity was also investigated among inoculated and uninoculated plants within the same cultivar. The activity of both PAL and peroxidase were more significant in inoculated ones. The results have shown that the after 72 hours of inoculation Inqilab-91 had more PAL activity i.e., 5.47 IU/ml/min than in Kirin-95 i.e., 2.08 IU/ml/min at 270 nm. While peroxidase activity in Inqilab-91 was 6.41 IU/ml/min and in Kirin-95, 3.66 IU/ml/min after 72 hours of inoculation, observed under 470 nm wavelength. Increase in one's activity increases the other enzyme's activity. The activity was more prominent after 72 hours of infection as pathogen had successfully established itself in the host plant tissue. The activities of these enzymes act as plants active defense mechanism against the attack of pathogen. (author)

  18. Role of Cystathionine Gamma-Lyase in Immediate Renal Impairment and Inflammatory Response in Acute Ischemic Kidney Injury.

    Science.gov (United States)

    Markó, Lajos; Szijártó, István A; Filipovic, Milos R; Kaßmann, Mario; Balogh, András; Park, Joon-Keun; Przybyl, Lukasz; N'diaye, Gabriele; Krämer, Stephanie; Anders, Juliane; Ishii, Isao; Müller, Dominik N; Gollasch, Maik

    2016-01-01

    Hydrogen sulfide (H2S) is known to act protectively during renal ischemia/reperfusion injury (IRI). However, the role of the endogenous H2S in acute kidney injury (AKI) is largely unclear. Here, we analyzed the role of cystathionine gamma-lyase (CTH) in acute renal IRI using CTH-deficient (Cth(-/-)) mice whose renal H2S levels were approximately 50% of control (wild-type) mice. Although levels of serum creatinine and renal expression of AKI marker proteins were equivalent between Cth(-/-) and control mice, histological analysis revealed that IRI caused less renal tubular damage in Cth(-/-) mice. Flow cytometric analysis revealed that renal population of infiltrated granulocytes/macrophages was equivalent in these mice. However, renal expression levels of certain inflammatory cytokines/adhesion molecules believed to play a role in IRI were found to be lower after IRI only in Cth(-/-) mice. Our results indicate that the systemic CTH loss does not deteriorate but rather ameliorates the immediate AKI outcome probably due to reduced inflammatory responses in the kidney. The renal expression of CTH and other H2S-producing enzymes was markedly suppressed after IRI, which could be an integrated adaptive response for renal cell protection. PMID:27273292

  19. Activation and stabilization of the hydroperoxide lyase enzymatic extract from mint leaves (Mentha spicata) using selected chemical additives.

    Science.gov (United States)

    Akacha, Najla B; Karboune, Salwa; Gargouri, Mohamed; Kermasha, Selim

    2010-03-01

    The effects of selected lyoprotecting excipients and chemical additives on the specific activity and the thermal stability of the hydroperoxide lyase (HPL) enzymatic extract from mint leaves were investigated. The addition of KCl (5%, w/w) and dextran (2.5%, w/w) to the enzymatic extract, prior to lyophilization, increased the HPL specific activity by 2.0- and 1.2-fold, respectively, compared to the control lyophilized extract. From half-life time (t (1/2)), it can be seen that KCl has enhanced the HPL stability by 1.3- to 2.3-fold, during long-period storage at -20 degrees Celsius and 4 degrees Celsius. Among the selected additives used throughout this study, glycine appeared to be the most effective one. In addition to the activation effect conferred by glycine, it also enhanced the HPL thermal stability. In contrast, polyhydroxyl-containing additives were not effective for stabilizing the HPL enzymatic extract. On the other hand, there was no signification increase in HPL activity and its thermal stability with the presence of Triton X-100. The results also showed that in the presence of glycine (10%), the catalytic efficiency of HPL was increased by 2.45-fold than that without additive. PMID:19430937

  20. Bacterial versus human sphingosine-1-phosphate lyase (S1PL) in the design of potential S1PL inhibitors.

    Science.gov (United States)

    Sanllehí, Pol; Abad, José-Luis; Casas, Josefina; Bujons, Jordi; Delgado, Antonio

    2016-09-15

    A series of potential active-site sphingosine-1-phosphate lyase (S1PL) inhibitors have been designed from scaffolds 1 and 2, arising from virtual screening using the X-ray structures of the bacterial (StS1PL) and the human (hS1PL) enzymes. Both enzymes are very similar at the active site, as confirmed by the similar experimental kinetic constants shown by the fluorogenic substrate RBM13 in both cases. However, the docking scoring functions used probably overestimated the weight of electrostatic interactions between the ligands and key active-site residues in the protein environment, which may account for the modest activity found for the designed inhibitors. In addition, the possibility that the inhibitors do not reach the enzyme active site should not be overlooked. Finally, since both enzymes show remarkable structural differences at the access channel and in the proximity to the active site cavity, caution should be taken when designing inhibitors acting around that area, as evidenced by the much lower activity found in StS1PL for the potent hS1PL inhibitor D. PMID:27475537

  1. Reduced photoinhibition under low irradiance enhanced Kacip Fatimah (Labisia pumila Benth) secondary metabolites, phenyl alanine lyase and antioxidant activity.

    Science.gov (United States)

    Ibrahim, Mohd Hafiz; Jaafar, Hawa Z E

    2012-01-01

    A randomized complete block design experiment was designed to characterize the relationship between production of total flavonoids and phenolics, anthocyanin, photosynthesis, maximum efficiency of photosystem II (Fv/Fm), electron transfer rate (Fm/Fo), phenyl alanine lyase activity (PAL) and antioxidant (DPPH) in Labisia pumila var. alata, under four levels of irradiance (225, 500, 625 and 900 μmol/m(2)/s) for 16 weeks. As irradiance levels increased from 225 to 900 μmol/m(2)/s, the production of plant secondary metabolites (total flavonoids, phenolics and antocyanin) was found to decrease steadily. Production of total flavonoids and phenolics reached their peaks under 225 followed by 500, 625 and 900 μmol/m(2)/s irradiances. Significant positive correlation of production of total phenolics, flavonoids and antocyanin content with Fv/Fm, Fm/Fo and photosynthesis indicated up-regulation of carbon-based secondary metabolites (CBSM) under reduced photoinhibition on the under low light levels condition. At the lowest irradiance levels, Labisia pumila extracts also exhibited a significantly higher antioxidant activity (DPPH) than under high irradiance. The improved antioxidative activity under low light levels might be due to high availability of total flavonoids, phenolics and anthocyanin content in the plant extract. It was also found that an increase in the production of CBSM was due to high PAL activity under low light, probably signifying more availability of phenylalanine (Phe) under this condition. PMID:22754297

  2. Reduced Photoinhibition under Low Irradiance Enhanced Kacip Fatimah (Labisia pumila Benth Secondary Metabolites, Phenyl Alanine Lyase and Antioxidant Activity

    Directory of Open Access Journals (Sweden)

    Hawa Z.E. Jaafar

    2012-04-01

    Full Text Available A randomized complete block design experiment was designed to characterize the relationship between production of total flavonoids and phenolics, anthocyanin, photosynthesis, maximum efficiency of photosystem II (Fv/Fm, electron transfer rate (Fm/Fo, phenyl alanine lyase activity (PAL and antioxidant (DPPH in Labisia pumila var. alata, under four levels of irradiance (225, 500, 625 and 900 µmol/m2/s for 16 weeks. As irradiance levels increased from 225 to 900 µmol/m2/s, the production of plant secondary metabolites (total flavonoids, phenolics and antocyanin was found to decrease steadily. Production of total flavonoids and phenolics reached their peaks under 225 followed by 500, 625 and 900 µmol/m2/s irradiances. Significant positive correlation of production of total phenolics, flavonoids and antocyanin content with Fv/Fm, Fm/Fo and photosynthesis indicated up-regulation of carbon-based secondary metabolites (CBSM under reduced photoinhibition on the under low light levels condition. At the lowest irradiance levels, Labisia pumila extracts also exhibited a significantly higher antioxidant activity (DPPH than under high irradiance. The improved antioxidative activity under low light levels might be due to high availability of total flavonoids, phenolics and anthocyanin content in the plant extract. It was also found that an increase in the production of CBSM was due to high PAL activity under low light, probably signifying more availability of phenylalanine (Phe under this condition.

  3. Sphingosine 1-phosphate lyase inhibition by 2-acetyl-4-(tetrahydroxybutyl)imidazole (THI) under conditions of vitamin B6 deficiency.

    Science.gov (United States)

    Ohtoyo, Mamoru; Tamura, Masakazu; Machinaga, Nobuo; Muro, Fumihito; Hashimoto, Ryuji

    2015-02-01

    Caramel food colorant 2-acetyl-4-(tetrahydroxybutyl)imidazole (THI) causes lymphopenia in animals through sphingosine 1-phosphate lyase (SPL) inhibition. However, this mechanism of action is partly still controversial because THI did not inhibit SPL in vitro either in cell-free or in cell-based systems. It is thought that the in vitro experimental conditions which have been used so far were not suitable for the evaluation of SPL inhibition, especially in case of cell-based experiments. We speculated that the key factor might be the coenzyme pyridoxal 5'-phosphate (PLP), an active form of vitamin B6 (VB6), because media used in cell-based assays usually contain an excess amount of VB6 which leads to the activation of SPL. By the use of VB6-deficient culture medium, we could regulate apo- (without PLP) and holo- (with PLP) SPL enzyme in cultured cells, resulting in the successful detection of SPL inhibition by THI. Although the observed inhibitory effect was not as strong as that of 4-deoxypyridoxine (a VB6 analog SPL inhibitor), these findings may be useful for further understanding the mechanism of action of THI. PMID:25381637

  4. Purification and simultaneous immobilization of Arabidopsis thaliana hydroxynitrile lyase using a family 2 carbohydrate-binding module.

    Science.gov (United States)

    Kopka, Benita; Diener, Martin; Wirtz, Astrid; Pohl, Martina; Jaeger, Karl-Erich; Krauss, Ulrich

    2015-05-01

    Tedious, time- and labor-intensive protein purification and immobilization procedures still represent a major bottleneck limiting the widespread application of enzymes in synthetic chemistry and industry. We here exemplify a simple strategy for the direct site-specific immobilization of proteins from crude cell extracts by fusion of a family 2 carbohydrate-binding module (CBM) derived from the exoglucanase/xylanase Cex from Cellulomonas fimi to a target enzyme. By employing a tripartite fusion protein consisting of the CBM, a flavin-based fluorescent protein (FbFP), and the Arabidopsis thaliana hydroxynitrile lyase (AtHNL), binding to cellulosic carrier materials can easily be monitored via FbFP fluorescence. Adsorption properties (kinetics and quantities) were studied for commercially available Avicel PH-101 and regenerated amorphous cellulose (RAC) derived from Avicel. The resulting immobilizates showed similar activities as the wild-type enzyme but displayed increased stability in the weakly acidic pH range. Finally, Avicel, RAC and cellulose acetate (CA) preparations were used for the synthesis of (R)-mandelonitrile in micro-aqueous methyl tert-butyl ether (MTBE) demonstrating the applicability and stability of the immobilizates for biotransformations in both aqueous and organic reaction systems. PMID:25755120

  5. Electrochemical sensing platform amplified with a nanobiocomposite of L-phenylalanine ammonia-lyase enzyme for the detection of capsaicin.

    Science.gov (United States)

    Sabela, Myalowenkosi I; Mpanza, Thabani; Kanchi, Suvardhan; Sharma, Deepali; Bisetty, Krishna

    2016-09-15

    The present study involves the development of a sensitive electrochemical biosensor for the determination of capsaicin extracted from chilli fruits, based on a novel signal amplification strategy using enzyme technology. For the first time, platinum electrode modified with multiwalled carbon nanotubes where phenylalanine ammonia-lyase enzyme was immobilized using nafion was characterized by attenuated total reflectance infrared spectroscopy, transmittance electron microscopy and thermo-gravimetric analysis supported by computational methods. Cyclic and differential pulse voltammetry measurements were performed to better understand the redox mechanism of capsaicin. The performance of the developed electrochemical biosensor was tested using spiked samples with recoveries ranging from 98.9 to 99.6%. The comparison of the results obtained from bare and modified platinum electrodes revealed the sensitivity of the developed biosensor, having a detection limit (S/N=3) of 0.1863µgmL(-1) and electron transfer rate constant (ks) of 3.02s(-1). Furthermore, adsorption and ligand-enzyme docking studies were carried out to better understand the redox mechanisms supported by density functional theory calculations. These results revealed that capsaicin forms hydrogen bonds with GLU355, GLU541, GLU586, ARG and other amino acids of the hydrophobic channel of the binding sites thereby facilitating the redox reaction for the detection of capsaicin. PMID:27104584

  6. Mechanism of tryptophan indole-lyase: Insights from pre-steady-state kinetics and substrate and solvent isotope effects

    International Nuclear Information System (INIS)

    The pre-steady-state kinetics of the reaction of Escherichia coli tryptophan indole-lyase have been examined with L-tryptophan, 7-aza-DL-tryptophan, and S-benzyl-L-cysteine. L-Tryptophan and 7-aza-DL-tryptophan exhibit three relaxations when the reactions are monitored at 506 nm. With L-tryptophan, α-deuteriation results in an estimated isotope effect of 3.6 on the first phase, while 2H2O produces apparent isotope effects of 2.5 and 2.7 on the second and third phases, respectively. On the basis of the substrate and solvent isotope effects and the effects of aza substitution, these three processes have been assigned to (1) deprotonation of the α-carbon, (2) an enzyme conformational change, and (3) indole tautomerization. In contrast, S-benzyl-L-cysteine exhibits only one catalytically competent relaxation, monitored at 512 nm. The intrinsic isotope effect for the reaction of α-(2H)-S-benzyl-L-cysteine is estimated to be 7.9. α-Proton abstraction is 10 to 100-fold faster than catalytic turnover in these reactions; thus, tautomerization of the indole ring of L-tryptophan may be partially rate-determining. 27 references, 3 figures

  7. Mechanistic deductions from multiple kinetic and solvent deuterium isotope effects and pH studies of pyridoxal phosphate dependent carbon-carbon lyases: escherichia coli tryptophan indole-lyase

    International Nuclear Information System (INIS)

    Analysis of the pH dependence of the kinetic parameters and competitive inhibitor Ki values for tryptophan indole-lyase suggests two enzymic groups must be unprotonated in order to facilitate binding and catalysis of tryptophan. The V/K for tryptophan and the pKi for oxindolyl-L-alanine, a putative transition state analogue and competitive inhibitor, decrease below two pK values of 7.6 and 6.0, while the Ki for L-alanine, also a competitive inhibitor, is 3300-fold larger (20 mM) than that for oxindolyl-L-alanine and increases below a single pK of 7.6. A single pK of 7.6 is also observed in the V/K profile for the alternate substrate, S-methyl-L-cysteine. Therefore, the enzymic group with a pK of 7.6 is responsible for proton abstraction at the 2-position of tryptophan, while the enzymic group with a pK of 6.0 interacts with the indole portion of tryptophan and probably catalyzes formation of the indolenine tautomer of tryptophan (in concert with proton transfer to C-3 of indole from the group with pK 7.6) to facilitate carbon-carbon bond cleavage and elimination of indole. The pH variation of the primary deuterium isotope effects for proton abstraction at the 2-position of tryptophan (DV = 2.5 and D(V/Ktrp) = 2.8) are pH independent, while the Vmax for tryptophan or S-methyl-L-cysteine is the same and also pH independent. Thus, substrates bind only to the correctly protonated form of the enzyme. Further, tryptophan is not sticky, and the pK values observed in both V/K profiles are the correct ones

  8. Action spectrum for induction of promoter activity of phenylalanine ammonia-lyase gene by UV in carrot suspension cells

    Energy Technology Data Exchange (ETDEWEB)

    Takeda, Junko [Kyoto Univ., Agricultural Chemistry Dept., Kyoto (Japan); Ozeki, Yoshihiro [Tokyo Univ. of Agriculture and Technology, Biotechnology Dept., Tokyo (Japan); Yoshida, Kazuichi [Kyoto Univ., Botany Dept., Kyoto (Japan)

    1997-10-01

    The full-length promoter (-2335) of the carrot (Daucus carota) phenylalanine ammonia-lyase gene (gDcPALLI) fused to the luciferase reporter gene was transiently transformed to carrot protoplasts by electroporation, and the promoter activity induced by monochromatic UV light of various wavelengths was examined. The action spectrum constructed from the fluence-response curves showed a single peak at around 280 nm, suggesting that the activation of the gDcPALI promoter is categorizable as one of the UVB light responses. The same assay system was applied to variously truncated gDcPALI promoters and to CaMV35S promoter fusion with various parts 5` - upstream of the gDcPALI promoter. The region from -396 to -190 (relative to the transcription start site) fused to the CaMV35S core (-90) promoter showed a 280 nm-dominant responses. However, gDCPALI promoters truncated above -570 and -396, although they contain the region between -396 and -190, did not show such a typical UVB response, i.e. they responded to 260 nm light as much as to 280 nm light. The promoter truncated to below -190 also responded to 260 nm light as much as to 280 nm light. Therefore we assumed that the gDcPALI promoter is composed of three functionally different parts: the upstream above -570 (modulator), the region from -396 to -190 (UVB responsive) and the downstream below -190 (UVB and C responsive). The overall UVB response of the gDcPALI full-length promoter is explained as the result of interaction of these three components. (Author).

  9. Dissimilation of cysteate via 3-sulfolactate sulfo-lyase and a sulfate exporter in Paracoccus pantotrophus NKNCYSA.

    Science.gov (United States)

    Rein, Ulrike; Gueta, Ronnie; Denger, Karin; Ruff, Jürgen; Hollemeyer, Klaus; Cook, Alasdair M

    2005-03-01

    Paracoccus pantotrophus NKNCYSA utilizes (R)-cysteate (2-amino-3-sulfopropionate) as a sole source of carbon and energy for growth, with either nitrate or molecular oxygen as terminal electron acceptor, and the specific utilization rate of cysteate is about 2 mkat (kg protein)(-1). The initial degradative reaction is catalysed by an (R)-cysteate : 2-oxoglutarate aminotransferase, which yields 3-sulfopyruvate. The latter was reduced to 3-sulfolactate by an NAD-linked sulfolactate dehydrogenase [3.3 mkat (kg protein)(-1)]. The inducible desulfonation reaction was not detected initially in cell extracts. However, a strongly induced protein with subunits of 8 kDa (alpha) and 42 kDa (beta) was found and purified. The corresponding genes had similarities to those encoding altronate dehydratases, which often require iron for activity. The purified enzyme could then be shown to convert 3-sulfolactate to sulfite and pyruvate and it was termed sulfolactate sulfo-lyase (Suy). A high level of sulfite dehydrogenase was also induced during growth with cysteate, and the organism excreted sulfate. A putative regulator, OrfR, was encoded upstream of suyAB on the reverse strand. Downstream of suyAB was suyZ, which was cotranscribed with suyB. The gene, an allele of tauZ, encoded a putative membrane protein with transmembrane helices (COG2855), and is a candidate to encode the sulfate exporter needed to maintain homeostasis during desulfonation. suyAB-like genes are widespread in sequenced genomes and environmental samples where, in contrast to the current annotation, several presumably encode the desulfonation of 3-sulfolactate, a component of bacterial spores. PMID:15758220

  10. Cystathionine-γ lyase-derived hydrogen sulfide mediates the cardiovascular protective effects of moxonidine in diabetic rats.

    Science.gov (United States)

    El-Sayed, Shaimaa S; Zakaria, Mohamed N M; Abdel-Ghany, Rasha H; Abdel-Rahman, Abdel A

    2016-07-15

    Blunted cystathionine-γ lyase (CSE) activity (reduced endogenous H2S-level) is implicated in hypertension and myocardial dysfunction in diabetes. Here, we tested the hypothesis that CSE derived H2S mediates the cardiovascular protection conferred by the imidazoline I1 receptor agonist moxonidine in a diabetic rat model. We utilized streptozotocin (STZ; 55mg/kg i.p) to induce diabetes in male Wistar rats. Four weeks later, STZ-treated rats received vehicle, moxonidine (2 or 6mg/kg; gavage), CSE inhibitor DL-propargylglycine, (37.5mg/kg i.p) or DL-propargylglycine with moxonidine (6mg/kg) for 3 weeks. Moxonidine improved the glycemic state, and reversed myocardial hypertrophy, hypertension and baroreflex dysfunction in STZ-treated rats. Ex vivo studies revealed that STZ caused reductions in CSE expression/activity, H2S and nitric oxide (NO) levels and serum adiponectin and elevations in myocardial imidazoline I1 receptor expression, p38 and extracellular signal-regulated kinase, ERK1/2, phosphorylation and lipid peroxidation (expressed as malondialdehyde). Moxonidine reversed these biochemical responses, and suppressed the expression of death associated protein kinase-3. Finally, pharmacologic CSE inhibition (DL-propargylglycine) abrogated the favorable cardiovascular, glycemic and biochemical responses elicited by moxonidine. These findings present the first evidence for a mechanistic role for CSE derived H2S in the glycemic control and in the favorable cardiovascular effects conferred by imidazoline I1 receptor activation (moxonidine) in a diabetic rat model. PMID:27138707

  11. Phenolics and flavonoids compounds, phenylanine ammonia lyase and antioxidant activity responses to elevated CO₂ in Labisia pumila (Myrisinaceae).

    Science.gov (United States)

    Jaafar, Hawa Z E; Ibrahim, Mohd Hafiz; Karimi, Ehsan

    2012-01-01

    A split plot 3 × 3 experiment was designed to examine the impact of three concentrations of CO₂ (400, 800 and 1,200 μmol·mol⁻¹) on the phenolic and flavonoid compound profiles, phenylalanine ammonia lyase (PAL) and antioxidant activity in three varieties of Labisia pumila Benth. (var. alata, pumila and lanceolata) after 15 weeks of exposure. HPLC analysis revealed a strong influence of increased CO₂ concentration on the modification of phenolic and flavonoid profiles, whose intensity depended on the interaction between CO₂ levels and L. pumila varieties. Gallic acid and quercetin were the most abundant phenolics and flavonoids commonly present in all the varieties. With elevated CO₂ (1,200 μmol·mol⁻¹) exposure, gallic acid increased tremendously, especially in var. alata and pumila (101-111%), whilst a large quercetin increase was noted in var. lanceolata (260%), followed closely by alata (201%). Kaempferol, although detected under ambient CO₂ conditions, was undetected in all varieties after exposure. Instead, caffeic acid was enhanced tremendously in var. alata (338~1,100%) and pumila (298~433%). Meanwhile, pyragallol and rutin were only seen in var. alata (810 μg·g⁻¹ DW) and pumila (25 μg·g⁻¹ DW), respectively, under ambient conditions; but the former compound went undetected in all varieties while rutin continued to increase by 262% after CO₂ enrichment. Interestingly, naringenin that was present in all varieties under ambient conditions went undetected under enrichment, except for var. pumila where it was enhanced by 1,100%. PAL activity, DPPH and FRAP also increased with increasing CO₂ levels implying the possible improvement of health-promoting quality of Malaysian L. pumila under high CO₂ enrichment conditions. PMID:22634843

  12. Probing the reaction mechanism of spore photoproduct lyase (SPL) via diastereoselectively labeled dinucleotide SP TpT substrates.

    Science.gov (United States)

    Yang, Linlin; Lin, Gengjie; Liu, Degang; Dria, Karl J; Telser, Joshua; Li, Lei

    2011-07-13

    5-Thyminyl-5,6-dihydrothymine (commonly called spore photoproduct or SP) is the exclusive DNA photodamage product in bacterial endospores. It is generated in the bacterial sporulation phase and repaired by a radical SAM enzyme, spore photoproduct lyase (SPL), at the early germination phase. SPL utilizes a special [4Fe-4S] cluster to reductively cleave S-adenosylmethionine (SAM) to generate a reactive 5'-dA radical. The 5'-dA radical is proposed to abstract one of the two H-atoms at the C6 carbon of SP to initiate the repair process. Via organic synthesis and DNA photochemistry, we selectively labeled the 6-H(proS) or 6-H(proR) position with a deuterium in a dinucleotide SP TpT substrate. Monitoring the deuterium migration in enzyme catalysis (employing Bacillus subtilis SPL) revealed that it is the 6-H(proR) atom of SP that is abstracted by the 5'-dA radical. Surprisingly, the abstracted deuterium was not returned to the resulting TpT after enzymatic catalysis; an H-atom from the aqueous buffer was incorporated into TpT instead. This result questions the currently hypothesized SPL mechanism which excludes the involvement of protein residue(s) in SPL reaction, suggesting that some protein residue(s), which is capable of exchanging a proton with the aqueous buffer, is involved in the enzyme catalysis. Moreover, evidence has been obtained for a possible SAM regeneration after each catalytic cycle; however, such a regeneration process is more complex than currently thought, with one or even more protein residues involved as well. These observations have enabled us to propose a modified reaction mechanism for this intriguing DNA repair enzyme. PMID:21671623

  13. Anti-atherogenic effect of hydrogen sulfide by over-expression of cystathionine gamma-lyase (CSE gene.

    Directory of Open Access Journals (Sweden)

    Sau Ha Cheung

    Full Text Available Hydrogen sulfide (H2S is an important gaseous signaling molecule that functions in physiological and pathological conditions, such as atherosclerosis. H2S dilates vessels and therefore has been suggested as an anti-atherogenic molecule. Since cystathionine gamma-lyase (CSE enzyme is responsible for producing H2S in the cardiovascular system, we hypothesized that up-regulation of CSE expression in vivo with preservation of H2S bioactivity can slow down plaque formation and, can serve as a therapeutic strategy against atherosclerosis. In this study, C57BL/6 wild type mice (WT, ApoE knockout mice (KO and transgenic ApoE knockout mice overexpressing CSE (Tg/KO at four weeks of age were weaned. They were then fed with either normal or atherogenic diet for 12 weeks. At week 16, serial plasma lipid levels, body weight, and blood pressure were measured prior to euthanization of the mice and the size of atherosclerotic plaques at their aortic roots was measured. Tg/KO mice showed an increase in endogenous H2S production in aortic tissue, reduced atherosclerotic plaque sizes and attenuation in plasma lipid profiles. We also showed an up-regulation in plasma glutathionine peroxidase that could indicate reduced oxidative stress. Furthermore, there was an increase in expression of p-p53 and down regulation of inflammatory nuclear factor-kappa B (NF-κB in aorta. To conclude, alteration of endogenous H2S by CSE gene activation was associated with reduced atherosclerosis in ApoE-deficient mice. Up-regulation of CSE/H2S pathway attenuates atherosclerosis and this would be a potential target for therapeutic intervention against its formation.

  14. Induction of phenylalanine ammonia-lyase and lipoxygenase in cotton seedlings by mechanical wounding and aphid infestation

    Institute of Scientific and Technical Information of China (English)

    QIN Qiuju; SHI Xueyan; LIANG Pei; GAO Xiwu

    2005-01-01

    It has been suggested that infestation of plants causes increases in the activities of phenylalanine ammonia-lyase (PAL)and lipoxygenase (LOX), key enzymes in the phenolic compounds synthesis pathway and the octadecanoid pathway, respectively. The purpose of this work is to investigate whether the infestation of cotton aphid (Aphis gossypii ) and mechanical wound can cause the induction of PAL and LOX activities in cotton seedlings, and whether the induction occurs in healthy seedlings growing nearby the attacked ones. The specific activities of PAL and LOX were measured using spectrophotometric method after aphid infestation and mechanical wounding. Result indicated that PAL activity and LOX activity were greatly induced by mechanical wounding and aphid infestation in cotton seedlings. The induction of PAL and LOX occurred not only in wounded and infested seedlings but also in intact healthy seedlings growing nearby. After exposed to the aphid infestation-induced volatiles, the specific activity of PAL in cotton seedlings increased by 6 % at 24 h, 80 % at 48 h, 235 % at 72 h compared to the control, and the specific activity of LOX increased by 18 % at 24 h, 34 % at 48 h,24 % at 72 h, respectively. In comparison, the specific activity of PAL in unwounded seedlings exposed to wound-induced volatiles increased by 0.0 at 24 h, 200% at 48 h, 164% at 72 h, respectively and the specific activity of LOX increased by 28% at 24 h, 37% at 48 h, 8 % at 72 h, respectively. It suggests that the induced volatiles are involved in plant-plant communication as airborne transferred signals.

  15. Modeling and Re-Engineering of Azotobacter vinelandii Alginate Lyase to Enhance Its Catalytic Efficiency for Accelerating Biofilm Degradation

    Science.gov (United States)

    Jang, Chul Ho; Piao, Yu Lan; Huang, Xiaoqin; Yoon, Eun Jeong; Park, So Hee; Lee, Kyoung; Zhan, Chang-Guo; Cho, Hoon

    2016-01-01

    Alginate is known to prevent elimination of Pseudomonas aeruginosa biofilms. Alginate lyase (AlgL) might therefore facilitate treatment of Pseudomonas aeruginosa-infected cystic fibrosis patients. However, the catalytic activity of wild-type AlgL is not sufficiently high. Therefore, molecular modeling and site-directed mutagenesis of AlgL might assist in enzyme engineering for therapeutic development. AlgL, isolated from Azotobacter vinelandii, catalyzes depolymerization of alginate via a β-elimination reaction. AlgL was modeled based on the crystal structure template of Sphingomonas AlgL species A1-III. Based on this computational analysis, AlgL was subjected to site-directed mutagenesis to improve its catalytic activity. The kcat/Km of the K194E mutant showed a nearly 5-fold increase against the acetylated alginate substrate, as compared to the wild-type. Double and triple mutants (K194E/K245D, K245D/K319A, K194E/K245D/E312D, and K194E/K245D/K319A) were also prepared. The most potent mutant was observed to be K194E/K245D/K319A, which has a 10-fold improved kcat value (against acetylated alginate) compared to the wild-type enzyme. The antibiofilm effect of both AlgL forms was identified in combination with piperacillin/tazobactam (PT) and the disruption effect was significantly higher in mutant AlgL combined with PT than wild-type AlgL. However, for both the wild-type and K194E/K245D/K319A mutant, the use of the AlgL enzyme alone did not show significant antibiofilm effect. PMID:27253324

  16. Phycourobilin in trichromatic phycocyanin from oceanic cyanobacteria is formed post-translationally by a phycoerythrobilin lyase-isomerase.

    Science.gov (United States)

    Blot, Nicolas; Wu, Xian-Jun; Thomas, Jean-Claude; Zhang, Juan; Garczarek, Laurence; Böhm, Stephan; Tu, Jun-Ming; Zhou, Ming; Plöscher, Matthias; Eichacker, Lutz; Partensky, Frédéric; Scheer, Hugo; Zhao, Kai-Hong

    2009-04-01

    Most cyanobacteria harvest light with large antenna complexes called phycobilisomes. The diversity of their constituting phycobiliproteins contributes to optimize the photosynthetic capacity of these microorganisms. Phycobiliprotein biosynthesis, which involves several post-translational modifications including covalent attachment of the linear tetrapyrrole chromophores (phycobilins) to apoproteins, begins to be well understood. However, the biosynthetic pathway to the blue-green-absorbing phycourobilin (lambda(max) approximately 495 nm) remained unknown, although it is the major phycobilin of cyanobacteria living in oceanic areas where blue light penetrates deeply into the water column. We describe a unique trichromatic phycocyanin, R-PC V, extracted from phycobilisomes of Synechococcus sp. strain WH8102. It is evolutionarily remarkable as the only chromoprotein known so far that absorbs the whole wavelength range between 450 and 650 nm. R-PC V carries a phycourobilin chromophore on its alpha-subunit, and this can be considered an extreme case of adaptation to blue-green light. We also discovered the enzyme, RpcG, responsible for its biosynthesis. This monomeric enzyme catalyzes binding of the green-absorbing phycoerythrobilin at cysteine 84 with concomitant isomerization to phycourobilin. This reaction is analogous to formation of the orange-absorbing phycoviolobilin from the red-absorbing phycocyanobilin that is catalyzed by the lyase-isomerase PecE/F in some freshwater cyanobacteria. The fusion protein, RpcG, and the heterodimeric PecE/F are mutually interchangeable in a heterologous expression system in Escherichia coli. The novel R-PC V likely optimizes rod-core energy transfer in phycobilisomes and thereby adaptation of a major phytoplankton group to the blue-green light prevailing in oceanic waters. PMID:19182270

  17. Action spectrum for induction of promoter activity of phenylalanine ammonia-lyase gene by UV in carrot suspension cells

    International Nuclear Information System (INIS)

    The full-length promoter (-2335) of the carrot (Daucus carota) phenylalanine ammonia-lyase gene (gDcPALLI) fused to the luciferase reporter gene was transiently transformed to carrot protoplasts by electroporation, and the promoter activity induced by monochromatic UV light of various wavelengths was examined. The action spectrum constructed from the fluence-response curves showed a single peak at around 280 nm, suggesting that the activation of the gDcPALI promoter is categorizable as one of the UVB light responses. The same assay system was applied to variously truncated gDcPALI promoters and to CaMV35S promoter fusion with various parts 5' - upstream of the gDcPALI promoter. The region from -396 to -190 (relative to the transcription start site) fused to the CaMV35S core (-90) promoter showed a 280 nm-dominant responses. However, gDCPALI promoters truncated above -570 and -396, although they contain the region between -396 and -190, did not show such a typical UVB response, i.e. they responded to 260 nm light as much as to 280 nm light. The promoter truncated to below -190 also responded to 260 nm light as much as to 280 nm light. Therefore we assumed that the gDcPALI promoter is composed of three functionally different parts: the upstream above -570 (modulator), the region from -396 to -190 (UVB responsive) and the downstream below -190 (UVB and C responsive). The overall UVB response of the gDcPALI full-length promoter is explained as the result of interaction of these three components. (Author)

  18. Hydrogen sulfide (H2S)/cystathionine γ-lyase (CSE) pathway contributes to the proliferation of hepatoma cells

    International Nuclear Information System (INIS)

    Highlights: • Inhibition of H2S/CSE pathway strongly stimulates cellular apoptosis. • Inhibition of H2S/CSE pathway suppresses cell growth by blocking EGFR pathway. • H2S/CSE pathway is critical for maintaining the proliferation of hepatoma cells. - Abstract: Hydrogen sulfide (H2S)/cystathionine γ-lyase (CSE) pathway has been demonstrated to play vital roles in physiology and pathophysiology. However, its role in tumor cell proliferation remains largely unclear. Here we found that CSE over-expressed in hepatoma HepG2 and PLC/PRF/5 cells. Inhibition of endogenous H2S/CSE pathway drastically decreased the proliferation of HepG2 and PLC/PRF/5 cells, and it also enhanced ROS production and mitochondrial disruption, pronounced DNA damage and increased apoptosis. Moreover, this increase of apoptosis was associated with the activation of p53 and p21 accompanied by a decreased ratio of Bcl-2/Bax and up-regulation of phosphorylated c-Jun N-terminal kinase (JNK) and caspase-3 activity. In addition, the negative regulation of cell proliferation by inhibition of H2S/CSE system correlated with the blockage of cell mitogenic and survival signal transduction of epidermal growth factor receptor (EGFR) via down-regulating the extracellular-signal-regulated kinase 1/2 (ERK1/2) activation. These results demonstrate that H2S/CSE and its downstream pathway contribute to the proliferation of hepatoma cells, and inhibition of this pathway strongly suppress the excessive growth of hepatoma cells by stimulating mitochondrial apoptosis and suppressing cell growth signal transduction

  19. Biochemical characteristics of an alkaline pectate lyase PelA from Volvariella volvacea: roles of the highly conserved N-glycosylation site in its secretion and activity.

    Science.gov (United States)

    Shi, Aiqin; Hu, Hang; Zheng, Fei; Long, Liangkun; Ding, Shaojun

    2015-04-01

    Alkaline pectate lyases have great application potential in the bioscouring of textiles. They are isolated predominantly from bacteria and a few fungi. Here, we report the biochemical characteristics of a novel alkaline pectate lyase PelA from the basidiomycete Volvariella volvacea. The full-length pelA encodes a 321-amino-acid polypeptide containing a putative 18-residue signal peptide and a pectate lyase family 1 catalytic domain. It contains one conserved and one non-conserved potential N-glycosylation site (N-X-S/T) at the residues N95 and N198, respectively. The enzyme showed optimal activity at 60 °C and pH 10, although it was stable between pH 4 and pH 11. Additional Ca(2+) was not required to measure PelA activity in vitro, but it could significantly enhance its activity and thermal stability. The V max values using polygalacturonic acid as substrate were increased from 50.71 to 89.96 IU mg(-1) by the addition of 0.1 mM Ca(2+), whereas the K m values were decreased from 0.681 to 0.514 mg ml(-1). Site-directed mutagenesis revealed PelA has only one N-glycan attached to the residue N95. This N-glycan is crucial to its efficient secretion and activity possibly due to its role in maintaining the secondary structure of PelA. Amino acid substitution at the residue N198 had no effect on PelA secretion, but resulted in a slight (5.16 %) to modest (27.37 %) decrease in specific activity and less thermal stability, indicating the amino acid itself is also important for activity due to it being highly conserved and because of its proximity to the catalytic site. PMID:25341402

  20. Host-pathogen interactions. XXIX. Oligogalacturonides released from sodium polypectate by endopolygalacturonic acid lyase are elicitors of phytoalexins in soybean. [Glycine max L

    Energy Technology Data Exchange (ETDEWEB)

    Davis, K.R.; Darvill, A.G.; Albersheim, P.; Dell, A.

    1986-02-01

    Recent studies have demonstrated that an apparently homogeneous preparation of an ..cap alpha..-1,4-D-endopolygalacturonic acid lyase (EC 4.2,2.2) isolated from the phytopathogenic bacterium Erwinia carotovora induced phytoalexin accumulation in cotyledons of soybean (Glycine max (L.) Merr. cv Wayne) and that this pectin-degrading enzyme released heat-stable elicitors of phytoalexins from soybean cell walls, citrus pectin, and sodium polypectate. The present paper reports the purification, by anion-exchange chromatography on QAE-Sephadex columns followed by gel-permeation chromatography on a Bio-Gel P-6 column, of the two fractions with highest specific elicitor activity present in a crude elicitor-preparation obtained by lyase treatment of sodium polypectate. Structural analysis of the fraction with highest specific elicitor activity indicated that the major, if not only, component was a decasaccharide of ..cap alpha..-1,4-D-galactosyluronic acid that contained the expected product of lyase cleavage, 4-deoxy-..beta..-L-5-threo-hexopyranos-4-enyluronic acid (4,5-unsaturated galactosyluronic acid), at the nonreducing terminus. This modified decagalacturonide fraction exhibited half-maximum and maximum elicitor activity at 1 microgram/cotyledon (6 micromolar) and 5 micrograms/cotyledon (32 micromolar) galactosyluronic acid equivalents, respectively. Reducing 90 to 95% of the carboxyl groups of the galactosyluronic acid residues abolished the elicitor activity of the decagalacturonide fraction. The second most elicitor-active fraction contained mostly undeca-..cap alpha..-1,4-D-galactosyluronic acid that contained 4,5-unsaturated galactosyluronic acid at the nonreducing termini. This fraction exhibited half-maximum and maximum elicitor activity at approximately 3 micrograms/cotyledon (17 micromolar) and 6 micrograms/cotyledon (34 micromolar) galactosyluronic acid equivalents, respectively.

  1. Molecular Cloning of cpcU and Heterodimeric Bilin Lyase Activity Analysis of CpcU and CpcS for Attachment of Phycocyanobilin to Cys-82 on the β-Subunit of Phycocyanin in Arthrospira platensis FACHB314.

    Science.gov (United States)

    Wu, Fei; Zang, Xiaonan; Zhang, Xuecheng; Zhang, Ran; Huang, Xiaoyun; Hou, Lulu; Jiang, Minjie; Liu, Chang; Pang, Chunhong

    2016-01-01

    A new bilin lyase gene cpcU was cloned from Arthrospira platensis FACHB314 to study the assembly of the phycocyanin β-Subunit. Two recombinant plasmids, one contained the phycocyanobilin (PCB) producing genes (hoxI and pcyA), while the other contained the gene of the β-Subunit of phycobiliprotein (cpcB) and the lyase gene (cpcU, cpcS, or cpcU/S) were constructed and separately transferred into Escherichia coli in order to test the activities of relevant lyases for catalyzing PCB addition to CpcB during synthesizing fluorescent β-PC of A. platensis FACHB314. The fluorescence intensity examination showed that Cys-82 maybe the active site for the β-Subunit binding to PCBs and the attachment could be carried out by CpcU, CpcS, or co-expressed cpcU/S in A. platensis FACHB314. PMID:26999083

  2. Molecular Cloning of cpcU and Heterodimeric Bilin Lyase Activity Analysis of CpcU and CpcS for Attachment of Phycocyanobilin to Cys-82 on the β-Subunit of Phycocyanin in Arthrospira platensis FACHB314

    Directory of Open Access Journals (Sweden)

    Fei Wu

    2016-03-01

    Full Text Available A new bilin lyase gene cpcU was cloned from Arthrospira platensis FACHB314 to study the assembly of the phycocyanin β-Subunit. Two recombinant plasmids, one contained the phycocyanobilin (PCB producing genes (hoxI and pcyA, while the other contained the gene of the β-Subunit of phycobiliprotein (cpcB and the lyase gene (cpcU, cpcS, or cpcU/S were constructed and separately transferred into Escherichia coli in order to test the activities of relevant lyases for catalyzing PCB addition to CpcB during synthesizing fluorescent β-PC of A. platensis FACHB314. The fluorescence intensity examination showed that Cys-82 maybe the active site for the β-Subunit binding to PCBs and the attachment could be carried out by CpcU, CpcS, or co-expressed cpcU/S in A. platensis FACHB314.

  3. Under light limiting growth, CpcB lyase null mutants of the Cyanobacterium Synechococcus sp. PCC 7002 are capable of producing pigmented beta phycocyanin but with altered chromophore function.

    Science.gov (United States)

    Derks, Allen K; Vasiliev, Serguei; Bruce, Doug

    2008-11-11

    Phycobilisomes are the major light-harvesting complexes for cyanobacteria, and phycocyanin is the primary phycobiliprotein of the phycobilisome rod. Phycocyanobilin chromophores are covalently bonded to the phycocyanin beta subunit (CpcB) by specific lyases which have been recently identified in the cyanobacterium Synechococcus sp. PCC 7002. Surprisingly, we found that mutants missing the CpcB lyases were nevertheless capable of producing pigmented phycocyanin when grown under low-light conditions. Absorbance measurements at 10 K revealed the energy states of the beta phycocyanin chromophores to be slightly shifted, and 77 K steady state fluorescence emission spectroscopy showed that excitation energy transfer involving the targeted chromophores was disrupted. This evidence indicates that the position of the phycocyanobilin chromophore within the binding domain of the phycocyanin beta subunit had been modified. We hypothesize that alternate, less specific lyases are able to add chromophores, with varying effectiveness, to the beta binding sites. PMID:18925744

  4. Involvement of Carbohydrate, Protein and Phenylanine Ammonia Lyase in Up-Regulation of Secondary Metabolites in Labisia pumila under Various CO2 and N2 Level

    OpenAIRE

    Mohd Hafiz Ibrahim; Jaafar, Hawa Z. E.

    2011-01-01

    A split plot factorial 2 × 3 experiment was designed to examine and characterize the relationships among secondary metabolites (total phenolics, TP; total flavonoids, TF), carbohydrate content, C/N ratio, protein synthesis and L–phenylalanine ammonia lyase (PAL; EC 4.3.1.5) activity in the Malaysian medicinal herb Labisia pumila (Blume) Fern-Vill. under different CO2 concentrations (400 = ambient and 1,200 µmol mol−1 CO2) and three levels of nitrogen fertilization (0, 90 and 270 kg N ha−1) fo...

  5. Residues C123 and D58 of the 2-Methylisocitrate Lyase (PrpB) Enzyme of Salmonella enterica Are Essential for Catalysis

    OpenAIRE

    Grimek, T. L.; Holden, H.; Rayment, I; Escalante-Semerena, J. C.

    2003-01-01

    The prpB gene of Salmonella enterica serovar Typhimurium LT2 encodes a protein with 2-methylisocitrate (2-MIC) lyase activity, which cleaves 2-MIC into pyruvate and succinate during the conversion of propionate to pyruvate via the 2-methylcitric acid cycle. This paper reports the isolation and kinetic characterization of wild-type and five mutant PrpB proteins. Wild-type PrpB protein had a molecular mass of approximately 32 kDa per subunit, and the biologically active enzyme was comprised of ...

  6. Cloning and Random Mutagenesis of the Erwinia herbicola tyrR Gene for High-Level Expression of Tyrosine Phenol-Lyase

    OpenAIRE

    Katayama, Takane; Suzuki, Hideyuki; Koyanagi, Takashi; Kumagai, Hidehiko

    2000-01-01

    Tyrosine phenol-lyase (Tpl), which can synthesize 3,4-dihydroxyphenylalanine from pyruvate, ammonia, and catechol, is a tyrosine-inducible enzyme. Previous studies demonstrated that the tpl promoter of Erwinia herbicola is activated by the TyrR protein of Escherichia coli. In an attempt to create a high-Tpl-expressing strain, we cloned the tyrR gene of E. herbicola and then randomly mutagenized it. Mutant TyrR proteins with enhanced ability to activate tpl were screened for by use of the lac ...

  7. Analysis of different de-esterification mechanisms for pectin by enzymatic fingerprinting using endopectin lyase and endopolygalacturonase II from A. niger

    DEFF Research Database (Denmark)

    Limberg, G; Körner, R; Buchholt, H C;

    2000-01-01

    with either endopectin lyase (PL) or endopolygalacturonase II (PG II) from Aspergillus niger were analysed using matrix assisted laser desorption ionisation mass spectrometry (MALDIMS) and high-performance anion-exchange chromatography with pulsed amperometric or UV detection (HPAEC-PAD/UV). Time...... course analysis using MALDIMS was used to identify the most preferred substrate for each enzyme. For PL, this was shown to be fully methyl esterified HG whereas for PG II, long regions of HG without any methyl esterification, as produced by p-PME was the optimal substrate. The blockwise de...... ester groups by f-PME is the most reasonable explanation for the detected differences....

  8. A rationale for autoinduction of a transcriptional activator: ethanolamine ammonia-lyase (EutBC) and the operon activator (EutR) compete for adenosyl-cobalamin in Salmonella typhimurium.

    OpenAIRE

    Sheppard, D E; Roth, J R

    1994-01-01

    The ethanolamine utilization (eut) operon of Salmonella typhimurium is controlled by a positive regulatory protein (EutR) which stimulates eut operon expression in response to the simultaneous presence of two effectors, ethanolamine and adenosyl-cobalamin (Ado-B12). Ado-B12 is a cofactor for ethanolamine ammonia-lyase (lyase), the first enzyme in the ethanolamine-degradative pathway. The dependence of this pathway on the use of Ado-B12 as an effector in eut operon induction may be explained b...

  9. Kinetic Parameters and Cytotoxic Activity of Recombinant Methionine γ-Lyase from Clostridium tetani, Clostridium sporogenes, Porphyromonas gingivalis and Citrobacter freundii.

    Science.gov (United States)

    Morozova, E A; Kulikova, V V; Yashin, D V; Anufrieva, N V; Anisimova, N Y; Revtovich, S V; Kotlov, M I; Belyi, Y F; Pokrovsky, V S; Demidkina, T V

    2013-07-01

    The steady-state kinetic parameters of pyridoxal 5'-phosphate-dependent recombinant methionine γ -lyase from three pathogenic bacteria, Clostridium tetani, Clostridium sporogenes, and Porphyromonas gingivalis, were determined in β- and γ-elimination reactions. The enzyme from C. sporogenes is characterized by the highest catalytic efficiency in the γ-elimination reaction of L-methionine. It was demonstrated that the enzyme from these three sources exists as a tetramer. The N-terminal poly-histidine fragment of three recombinant enzymes influences their catalytic activity and facilitates the aggregation of monomers to yield dimeric forms under denaturing conditions. The cytotoxicity of methionine γ-lyase from C. sporogenes and C. tetani in comparison with Citrobacter freundii was evaluated using K562, PC-3, LnCap, MCF7, SKOV-3, and L5178y tumor cell lines. K562 (IC50=0.4-1.3 U/ml), PC-3 (IC50=0.1-0.4 U/ml), and MCF7 (IC50=0.04-3.2 U/ml) turned out to be the most sensitive cell lines. PMID:24303205

  10. Lyase activities of heterologous CpcS and CpcT for phycocyanin holo-β-subunit from Arthrospira platensis in Escherichia coli

    Science.gov (United States)

    Yi, Junjie; Xu, Di; Zang, Xiaonan; Yuan, Dingyang; Zhao, Bingran; Tang, Li; Tan, Yanning; Zhang, Xuecheng

    2014-06-01

    Arthrospira platensis is an economically important cyanobacterium; and it has been used widely in food and pharmaceutical industries. The phycocyanin (PC) from A. platensis is extremely valuable in medicine and molecular biology due to its antioxidation and anti-tumoring activity and applicability as fluorescence protein tag. In present study, two recombinant plasmids, one contained the phycocyanobilin (PCB)-producing genes ( hox1 and pcyA) while the other contained the phycobiliprotein gene ( cpcB) and the lyase gene (either cpcS/U or cpcT), were constructed and synchronically transferred into E. coli in order to test the the activities of relevant lyases for catalysing PCB addition to CpcB during synthesizing fluorescent PC holo-β-subunit (β-PC) of A. platensis. As was evidenced by the fluorescence emitted at a peak specific for PC, CpcB was successfully synthesized in E. coli, to which co-expressed PCBs attached though at a relatively low efficiency. The results showed that the attachment of PCBs to CpcB were carried out mainly by co-expressed CpcS/U but CpcB also showed some autocatalytic activity. Currently, no CpcT activity was detected in this E. coli expression system. Further studies will be conducted to improve the efficiency of fluorescent PC synthesis in E. coli.

  11. Reduced Lignin Content and Altered Lignin Composition in Transgenic Tobacco Down-Regulated in Expression of L-Phenylalanine Ammonia-Lyase or Cinnamate 4-Hydroxylase.

    Science.gov (United States)

    Sewalt, VJH.; Ni, W.; Blount, J. W.; Jung, H. G.; Masoud, S. A.; Howles, P. A.; Lamb, C.; Dixon, R. A.

    1997-09-01

    We analyzed lignin content and composition in transgenic tobacco (Nicotiana tabacum) lines altered in the expression of the early phenylpropanoid biosynthetic enzymes L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase (C4H). The reduction of C4H activity by antisense expression or sense suppression resulted in reduced levels of Klason lignin, accompanied by a decreased syringyl/guaiacyl monomer ratio as determined by pyrolysis gas chromatography/mass spectrometry Similar reduction of lignin levels by down -regulation of L-phenylalanine ammonia-lyase, the enzyme preceding C4H in the central phenylpropanoid pathway, did not result in a decreased syringyl/guaiacyl ratio. Rather, analysis of lignin methoxyl content and pyrolysis suggested an increased syringyl/guaiacyl ratio. One possible explanation of these results is that monolignol biosynthesis from L-phenylalanine might occur by more than one route, even at the early stages of the core phenylpropanoid pathway, prior to the formation of specific monolignol precursors. PMID:12223790

  12. Lyase Activities of Heterologous CpcS and CpcT for Phycocyanin Holo-β-subunit from Arthrospira platensis in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    YI Junjie; XU Di; ZANG Xiaonan; YUAN Dingyang; ZHAO Bingran; TANG Li; TAN Yanning; ZHANG Xuecheng

    2014-01-01

    Arthrospira platensis is an economically important cyanobacterium;and it has been used widely in food and pharmaceu-tical industries. The phycocyanin (PC) from A. platensis is extremely valuable in medicine and molecular biology due to its anti-oxidation and anti-tumoring activity and applicability as fluorescence protein tag. In present study, two recombinant plasmids, one contained the phycocyanobilin (PCB)-producing genes (hox1 and pcyA) while the other contained the phycobiliprotein gene (cpcB) and the lyase gene (either cpcS/U or cpcT), were constructed and synchronically transferred into E. coli in order to test the the activi-ties of relevant lyases for catalysing PCB addition to CpcB during synthesizing fluorescent PC holo-β-subunit (β-PC) of A. platensis. As was evidenced by the fluorescence emitted at a peak specific for PC, CpcB was successfully synthesized in E. coli, to which co-expressed PCBs attached though at a relatively low efficiency. The results showed that the attachment of PCBs to CpcB were carried out mainly by co-expressed CpcS/U but CpcB also showed some autocatalytic activity. Currently, no CpcT activity was de-tected in this E. coli expression system. Further studies will be conducted to improve the efficiency of fluorescent PC synthesis in E. coli.

  13. Biochemical Stability and Molecular Dynamic Characterization of Aspergillus fumigatus Cystathionine γ-Lyase in Response to Various Reaction Effectors

    KAUST Repository

    El-Sayed, Ashraf S.A.

    2015-08-11

    Cystathionine γ-lyase (CGL) is a key enzyme in the methionine-cysteine cycle in all living organisms forming cysteine, α-ketobutyrate and ammonia via homocysteine and cystathionine intermediates. Although, human and plant CGLs have been extensively studied at the molecular and mechanistic levels, there has been little work on the molecular and catalytic properties of fungal CGL. Herein, we studied in detail for the first time the molecular and catalytic stability of Aspergillus fumigatus CGL, since conformational instability, inactivation and structural antigenicity are the main limitations of the PLP-dependent enzymes on various therapeutic uses. We examined these properties in response to buffer compositions, stabilizing and destabilizing agents using Differential Scanning Fluorometery (DSF), steady state and gel-based fluorescence of the intrinsic hydrophobic core, stability of internal aldimine linkage and catalytic properties. The activity of the recombinant A. fumigatus CGL was 13.8 U/mg. The melting temperature (Tm) of CGL in potassium phosphate buffer (pH 7.0-8.0) was 73.3 °C, with ∼3 °C upshifting in MES and sodium phosphate buffers (pH 7.0). The conformational thermal stability was increased in potassium phosphate, sodium phosphate and MES buffers, in contrast to Tris-HCl, HEPES (pH 7.0) and CAPS (pH 9.0-10.0). The thermal stability and activity of CGL was slightly increased in the presence of trehalose and glycerol that might be due to hydration of the enzyme backbone, unlike the denaturing effect of GdmCl and urea. Modification of surface CGL glutamic and aspartic acids had no significant effect on the enzyme conformational and catalytic stability. Molecular modeling and dynamics simulations unveil the high conformational stability of the overall scaffold of CGL with high flexibility at the non-structural regions. CGL structure has eight buried Trp residues, which are reoriented to the enzyme surface and get exposed to the solvent under

  14. Cystathionine γ-lyase

    Directory of Open Access Journals (Sweden)

    Halina Jurkowska

    2014-01-01

    Full Text Available γ-Cystathionase (CTH, EC: 4.4.1.1, an enzyme widely distributed in the world of prokaryotic and eukaryotic organisms, catalyzes the formation and transformations of sulfane sulfur-containing compounds and plays a pivotal role in the L-cysteine desulfuration pathway. Human, tetrameric CTH is composed of two dimers and each monomer binds pyridoxal phosphate (PLP. The gene, located on the short arm of chromosome 1, consists of 13 exons and 12 introns. As a result of alternative splicing, three isoforms of human CTH arise. Analysis of genetic variations of the CTH encoding gene showed a large number of polymorphisms. A decrease of the expression of CTH entails a drop in the level of cysteine , glutathione (GSH, taurine and hydrogen sulfide (H2S in the cells and, more importantly, leads to cystathioninuria. H2S, endogenously formed by CTH, affects the vasodilation and regulation of blood pressure. CTH knockout mice have decreased levels of H2S, hypertension, and reduced capacity for vascular endothelium relaxation. Overexpression of the gene encoding CTH in the cells leads to increased production of H2S. H2S plays a role in protection of neurons against oxidative stress, and stimulates an increase in γ-glutamylcysteine synthetase and thereby an increase in the level of GSH. Sulfurtransferases, including CTH, can locally prevent oxidative stress due to reversible oxidation of – SH groups in the presence of increased levels of reactive oxygen species, and reduction in the presence of GSH and/or reduced thioredoxin.

  15. Cystathionine γ-lyase

    OpenAIRE

    Halina Jurkowska; Marta Kaczor-Kamińska; Patrycja Bronowicka-Adamska; Maria Wróbel; Katarzyna Leszczyńska; Dorota Cibor; Tomasz Mach; Zofia Dzierżewicz

    2014-01-01

    γ-Cystathionase (CTH, EC: 4.4.1.1), an enzyme widely distributed in the world of prokaryotic and eukaryotic organisms, catalyzes the formation and transformations of sulfane sulfur-containing compounds and plays a pivotal role in the L-cysteine desulfuration pathway. Human, tetrameric CTH is composed of two dimers and each monomer binds pyridoxal phosphate (PLP). The gene, located on the short arm of chromosome 1, consists of 13 exons and 12 introns. As a result of alternative splicing, three...

  16. Common sequence motifs coding for higher-plant and prokaryotic O-acetylserine (thiol)-lyases: bacterial origin of a chloroplast transit peptide?

    Science.gov (United States)

    Rolland, N; Job, D; Douce, R

    1993-08-01

    A comparison of the amino acid sequence of O-acetylserine (thiol)-lyase (EC 4.2.99.8) from Escherichia coli and the isoforms of this enzyme found in the cytosolic and chloroplastic compartments of spinach (Spinacia oleracea) leaf cells allows the essential lysine residue involved in the binding of the pyridoxal 5'-phosphate cofactor to be identified. The results of further sequence comparison of cDNAs coding for these proteins are discussed in the frame of the endosymbiotic theory of chloroplast evolution. The results are compatible with a mechanism in which the chloroplast enzyme originated from the cytosolic enzyme and both plant genes originated from a common prokaryotic ancestor. The comparison also suggests that the 5'-non-coding sequence of the bacterial gene was transferred to the plant cell nucleus and that it has been used to create the N-terminal portions of both plant enzymes, and possibly the transit peptide of the chloroplast enzyme. PMID:7916619

  17. Multiple rewards from a treasure trove of novel glycoside hydrolase and polysaccharide lyase structures: new folds, mechanistic details, and evolutionary relationships.

    Science.gov (United States)

    Fushinobu, Shinya; Alves, Victor D; Coutinho, Pedro M

    2013-10-01

    Recent progress in three-dimensional structure analyses of glycoside hydrolases (GHs) and polysaccharide lyases (PLs), the historically relevant enzyme classes involved in the cleavage of glycosidic bonds of carbohydrates and glycoconjugates, is reviewed. To date, about 80% and 95% of the GH and PL families, respectively, have a representative crystal structure. New structures have been determined for enzymes acting on plant cell wall polysaccharides, sphingolipids, blood group antigens, milk oligosaccharides, N-glycans, oral biofilms and dietary seaweeds. Some GH enzymes have very unique catalytic residues such as the Asp-His dyad. New methods such as high-speed atomic force microscopy and computational simulation have opened up a path to investigate both the dynamics and the detailed molecular interactions displayed by these enzymes. PMID:23816329

  18. Cis-and Trans-Cinnamic Acids Have Different Effects on the Catalytic Properties of Arabidopsis Phenylalanine Ammonia Lyases PAL1, PAL2, PAL4

    Institute of Scientific and Technical Information of China (English)

    Ming-Jie CHEN; Veerappan VIJAYKUMAR; Bing-Wen LU; Bing XIA; Ning LI

    2005-01-01

    Cis-cinnamic acid (CA) is a naturally occurring compound, presumably converted from transCA in higher plants. To investigate the effect of cis-CA on the activity of Arabidopsis phenylalanine ammonia lyase (PAL), AtPAL1, AtPAL2, and AtPAL4 genes were isolated using reverse transcription polymerase chain reaction. These genes were fused to a glutathione S-transferase gene and overexpressed in a heterologous prokaryotic system of Escherichia coli. The purified PAL1, PAL2 and PAL4 enzymes were characterized biochemically to determine the effects of cis-CA on the kinetic parameter Km. The results showed that cis-CA is a competitive inhibitor for PAL1, but not PAL2 and PAL4, whereas trans-CA acts as a competitive inhibitor for all three PAL isomers, suggesting that cis- and trans-CA have different effects on the catalytic activity of PAL.

  19. Anaerobic Induction of Isocitrate Lyase and Malate Synthase in Submerged Rice Seedlings Indicates the Important Metabolic Role of the Glyoxylate Cycle

    Institute of Scientific and Technical Information of China (English)

    Ying LU; Yong-Rui WU; Bin HAN

    2005-01-01

    The glyoxylate cycle is a modified form of the tricarboxylic acid cycle that converts C2compounds into C4 dicarboxylic acids at plant developmental stages. By studying submerged rice seedlings,we revealed the activation of the glyoxylate cycle by identifying the increased transcripts of mRNAs of the genes of isocitrate lyase (ICL) and malate synthase (MS), two characteristic enzymes of the glyoxylate cycle. Northern blot analysis showed that ICL and MS were activated in the prolonged anaerobic environment.The activity assay of pyruvate decarboxylase and ICL in the submerged seedlings indicated an 8.8-fold and 3.5-fold increase over that in the unsubmerged seedlings, respectively. The activity assay of acetyl-coenzyme A synthetase in the submerged seedlings indicated a 3-fold increase over that in the unsubmerged seedlings, which is important for initiating acetate metabolism. Consequently, we concluded that the glyoxylate cycle was involved in acetate metabolism under anaerobic conditions.

  20. Change in the Content of Salicylic Acid and in the Activities of Phenylalanine Ammonia-Lyase and Catalase in Wheat Seedling Roots Under the Effect of Azospirillum Lectins

    Directory of Open Access Journals (Sweden)

    Alen'kina S.A.

    2012-05-01

    Full Text Available We investigated the time course of changes in the endogenous content of salicylic acid, the ratio between the acid's free and bound forms, and changes in the activities of phenylalanine ammonia-lyase and catalase in wheat seedling roots under the effect of the lectins of two strains of the associative nitrogen-fixing bacterium Azospirillum: A. brasilense Sp7 and its mutant defective in lectin activity, A. brasilense Sp7.2.3. Differences in plant response to the action of the lectins from these two strains were established. On the basis of the obtained data, a model was proposed for lectin-assisted induction of resistance, according to which the lectin effect on the roots of seedlings results in accumulation of free salicylic acid, which inhibits catalase activity, ultimately leading to accumulation of hydrogen peroxide and to formation of induced resistance.

  1. The chemolithoautotroph Acidithiobacillus ferrooxidans can survive under phosphate-limiting conditions by expressing a C-P lyase operon that allows it to grow on phosphonates.

    Science.gov (United States)

    Vera, Mario; Pagliai, Fernando; Guiliani, Nicolas; Jerez, Carlos A

    2008-03-01

    The chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans is of great importance in biomining operations. During the bioleaching of ores, microorganisms are subjected to a variety of environmental stresses and to the limitations of some nutrients, such as inorganic phosphate (P(i)), which is an essential component for all living cells. Although the primary source of phosphorus for microorganisms is P(i), some bacteria are also able to metabolize P(i) esters (with a C-O-P bond) and phosphonates (with a very inert C-P bond). By using bioinformatic analysis of genomic sequences of the type strain of A. ferrooxidans (ATCC 23270), we found that as part of a Pho regulon, this bacterium has a complete gene cluster encoding C-P lyase, which is the main bacterial enzyme involved in phosphonate (Pn) degradation in other microorganisms. A. ferrooxidans was able to grow in the presence of methyl-Pn or ethyl-Pn as an alternative phosphorus source. Under these growth conditions, a great reduction in inorganic polyphosphate (polyP) levels was seen compared with the level for cells grown in the presence of P(i). By means of reverse transcription-PCR (RT-PCR), DNA macroarrays, and real-time RT-PCR experiments, it was found that A. ferrooxidans phn genes were cotranscribed and their expression was induced when the microorganism was grown in methyl-Pn as the only phosphorus source. This is the first report of phosphonate utilization in a chemolithoautotrophic microorganism. The existence of a functional C-P lyase system is a clear advantage for the survival under P(i) limitation, a condition that may greatly affect the bioleaching of ores. PMID:18203861

  2. Impact of Soil Field Water Capacity on Secondary Metabolites, Phenylalanine Ammonia-lyase (PAL), Maliondialdehyde (MDA) and Photosynthetic Responses of Malaysian Kacip Fatimah (Labisia pumila Benth)

    OpenAIRE

    Jaafar, Hawa Z. E.; Nur Farhana Mohamad Fakri; Mohd Hafiz Ibrahim

    2012-01-01

    A randomized complete block design 2 × 4 experiment was designed and conducted for 15 weeks to characterize the relationships between production of total phenolics, flavonoid, anthocyanin, leaf gas exchange, total chlorophyll, phenylalanine ammonia-lyase (PAL) and malondialdehyde (MDA) activity in two varieties of Labisia pumila Benth, namely the var. alata and pumila, under four levels of evapotranspiration replacement (ER) (100%...

  3. The R46Q, R131Q and R154H Polymorphs of Human DNA Glycosylase/β-Lyase hOgg1 Severely Distort the Active Site and DNA Recognition Site but do not Cause Unfolding†

    OpenAIRE

    Anderson, Peter C.; Daggett, Valerie

    2009-01-01

    Reactive oxygen species can cause widespread cellular damage, including base alterations and strand breaks in DNA. An array of DNA-repair enzymes constitutes an essential part of the line of defense that cells use against oxidative damage to the genome. A DNA glycosylase/β-lyase enzyme, Ogg1, scavenges the genome for 8-oxoguanine, a major mutagenic DNA adduct induced by reactive oxygen species, and catalyzes its excision and subsequent cleavage of the DNA phosphate backbone. Several polymorph...

  4. 3- and 4-pyridylalkyl adamantanecarboxylates: inhibitors of human cytochrome P450(17 alpha) (17 alpha-hydroxylase/C17,20-lyase). Potential nonsteroidal agents for the treatment of prostatic cancer.

    Science.gov (United States)

    Chan, F C; Potter, G A; Barrie, S E; Haynes, B P; Rowlands, M G; Houghton, J; Jarman, M

    1996-08-16

    Various 3- and 4-pyridylalkyl 1-adamantanecarboxylates have been synthesized and tested for inhibitory activity toward the 17 alpha-hydroxylase and C17,20-lyase activities of human testicular cytochrome P450(17 alpha). The 4-pyridylalkyl esters were much more inhibitory than their 3-pyridylalkyl counterparts. The most potent was (S)-1-(4-pyridyl)ethyl 1-adamantanecarboxylate (3b; IC50 for lyase, 1.8 nM), whereas the (R)-enantiomer 3a was much less inhibitory (IC50 74 nM). Nearly as potent as 3b was the dimethylated counterpart, the 2-(4-pyridylpropan-2-yl) ester 5 (IC50 2.7 nM), which was also more resistant to degradation by esterases. In contrast to their 4-pyridyl analogs, the enantiomers of the 1-(3-pyridyl)ethyl ester were similarly inhibitory (IC50 for lyase; (R)-isomer 8a 150 nM, (S)-isomer 8b 230 nM). Amides corresponding to the 4-pyridylmethyl ester 1 and the (S)-1-(4-pyridyl)ethyl ester 3b, respectively 11 and 15b, were much less inhibitory than their ester counterparts. On the basis of a combination of inhibitory potency and resistance to esterases, the ester 5 was the best candidate for further development as a potential nonsteroidal inhibitor of cytochrome P450(17 alpha) for the treatment of prostate cancer. PMID:8765515

  5. NMDA Receptors and Oxidative Stress Induced by the Major Metabolites Accumulating in HMG Lyase Deficiency Mediate Hypophosphorylation of Cytoskeletal Proteins in Brain From Adolescent Rats: Potential Mechanisms Contributing to the Neuropathology of This Disease.

    Science.gov (United States)

    Fernandes, Carolina Gonçalves; Pierozan, Paula; Soares, Gilberto Machado; Ferreira, Fernanda; Zanatta, Ângela; Amaral, Alexandre Umpierrez; Borges, Clarissa Günther; Wajner, Moacir; Pessoa-Pureur, Regina

    2015-10-01

    Neurological symptoms and cerebral abnormalities are commonly observed in patients with 3-hydroxy-3-methylglutaryl-CoA lyase (HMG lyase) deficiency, which is biochemically characterized by predominant tissue accumulation of 3-hydroxy-3-methylglutaric (HMG), 3-methylglutaric (MGA), and 3-methylglutaconic (MGT) acids. Since the pathogenesis of this disease is poorly known, the present study evaluated the effects of these compounds on the cytoskeleton phosphorylating system in rat brain. HMG, MGA, and MGT caused hypophosphorylation of glial fibrillary acidic protein (GFAP) and of the neurofilament subunits NFL, NFM, and NFH. HMG-induced hypophosphorylation was mediated by inhibiting the cAMP-dependent protein kinase (PKA) on Ser55 residue of NFL and c-Jun kinase (JNK) by acting on KSP repeats of NFM and NFH subunits. We also evidenced that the subunit NR2B of NMDA receptor and Ca(2+) was involved in HMG-elicited hypophosphorylation of cytoskeletal proteins. Furthermore, the antioxidants L-NAME and TROLOX fully prevented both the hypophosphorylation and the inhibition of PKA and JNK caused by HMG, suggesting that oxidative damage may underlie these effects. These findings indicate that the main metabolites accumulating in HMG lyase deficiency provoke hypophosphorylation of cytoskeleton neural proteins with the involvement of NMDA receptors, Ca(2+), and reactive species. It is presumed that these alterations may contribute to the neuropathology of this disease. PMID:26174040

  6. Structural and molecular basis for the novel catalytic mechanism and evolution of DddP, an abundant peptidase-like bacterial Dimethylsulfoniopropionate lyase: a new enzyme from an old fold.

    Science.gov (United States)

    Wang, Peng; Chen, Xiu-Lan; Li, Chun-Yang; Gao, Xiang; Zhu, De-yu; Xie, Bin-Bin; Qin, Qi-Long; Zhang, Xi-Ying; Su, Hai-Nan; Zhou, Bai-Cheng; Xun, Lu-ying; Zhang, Yu-Zhong

    2015-10-01

    The microbial cleavage of dimethylsulfoniopropionate (DMSP) generates volatile dimethyl sulfide (DMS) and is an important step in global sulfur and carbon cycles. DddP is a DMSP lyase in marine bacteria, and the deduced dddP gene product is abundant in marine metagenomic data sets. However, DddP belongs to the M24 peptidase family according to sequence alignment. Peptidases hydrolyze C-N bonds, but DddP is deduced to cleave C-S bonds. Mechanisms responsible for this striking functional shift are currently unknown. We determined the structures of DMSP lyase RlDddP (the DddP from Ruegeria lacuscaerulensis ITI_1157) bound to inhibitory 2-(N-morpholino) ethanesulfonic acid or PO4 (3-) and of two mutants of RlDddP bound to acrylate. Based on structural, mutational and biochemical analyses, we characterized a new ion-shift catalytic mechanism of RlDddP for DMSP cleavage. Furthermore, we suggested the structural mechanism leading to the loss of peptidase activity and the subsequent development of DMSP lyase activity in DddP. This study sheds light on the catalytic mechanism and the divergent evolution of DddP, leading to a better understanding of marine bacterial DMSP catabolism and global DMS production. PMID:26154071

  7. Study of RNA interference inhibiting rat ovarian androgen biosynthesis by depressing 17alpha-hydroxylase/17, 20-lyase activity in vivo

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    Yang Xing

    2009-07-01

    Full Text Available Abstract Background 17alpha-hydroxylase/17, 20-lyase encoded by CYP17 is the key enzyme in androgen biosynthesis pathway. Previous studies demonstrated the accentuation of the enzyme in patients with polycystic ovary syndrome (PCOS was the most important mechanism of androgen excess. We chose CYP17 as the therapeutic target, trying to suppress the activity of 17alpha-hydroxylase/17, 20-lyase and inhibit androgen biosynthesis by silencing the expression of CYP17 in the rat ovary. Methods Three CYP17-targeting and one negative control oligonucleotides were designed and used in the present study. The silence efficiency of lentivirus shRNA was assessed by qRT-PCR, Western blotting and hormone assay. After subcapsular injection of lentivirus shRNA in rat ovary, the delivery efficiency was evaluated by GFP fluorescence and qPCR. Total RNA was extracted from rat ovary for CYP17 mRNA determination and rat serum was collected for hormone measurement. Results In total, three CYP17-targeting lentivirus shRNAs were synthesized. The results showed that all of them had a silencing effect on CYP17 mRNA and protein. Moreover, androstenedione secreted by rat theca interstitial cells (TIC in the RNAi group declined significantly compared with that in the control group. Two weeks after rat ovarian subcapsular injection of chosen CYP17 shRNA, the GFP fluorescence of frozen ovarian sections could be seen clearly under fluorescence microscope. It also showed that the GFP DNA level increased significantly, and its relative expression level was 7.42 times higher than that in the control group. Simultaneously, shRNA treatment significantly decreased CYP17 mRNA and protein levels at 61% and 54%, respectively. Hormone assay showed that all the levels of androstenedione, 17-hydroxyprogesterone and testosterone declined to a certain degree, but progesterone levels declined significantly. Conclusion The present study proves for the first time that ovarian androgen

  8. Phenylalanine Ammonia-Lyase-Catalyzed Deamination of an Acyclic Amino Acid: Enzyme Mechanistic Studies Aided by a Novel Microreactor Filled with Magnetic Nanoparticles.

    Science.gov (United States)

    Weiser, Diána; Bencze, László Csaba; Bánóczi, Gergely; Ender, Ferenc; Kiss, Róbert; Kókai, Eszter; Szilágyi, András; Vértessy, Beáta G; Farkas, Ödön; Paizs, Csaba; Poppe, László

    2015-11-01

    Phenylalanine ammonia-lyase (PAL), found in many organisms, catalyzes the deamination of l-phenylalanine (Phe) to (E)-cinnamate by the aid of its MIO prosthetic group. By using PAL immobilized on magnetic nanoparticles and fixed in a microfluidic reactor with an in-line UV detector, we demonstrated that PAL can catalyze ammonia elimination from the acyclic propargylglycine (PG) to yield (E)-pent-2-ene-4-ynoate. This highlights new opportunities to extend MIO enzymes towards acyclic substrates. As PG is acyclic, its deamination cannot involve a Friedel-Crafts-type attack at an aromatic ring. The reversibility of the PAL reaction, demonstrated by the ammonia addition to (E)-pent-2-ene-4-ynoate yielding enantiopure l-PG, contradicts the proposed highly exothermic single-step mechanism. Computations with the QM/MM models of the N-MIO intermediates from L-PG and L-Phe in PAL show similar arrangements within the active site, thus supporting a mechanism via the N-MIO intermediate. PMID:26345352

  9. Structural Basis for the Inhibition Mechanism of Human Cystathionine gamma-Lyase, an Enzyme Responsible for the Productin of H2S

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Q.; Collins, R; Huang, S; Holmberg-Schiavone, L; Anand, G; Tan, C; van-den-Berg, S; Deng, L; Moore, P; et. al.

    2009-01-01

    Impairment of the formation or action of hydrogen sulfide (H(2)S), an endogenous gasotransmitter, is associated with various diseases, such as hypertension, diabetes mellitus, septic and hemorrhagic shock, and pancreatitis. Cystathionine beta-synthase and cystathionine gamma-lyase (CSE) are two pyridoxal-5'-phosphate (PLP)-dependent enzymes largely responsible for the production of H(2)S in mammals. Inhibition of CSE by DL-propargylglycine (PAG) has been shown to alleviate disease symptoms. Here we report crystal structures of human CSE (hCSE), in apo form, and in complex with PLP and PLP.PAG. Structural characterization, combined with biophysical and biochemical studies, provides new insights into the inhibition mechanism of hCSE-mediated production of H(2)S. Transition from the open form of apo-hCSE to the closed PLP-bound form reveals large conformational changes hitherto not reported. In addition, PAG binds hCSE via a unique binding mode, not observed in PAG-enzyme complexes previously. The interaction of PAG-hCSE was not predicted based on existing information from known PAG complexes. The structure of hCSE.PLP.PAG complex highlights the particular importance of Tyr(114) in hCSE and the mechanism of PAG-dependent inhibition of hCSE. These results provide significant insights, which will facilitate the structure-based design of novel inhibitors of hCSE to aid in the development of therapies for diseases involving disorders of sulfur metabolism.

  10. Functional Analyses of Resurrected and Contemporary Enzymes Illuminate an Evolutionary Path for the Emergence of Exolysis in Polysaccharide Lyase Family 2.

    Science.gov (United States)

    McLean, Richard; Hobbs, Joanne K; Suits, Michael D; Tuomivaara, Sami T; Jones, Darryl R; Boraston, Alisdair B; Abbott, D Wade

    2015-08-28

    Family 2 polysaccharide lyases (PL2s) preferentially catalyze the β-elimination of homogalacturonan using transition metals as catalytic cofactors. PL2 is divided into two subfamilies that have been generally associated with secretion, Mg(2+) dependence, and endolysis (subfamily 1) and with intracellular localization, Mn(2+) dependence, and exolysis (subfamily 2). When present within a genome, PL2 genes are typically found as tandem copies, which suggests that they provide complementary activities at different stages along a catabolic cascade. This relationship most likely evolved by gene duplication and functional divergence (i.e. neofunctionalization). Although the molecular basis of subfamily 1 endolytic activity is understood, the adaptations within the active site of subfamily 2 enzymes that contribute to exolysis have not been determined. In order to investigate this relationship, we have conducted a comparative enzymatic analysis of enzymes dispersed within the PL2 phylogenetic tree and elucidated the structure of VvPL2 from Vibrio vulnificus YJ016, which represents a transitional member between subfamiles 1 and 2. In addition, we have used ancestral sequence reconstruction to functionally investigate the segregated evolutionary history of PL2 progenitor enzymes and illuminate the molecular evolution of exolysis. This study highlights that ancestral sequence reconstruction in combination with the comparative analysis of contemporary and resurrected enzymes holds promise for elucidating the origins and activities of other carbohydrate active enzyme families and the biological significance of cryptic metabolic pathways, such as pectinolysis within the zoonotic marine pathogen V. vulnificus. PMID:26160170

  11. Cystathionine-γ-lyase gene silencing with siRNA in monocytes/ macrophages attenuates inflammation in cecal ligation and puncture-induced sepsis in the mouse

    Indian Academy of Sciences (India)

    A Badiei; ST Chambers; RR Gaddam; M Bhatia

    2016-03-01

    Hydrogen sulphide is an endogenous inflammatory mediator produced by cystathionine-γ-lyase (CSE) in macrophages. To determine the role of H2S and macrophages in sepsis, we used small interference RNA (siRNA) to target the CSE gene and investigated its effect in a mouse model of sepsis. Cecal ligation puncture (CLP)-induced sepsis is characterized by increased levels of myeloperoxidase (MPO) activity, morphological changes in liver and pro-inflammatory cytokines and chemokines in the liver and lung. SiRNA treatment attenuated inflammation in the liver and lungs of mice following CLP-induced sepsis. Liver MPO activity increased in CLP-induced sepsis and treatment with siRNA significantly reduced this. Similarly, lung MPO activity increased following induction of sepsis with CLP while siRNA treatment significantly reduced MPO activity. Liver and lung cytokine and chemokine levels in CLP-induced sepsis reduced following treatment with siRNA. These findings show a crucial pro-inflammatory role for H2S synthesized by CSE in macrophages in sepsis and suggest CSE gene silencing with siRNA as a potential therapeutic approach for this condition.

  12. Rhodotorula glutinis Phenylalanine/Tyrosine Ammonia Lyase Enzyme Catalyzed Synthesis of the Methyl Ester of para-Hydroxycinnamic Acid and its Potential Antibacterial Activity

    Science.gov (United States)

    MacDonald, Marybeth C.; Arivalagan, Pugazhendhi; Barre, Douglas E.; MacInnis, Judith A.; D’Cunha, Godwin B.

    2016-01-01

    Biotransformation of L-tyrosine methyl ester (L-TM) to the methyl ester of para- hydroxycinnamic acid (p-HCAM) using Rhodotorula glutinis yeast phenylalanine/tyrosine ammonia lyase (PTAL; EC 4.3.1.26) enzyme was successfully demonstrated for the first time; progress of the reaction was followed by spectrophotometric determination at 315 nm. The following conditions were optimized for maximal formation of p-HCAM: pH (8.5), temperature (37°C), speed of agitation (50 rpm), enzyme concentration (0.080 μM), and substrate concentration (0.50 mM). Under these conditions, the yield of the reaction was ∼15% in 1 h incubation period and ∼63% after an overnight (∼18 h) incubation period. The product (p-HCAM) of the reaction of PTAL with L-TM was confirmed using Nuclear Magnetic Resonance spectroscopy (NMR). Fourier Transform Infra-Red spectroscopy (FTIR) was carried out to rule out potential hydrolysis of p-HCAM during overnight incubation. Potential antibacterial activity of p-HCAM was tested against several strains of Gram-positive and Gram-negative bacteria. This study describes a synthetically useful transformation, and could have future clinical and industrial applications. PMID:27014206

  13. IMMUNOHISTOCHEMICAL APPROACH REVEALS LOCALIZATION OF CYSTATHIONINE-?-LYASE AND CYSTATHIONINE-ß-SYNTHETASE IN ETHANOL-INDUCED GASTRIC MUCOSA DAMAGE IN MICE

    Directory of Open Access Journals (Sweden)

    Jand-Venes Rolim MEDEIROS

    2013-04-01

    Full Text Available Context Hydrogen sulphide (H2S has been proved to be a neuromodulator and contributes to the maintenance of gastric mucosal integrity in damage caused by anti-inflammatory nonsteroidal drugs. Previously, we demonstrated that H2S synthesis is essential to gastric protection against ethanol. Objective To better understanding the role of H2S and the detailed localization of its production in both normal and injured stomach due to ethanol injection, we studied the expression of cystathionine-γ-lyase (CSE and cystathionine-β-synthetase (CBS isoforms in gastric mucosa of mice treated with saline or 50% ethanol. Methods Mice were treated by gavage with saline or 50% ethanol (0.5 mL/25 g. After 1 hour, mice were sacrificed, and gastric tissue was evaluated by histological and immunohistochemical analysis specific for CSE and CBS. Results We have demonstrated a non-specific expression of CBS in the normal gastric mucosa and expression of CSE occurring mainly in the parietal cells of the animals treated with ethanol. Conclusion Thus, we demonstrated that the expression of CBS appears to be constitutive and diffuse across the gastric epithelium, while the expression of CSE appears to be induced in parietal cells by damage agents such as ethanol.

  14. Efficient preparation of enantiopure D-phenylalanine through asymmetric resolution using immobilized phenylalanine ammonia-lyase from Rhodotorula glutinis JN-1 in a recirculating packed-bed reactor.

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    Longbao Zhu

    Full Text Available An efficient enzymatic process was developed to produce optically pure D-phenylalanine through asymmetric resolution of the racemic DL-phenylalanine using immobilized phenylalanine ammonia-lyase (RgPAL from Rhodotorula glutinis JN-1. RgPAL was immobilized on a modified mesoporous silica support (MCM-41-NH-GA. The resulting MCM-41-NH-GA-RgPAL showed high activity and stability. The resolution efficiency using MCM-41-NH-GA-RgPAL in a recirculating packed-bed reactor (RPBR was higher than that in a stirred-tank reactor. Under optimal operational conditions, the volumetric conversion rate of L-phenylalanine and the productivity of D-phenylalanine reached 96.7 mM h⁻¹ and 0.32 g L⁻¹ h⁻¹, respectively. The optical purity (eeD of D-phenylalanine exceeded 99%. The RPBR ran continuously for 16 batches, the conversion ratio did not decrease. The reactor was scaled up 25-fold, and the productivity of D-phenylalanine (eeD>99% in the scaled-up reactor reached 7.2 g L⁻¹ h⁻¹. These results suggest that the resolution process is an alternative method to produce highly pure D-phenylalanine.

  15. AMP-activated protein kinase and ATP-citrate lyase are two distinct molecular targets for ETC-1002, a novel small molecule regulator of lipid and carbohydrate metabolism.

    Science.gov (United States)

    Pinkosky, Stephen L; Filippov, Sergey; Srivastava, Rai Ajit K; Hanselman, Jeffrey C; Bradshaw, Cheryl D; Hurley, Timothy R; Cramer, Clay T; Spahr, Mark A; Brant, Ashley F; Houghton, Jacob L; Baker, Chris; Naples, Mark; Adeli, Khosrow; Newton, Roger S

    2013-01-01

    ETC-1002 (8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel investigational drug being developed for the treatment of dyslipidemia and other cardio-metabolic risk factors. The hypolipidemic, anti-atherosclerotic, anti-obesity, and glucose-lowering properties of ETC-1002, characterized in preclinical disease models, are believed to be due to dual inhibition of sterol and fatty acid synthesis and enhanced mitochondrial long-chain fatty acid β-oxidation. However, the molecular mechanism(s) mediating these activities remained undefined. Studies described here show that ETC-1002 free acid activates AMP-activated protein kinase in a Ca(2+)/calmodulin-dependent kinase β-independent and liver kinase β 1-dependent manner, without detectable changes in adenylate energy charge. Furthermore, ETC-1002 is shown to rapidly form a CoA thioester in liver, which directly inhibits ATP-citrate lyase. These distinct molecular mechanisms are complementary in their beneficial effects on lipid and carbohydrate metabolism in vitro and in vivo. Consistent with these mechanisms, ETC-1002 treatment reduced circulating proatherogenic lipoproteins, hepatic lipids, and body weight in a hamster model of hyperlipidemia, and it reduced body weight and improved glycemic control in a mouse model of diet-induced obesity. ETC-1002 offers promise as a novel therapeutic approach to improve multiple risk factors associated with metabolic syndrome and benefit patients with cardiovascular disease. PMID:23118444

  16. Modification of potato cell wall pectin by the introduction of rhamnogalacturonan lyase and β-galactosidase transgenes and their side effects.

    Science.gov (United States)

    Huang, Jie-Hong; Kortstee, Anne; Dees, Dianka C T; Trindade, Luisa M; Schols, Henk A; Gruppen, Harry

    2016-06-25

    Genes encoding pectic enzymes were introduced to wild-type potato Karnico. Cell wall materials were extracted from Karnico and transgenic lines expressing β-galactosidase (β-Gal-14 mutant) or rhamnogalacturonan lyase (RGL-18 mutant). After sequential extraction, β-Gal-14 hot buffer-soluble solids (HBSS) of pectin contained 54% less galactose than Karnico HBSS, representing shorter galactan side chains. The individual pectin populations of β-Gal-14 HBSS showed different modifications extended to the two sub-populations as obtained by ion-exchange chromatography. Compared to wild-type, RGL-18 HBSS contained 27% more galacturonic acid and 55% less Gal on fresh potato weight basis, which was due to the removal of galactan-rich rhamnogalacturonan I (RG-I) segments. All pectin populations of RGL-18 showed consistently low levels of RG-I segments. Transgenic modification showed side effects on the methyl-esterification and acetyl substitution of RGL-18 HBSS (DM=53, DA=21), but not of the β-Gal-14 HBSS in comparison to wild-type (DM=29, DA=54). PMID:27083787

  17. Overexpression of Rice Sphingosine-1-Phoshpate Lyase Gene OsSPL1 in Transgenic Tobacco Reduces Salt and Oxidative Stress Tolerance

    Institute of Scientific and Technical Information of China (English)

    Huijuan Zhang; Jing Zhai; Jibo Mo; Dayong Li; Fengming Song

    2012-01-01

    Sphingolipids,including sphingosine-1-phosphate (S1P),have been shown to function as signaling mediators to regulate diverse aspects of plant growth,development,and stress response.In this study,we performed functional analysis of a rice (Oryza sativa) S1P lyase gene OsSPL1 in transgenic tobacco plants and explored its possible involvement in abiotic stress response.Overexpression of OsSPL1 in transgenic tobacco resulted in enhanced sensitivity to exogenous abscisic acid (ABA),and decreased tolerance to salt and oxidative stress,when compared with the wild type.Furthermore,the expression levels of some selected stress-related genes in OsSPL1-overexpressing plants were reduced after application of salt or oxidative stress,indicating that the altered responsiveness of stress-related genes may be responsible for the reduced tolerance in OsSPL1-overexpressing tobacco plants under salt and oxidative stress.Our results suggest that rice OsSPL1 plays an important role in abiotic stress responses.

  18. Impact of soil field water capacity on secondary metabolites, phenylalanine ammonia-lyase (PAL), maliondialdehyde (MDA) and photosynthetic responses of Malaysian kacip fatimah (Labisia pumila Benth).

    Science.gov (United States)

    Jaafar, Hawa Z E; Ibrahim, Mohd Hafiz; Mohamad Fakri, Nur Farhana

    2012-01-01

    A randomized complete block design 2 × 4 experiment was designed and conducted for 15 weeks to characterize the relationships between production of total phenolics, flavonoid, anthocyanin, leaf gas exchange, total chlorophyll, phenylalanine ammonia-lyase (PAL) and malondialdehyde (MDA) activity in two varieties of Labisia pumila Benth, namely the var. alata and pumila, under four levels of evapotranspiration replacement (ER) (100%; well watered), (75%, moderate water stress), (50%; high water stress) and (25%; severe water stress). The production of total phenolics, flavonoids, anthocyanin, soluble sugar and relative leaf water content was affected by the interaction between varieties and SWC. As the ER levels decreased from 100% to 25%, the production of PAL and MDA activity increased steadily. At the highest (100%) ER L. pumila exhibited significantly higher net photosynthesis, apparent quantum yield, maximum efficiency of photosystem II (f(v)/f(m)) and lower dark respiration rates compared to the other treatment. The production of total phenolics, flavonoids and anthocyanin was also found to be higher under high water stress (50% ER replacement) compared to severe water stress (25% ER). From this study, it was observed that as net photosynthesis, apparent quantum yield and chlorophyll content were downregulated under high water stress the production of total phenolics, flavonoids and anthocyanin were upregulated implying that the imposition of high water stress can enhance the medicinal properties of L. pumila Benth. PMID:22695235

  19. Prognostic Value of Malic Enzyme and ATP-Citrate Lyase in Non-Small Cell Lung Cancer of the Young and the Elderly.

    Directory of Open Access Journals (Sweden)

    Agnes Csanadi

    Full Text Available Lung cancer is the leading cause of death among malignancies worldwide. Understanding its biology is therefore of pivotal importance to improve patient's prognosis. In contrast to non-neoplastic tissues, cancer cells utilize glucose mainly for production of basic cellular modules '(i.e. nucleotides, aminoacids, fatty acids. In cancer, Malic enzyme (ME and ATP-citrate lyase (ACLY are key enzymes linking aerobic glycolysis and fatty acid synthesis and may therefore be of biological and prognostic significance in non-small cell lung cancer (NSCLC.ME and ACLY expression was analyzed in 258 NSCLC in correlation with clinico-pathological parameters including patient's survival.Though, overall expression of both enzymes correlated positively, ACLY was associated with local tumor stage, whereas ME correlated with occurrence of mediastinal lymph node metastases. Young patients overexpressing ACLY and/or ME had a significantly longer overall survival. This proved to be an independent prognostic factor. This contrasts older NSCLC patients, in whom overexpression of ACLY and/or ME appears to predict the opposite.In NSCLC, ME and ACLY show different enzyme expressions relating to local and mediastinal spread. Most important, we detected an inverse prognostic impact of ACLY and/or ME overexpression in young and elderly patients. It can therefore be expected, that treatment of NSCLC especially, if targeting metabolic pathways, requires different strategies in different age groups.

  20. Analysis of ATP-citrate lyase and malic enzyme mutants of Yarrowia lipolytica points out the importance of mannitol metabolism in fatty acid synthesis.

    Science.gov (United States)

    Dulermo, Thierry; Lazar, Zbigniew; Dulermo, Rémi; Rakicka, Magdalena; Haddouche, Ramedane; Nicaud, Jean-Marc

    2015-09-01

    The role of the two key enzymes of fatty acid (FA) synthesis, ATP-citrate lyase (Acl) and malic enzyme (Mae), was analyzed in the oleaginous yeast Yarrowia lipolytica. In most oleaginous yeasts, Acl and Mae are proposed to provide, respectively, acetyl-CoA and NADPH for FA synthesis. Acl was mainly studied at the biochemical level but no strain depleted for this enzyme was analyzed in oleaginous microorganisms. On the other hand the role of Mae in FA synthesis in Y. lipolytica remains unclear since it was proposed to be a mitochondrial NAD(H)-dependent enzyme and not a cytosolic NADP(H)-dependent enzyme. In this study, we analyzed for the first time strains inactivated for corresponding genes. Inactivation of ACL1 decreases FA synthesis by 60 to 80%, confirming its essential role in FA synthesis in Y. lipolytica. Conversely, inactivation of MAE1 has no effects on FA synthesis, except in a FA overaccumulating strain where it improves FA synthesis by 35%. This result definitively excludes Mae as a major key enzyme for FA synthesis in Y. lipolytica. During the analysis of both mutants, we observed a negative correlation between FA and mannitol level. As mannitol and FA pathways may compete for carbon storage, we inactivated YlSDR, encoding a mannitol dehydrogenase converting fructose and NADPH into mannitol and NADP+. The FA content of the resulting mutant was improved by 60% during growth on fructose, demonstrating that mannitol metabolism may modulate FA synthesis in Y. lipolytica. PMID:25959598

  1. Steroidal 5α-reductase and 17α-hydroxylase/17,20-lyase (CYP17) inhibitors useful in the treatment of prostatic diseases.

    Science.gov (United States)

    Salvador, Jorge A R; Pinto, Rui M A; Silvestre, Samuel M

    2013-09-01

    The role of steroidal inhibitors of androgen biosynthesis as potential weapons in the treatment of prostatic diseases, such as benign prostatic hyperplasia and prostatic cancer will be reviewed. Two enzymes have been targeted in the development of inhibitors that potentially could be useful in the management of such conditions. 5α-Reductase is primarily of interest in benign prostatic disease, though some role in the chemoprevention of prostatic carcinoma have been considered, whereas the 17α-hydroxylase/17,20-lyase (CYP17) enzyme is of interest in the treatment of malignant disease. An overview of the main achievements obtained during the past years will be presented, however special focus will be made on steroidal molecules that reached clinical trials or have been commercially launched. Relevant examples of such drugs are finasteride, dutasteride, abiraterone acetate and galeterone (TOK-001, formerly known as VN/124-1). This article is part of a Special Issue entitled "Synthesis and biological testing of steroid derivatives as inhibitors". PMID:23688836

  2. A prokaryotic S1P lyase degrades extracellular S1P in vitro and in vivo: implication for treating hyperproliferative disorders.

    Directory of Open Access Journals (Sweden)

    Andrea Huwiler

    Full Text Available Sphingosine-1-phosphate (S1P regulates a broad spectrum of fundamental cellular processes like proliferation, death, migration and cytokine production. Therefore, elevated levels of S1P may be causal to various pathologic conditions including cancer, fibrosis, inflammation, autoimmune diseases and aberrant angiogenesis. Here we report that S1P lyase from the prokaryote Symbiobacterium thermophilum (StSPL degrades extracellular S1P in vitro and in blood. Moreover, we investigated its effect on cellular responses typical of fibrosis, cancer and aberrant angiogenesis using renal mesangial cells, endothelial cells, breast (MCF-7 and colon (HCT 116 carcinoma cells as disease models. In all cell types, wild-type StSPL, but not an inactive mutant, disrupted MAPK phosphorylation stimulated by exogenous S1P. Functionally, disruption of S1P receptor signaling by S1P depletion inhibited proliferation and expression of connective tissue growth factor in mesangial cells, proliferation, migration and VEGF expression in carcinoma cells, and proliferation and migration of endothelial cells. Upon intravenous injection of StSPL in mice, plasma S1P levels rapidly declined by 70% within 1 h and then recovered to normal 6 h after injection. Using the chicken chorioallantoic membrane model we further demonstrate that also under in vivo conditions StSPL, but not the inactive mutant, inhibited tumor cell-induced angiogenesis as an S1P-dependent process. Our data demonstrate that recombinant StSPL is active under extracellular conditions and holds promise as a new enzyme therapeutic for diseases associated with increased levels of S1P and S1P receptor signaling.

  3. Isolation and Functional Characterization of a Phenylalanine Ammonia-Lyase Gene (SsPAL1 from Coleus (Solenostemon scutellarioides (L. Codd

    Directory of Open Access Journals (Sweden)

    Qinlong Zhu

    2015-09-01

    Full Text Available Phenylalanine ammonia-lyase (PAL is the first enzyme involved in the phenylpropanoid pathway and plays important roles in the secondary metabolisms, development and defense of plants. To study the molecular function of PAL in anthocyanin synthesis of Coleus (Solenostemon scutellarioides (L. Codd, a Coleus PAL gene designated as SsPAL1 was cloned and characterized using a degenerate oligonucleotide primer PCR and RACE method. The full-length SsPAL1 was 2450 bp in size and consisted of one intron and two exons encoding a polypeptide of 711 amino acids. The deduced SsPAL1 protein showed high identities and structural similarities with other functional plant PAL proteins. A series of putative cis-acting elements involved in transcriptional regulation, light and stress responsiveness were found in the upstream regulatory sequence of SsPAL1. Transcription pattern analysis indicated that SsPAL1 was constitutively expressed in all tissues examined and was enhanced by light and different abiotic factors. The recombinant SsPAL1 protein exhibited high PAL activity, at optimal conditions of 60 °C and pH 8.2. Although the levels of total PAL activity and total anthocyanin concentration have a similar variation trend in different Coleus cultivars, there was no significant correlation between them (r = 0.7529, p > 0.1, suggesting that PAL was not the rate-limiting enzyme for the downstream anthocyanin biosynthetic branch in Coleus. This study enables us to further understand the role of SsPAL1 in the phenylpropanoid (flavonoids, anthocyanins biosynthesis in Coleus at the molecular level.

  4. Cystathionine γ-lyase, a H2S-generating enzyme, is a GPBAR1-regulated gene and contributes to vasodilation caused by secondary bile acids.

    Science.gov (United States)

    Renga, Barbara; Bucci, Mariarosaria; Cipriani, Sabrina; Carino, Adriana; Monti, Maria Chiara; Zampella, Angela; Gargiulo, Antonella; d'Emmanuele di Villa Bianca, Roberta; Distrutti, Eleonora; Fiorucci, Stefano

    2015-07-01

    GPBAR1 is a bile acid-activated receptor (BAR) for secondary bile acids, lithocholic (LCA) and deoxycholic acid (DCA), expressed in the enterohepatic tissues and in the vasculature by endothelial and smooth muscle cells. Despite that bile acids cause vasodilation, it is unclear why these effects involve GPBAR1, and the vascular phenotype of GPBAR1 deficient mice remains poorly defined. Previous studies have suggested a role for nitric oxide (NO) in regulatory activity exerted by GPBAR1 in liver endothelial cells. Hydrogen sulfide (H2S) is a vasodilatory agent generated in endothelial cells by cystathionine-γ-lyase (CSE). Here we demonstrate that GPBAR1 null mice had increased levels of primary and secondary bile acids and impaired vasoconstriction to phenylephrine. In aortic ring preparations, vasodilation caused by chenodeoxycholic acid (CDCA), a weak GPBAR1 ligand and farnesoid-x-receptor agonist (FXR), was iberiotoxin-dependent and GPBAR1-independent. In contrast, vasodilation caused by LCA was GPBAR1 dependent and abrogated by propargyl-glycine, a CSE inhibitor, and by 5β-cholanic acid, a GPBAR1 antagonist, but not by N(5)-(1-iminoethyl)-l-ornithine (l-NIO), an endothelial NO synthase inhibitor, or iberiotoxin, a large-conductance calcium-activated potassium (BKCa) channels antagonist. In venular and aortic endothelial (HUVEC and HAEC) cells GPBAR1 activation increases CSE expression/activity and H2S production. Two cAMP response element binding protein (CREB) sites (CREs) were identified in the CSE promoter. In addition, TLCA stimulates CSE phosphorylation on serine residues. In conclusion we demonstrate that GPBAR1 mediates the vasodilatory activity of LCA and regulates the expression/activity of CSE. Vasodilation caused by CDCA involves BKCa channels. The GPBAR1/CSE pathway might contribute to endothelial dysfunction and hyperdynamic circulation in liver cirrhosis. PMID:25934094

  5. Hydrogen sulfide (H{sub 2}S)/cystathionine γ-lyase (CSE) pathway contributes to the proliferation of hepatoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Yan; Ye, Shuang; Yuan, Dexiao; Zhang, Jianghong; Bai, Yang; Shao, Chunlin, E-mail: clshao@shmu.edu.cn

    2014-05-15

    Highlights: • Inhibition of H{sub 2}S/CSE pathway strongly stimulates cellular apoptosis. • Inhibition of H{sub 2}S/CSE pathway suppresses cell growth by blocking EGFR pathway. • H{sub 2}S/CSE pathway is critical for maintaining the proliferation of hepatoma cells. - Abstract: Hydrogen sulfide (H{sub 2}S)/cystathionine γ-lyase (CSE) pathway has been demonstrated to play vital roles in physiology and pathophysiology. However, its role in tumor cell proliferation remains largely unclear. Here we found that CSE over-expressed in hepatoma HepG2 and PLC/PRF/5 cells. Inhibition of endogenous H{sub 2}S/CSE pathway drastically decreased the proliferation of HepG2 and PLC/PRF/5 cells, and it also enhanced ROS production and mitochondrial disruption, pronounced DNA damage and increased apoptosis. Moreover, this increase of apoptosis was associated with the activation of p53 and p21 accompanied by a decreased ratio of Bcl-2/Bax and up-regulation of phosphorylated c-Jun N-terminal kinase (JNK) and caspase-3 activity. In addition, the negative regulation of cell proliferation by inhibition of H{sub 2}S/CSE system correlated with the blockage of cell mitogenic and survival signal transduction of epidermal growth factor receptor (EGFR) via down-regulating the extracellular-signal-regulated kinase 1/2 (ERK1/2) activation. These results demonstrate that H{sub 2}S/CSE and its downstream pathway contribute to the proliferation of hepatoma cells, and inhibition of this pathway strongly suppress the excessive growth of hepatoma cells by stimulating mitochondrial apoptosis and suppressing cell growth signal transduction.

  6. Structure of the ArsI C-As Lyase: Insights into the Mechanism of Degradation of Organoarsenical Herbicides and Growth Promoters.

    Science.gov (United States)

    Nadar, Venkadesh Sarkarai; Yoshinaga, Masafumi; Pawitwar, Shashank S; Kandavelu, Palani; Sankaran, Banumathi; Rosen, Barry P

    2016-06-01

    Arsenic is a ubiquitous and carcinogenic environmental element that enters the biosphere primarily from geochemical sources, but also through anthropogenic activities. Microorganisms play an important role in the arsenic biogeochemical cycle by biotransformation of inorganic arsenic into organic arsenicals and vice versa. ArsI is a microbial non-heme, ferrous-dependent dioxygenase that transforms toxic methylarsenite [MAs(III)] to less toxic and carcinogenic inorganic arsenite [As(III)] by C-As bond cleavage. An ArsI ortholog, TcArsI, from the thermophilic bacterium Thermomonospora curvata was expressed, purified, and crystallized. The structure was solved in both the apo form and with Ni(II), Co(II), or Fe(III). The MAs(III) binding site is a vicinal cysteine pair in a flexible loop. A structure with the loop occupied with β-mercaptoethanol mimics binding of MAs(III). The structure of a mutant protein (Y100H/V102F) was solved in two different crystal forms with two other orientations of the flexible loop. These results suggest that a loop-gating mechanism controls the catalytic reaction. In the ligand-free open state, the loop is exposed to solvent, where it can bind MAs(III). The loop moves toward the active site, where it forms a closed state that orients the C-As bond for dioxygen addition and cleavage. Elucidation of the enzymatic mechanism of this unprecedented C-As lyase reaction will enhance our understanding of recycling of environmental organoarsenicals. PMID:27107642

  7. Redox homeostasis is compromised in vivo by the metabolites accumulating in 3-hydroxy-3-methylglutaryl-CoA lyase deficiency in rat cerebral cortex and liver.

    Science.gov (United States)

    da Rosa, M S; Seminotti, B; Amaral, A U; Fernandes, C G; Gasparotto, J; Moreira, J C F; Gelain, D P; Wajner, M; Leipnitz, G

    2013-12-01

    3-Hydroxy-3-methylglutaryl-CoA lyase (HL) deficiency is a disorder biochemically characterized by the predominant accumulation of 3-hydroxy-3-methylglutarate (HMG), 3-methylglutarate (MGA), 3-methylglutaconate and 3-hydroxyisovalerate in tissues and biological fluids of the affected patients. Neurological symptoms and hepatopathy are commonly found in HL deficiency, especially during metabolic crises. Since the mechanisms of tissue damage in this disorder are not well understood, in the present study we evaluated the ex vivo effects of acute administration of HMG and MGA on important parameters of oxidative stress in cerebral cortex and liver from young rats. In vivo administration of HMG and MGA provoked an increase of carbonyl and carboxy-methyl-lysine formation in cerebral cortex, but not in liver, indicating that these metabolites induce protein oxidative damage in the brain. We also verified that HMG and MGA significantly decreased glutathione concentrations in both cerebral cortex and liver, implying a reduction of antioxidant defenses. Furthermore, HMG and MGA increased 2',7'-dichlorofluorescin oxidation, but did not alter nitrate and nitrite content in cerebral cortex and liver, indicating that HMG and MGA effects are mainly mediated by reactive oxygen species. HMG and MGA also increased the activities of superoxide dismutase and catalase in cerebral cortex and liver, whereas MGA decreased glutathione peroxidase activity in cerebral cortex. Our present data showing a disruption of redox homeostasis in cerebral cortex and liver caused by in vivo administration of HMG and MGA suggest that this pathomechanism may possibly contribute to the brain and liver abnormalities observed in HL-deficient patients. PMID:24127998

  8. Genome-wide identification of citrus ATP-citrate lyase genes and their transcript analysis in fruits reveals their possible role in citrate utilization.

    Science.gov (United States)

    Hu, Xiao-Mei; Shi, Cai-Yun; Liu, Xiao; Jin, Long-Fei; Liu, Yong-Zhong; Peng, Shu-Ang

    2015-02-01

    ATP-citrate lyase (ACL, EC4.1.3.8) catalyzes citrate to oxaloacetate and acetyl-CoA in the cell cytosol, and has important roles in normal plant growth and in the biosynthesis of some secondary metabolites. We identified three ACL genes, CitACLα1, CitACLα2, and CitACLβ1, in the citrus genome database. Both CitACLα1 and CitACLα2 encode putative ACL α subunits with 82.5 % amino acid identity, whereas CitACLβ1 encodes a putative ACL β subunit. Gene structure analysis showed that CitACLα1 and CitACLα2 had 12 exons and 11 introns, and CitACLβ1 had 16 exons and 15 introns. CitACLα1 and CitACLβ1 were predominantly expressed in flower, and CitACLα2 was predominantly expressed in stem and fibrous roots. As fruits ripen, the transcript levels of CitACLα1, CitACLβ1, and/or CitACLα2 in cultivars 'Niuher' and 'Owari' increased, accompanied by significant decreases in citrate content, while their transcript levels decreased significantly in 'Egan No. 1' and 'Iyokan', although citrate content also decreased. In 'HB pummelo', in which acid content increased as fruit ripened, and in acid-free pummelo, transcript levels of CitACLα2, CitACLβ1, and/or CitACLα1 increased. Moreover, mild drought stress and ABA treatment significantly increased citrate contents in fruits. Transcript levels of the three genes were significantly reduced by mild drought stress, and the transcript level of only CitACLβ1 was significantly reduced by ABA treatment. Taken together, these data indicate that the effects of ACL on citrate use during fruit ripening depends on the cultivar, and the reduction in ACL gene expression may be attributed to citrate increases under mild drought stress or ABA treatment. PMID:25120169

  9. First evidence of sphingosine 1-phosphate lyase protein expression and activity downregulation in human neoplasm: implication for resistance to therapeutics in prostate cancer.

    Science.gov (United States)

    Brizuela, Leyre; Ader, Isabelle; Mazerolles, Catherine; Bocquet, Magalie; Malavaud, Bernard; Cuvillier, Olivier

    2012-09-01

    This is the first report of sphingosine 1-phosphate lyase (SPL) protein expression and enzymatic activity in human neoplasm. This enzyme drives irreversible degradation of sphingosine 1-phosphate (S1P), a bioactive lipid associated with resistance to therapeutics in various cancers, including prostate adenocarcinoma. In fresh human prostatectomy specimens, a remarkable decrease in SPL enzymatic activity was found in tumor samples, as compared with normal adjacent tissues. A significant relationship between loss of SPL expression and higher Gleason score was confirmed in tissue microarray (TMA) analysis. Moreover, SPL protein expression and activity were inversely correlated with those of sphingosine kinase-1 (SphK1), the enzyme producing S1P. SPL and SphK1 expressions were independently predictive of aggressive cancer on TMA, supporting the relevance of S1P in prostate cancer. In human C4-2B and PC-3 cell lines, silencing SPL enhanced survival after irradiation or chemotherapy by decreasing expression of proteins involved in sensing and repairing DNA damage or apoptosis, respectively. In contrast, enforced expression of SPL sensitized cancer cells to irradiation or docetaxel by tilting the ceramide/S1P balance toward cell death. Interestingly, the S1P degradation products failed to sensitize to chemo- and radiotherapy, supporting the crucial role of ceramide/S1P balance in cancer. Of note, the combination of SPL enforced expression with a SphK1 silencing strategy by further decreasing S1P content made prostate cancer cells even more sensitive to anticancer therapies, suggesting that a dual strategy aimed at stimulating SPL, and inhibiting SphK1 could represent a future approach to sensitize cancer cells to cancer treatments. PMID:22784711

  10. Reduced Photoinhibition under Low Irradiance Enhanced Kacip Fatimah (Labisia pumila Benth Secondary Metabolites, Phenyl Alanine Lyase and Antioxidant Activity

    Directory of Open Access Journals (Sweden)

    Hawa Z.E. Jaafar

    2012-04-01

    Full Text Available A randomized complete block design experiment was designed to characterize the relationship between production of total flavonoids and phenolics, anthocyanin, photosynthesis, maximum efficiency of photosystem II (Fv/Fm, electron transfer rate (Fm/Fo, phenyl alanine lyase activity (PAL and antioxidant (DPPH in Labisia pumila var. alata, under four levels of irradiance (225, 500, 625 and 900 µmol/m2/s for 16 weeks. As irradiance levels increased from 225 to 900 µmol/m2/s, the production of plant secondary metabolites (total flavonoids, phenolics and antocyanin was found to decrease steadily. Production of total flavonoids and phenolics reached their peaks under 225 followed by 500, 625 and 900 µmol/m2/s irradiances. Significant positive correlation of production of total phenolics, flavonoids and antocyanin content with Fv/Fm, Fm/Fo and photosynthesis indicated up-regulation of carbon-based secondary metabolites (CBSM under reduced photoinhibition on the under low light levels condition. At the lowest irradiance levels, Labisia pumila extracts also exhibited a significantly higher antioxidant activity (DPPH than under high irradiance. The improved antioxidative activity under low light levels might be due to high availability of total flavonoids, phenolics and anthocyanin content in the plant extract. It was also found that an increase in the production of CBSM was due to high PAL activity under low light, probably signifying more availability of phenylalanine (Phe under this condition.

  11. Involvement of Carbohydrate, Protein and Phenylanine Ammonia Lyase in Up-Regulation of Secondary Metabolites in Labisia pumila under Various CO2 and N2 Level

    Directory of Open Access Journals (Sweden)

    Mohd Hafiz Ibrahim

    2011-05-01

    Full Text Available A split plot factorial 2 × 3 experiment was designed to examine and characterize the relationships among secondary metabolites (total phenolics, TP; total flavonoids, TF, carbohydrate content, C/N ratio, protein synthesis and L–phenylalanine ammonia lyase (PAL; EC 4.3.1.5 activity in the Malaysian medicinal herb Labisia pumila (Blume Fern-Vill. under different CO2 concentrations (400 = ambient and 1,200 µmol mol−1 CO2 and three levels of nitrogen fertilization (0, 90 and 270 kg N ha−1 for 15 weeks. The interaction between CO2 and nitrogen levels imposed a significant impact on plant secondary metabolite production, protein, PAL activity and fructose levels. Highest TP and TF were recorded under 1,200 µmol mol−1 CO2 when N fertilizer was not applied; lowest values were obtained at 400 µmol mol−1 CO2 fertilized with the highest N level. Concurrently, fructose contents increased tremendously. Increase in fructose content might also enhance erythose-4-phosphate production (substrate for lignin and phenolic compounds, which shares a common precursor transdalolase in the pentose phosphate pathway. PAL activity was noted to be highest under 1,200 µmol mol−1 CO2 + 0 kg N ha−1 coinciding with subsequent recording of the lowest protein content. The results implied that the increase in plant secondary metabolites production under the tested conditions might be due to diversion of phenylalanine for protein synthesis to production of secondary metabolites. It was also found that the sucrose to starch ratio was also high under high levels of nitrogen fertilization, indicating an enhanced sucrose phosphate synthase activity (SPS; EC 2.4.1.14 under such condition.

  12. Cloning and random mutagenesis of the Erwinia herbicola tyrR gene for high-level expression of tyrosine phenol-lyase.

    Science.gov (United States)

    Katayama, T; Suzuki, H; Koyanagi, T; Kumagai, H

    2000-11-01

    Tyrosine phenol-lyase (Tpl), which can synthesize 3, 4-dihydroxyphenylalanine from pyruvate, ammonia, and catechol, is a tyrosine-inducible enzyme. Previous studies demonstrated that the tpl promoter of Erwinia herbicola is activated by the TyrR protein of Escherichia coli. In an attempt to create a high-Tpl-expressing strain, we cloned the tyrR gene of E. herbicola and then randomly mutagenized it. Mutant TyrR proteins with enhanced ability to activate tpl were screened for by use of the lac reporter system in E. coli. The most increased transcription of tpl was observed for the strain with the mutant tyrR allele involving amino acid substitutions of alanine, cysteine, and glycine for valine-67, tyrosine-72, and glutamate-201, respectively. A tyrR-deficient derivative of E. herbicola was constructed and transformed with a plasmid carrying the mutant tyrR allele (V67A Y72C E201G substitutions). The resultant strain expressed Tpl without the addition of tyrosine to the medium and produced as much of it as was produced by the wild-type strain grown under tyrosine-induced conditions. The regulatory properties of the mutant TyrR(V67A), TyrR(Y72C), TyrR(E201G), and TyrR(V67A Y72C E201G) proteins were examined in vivo. Interestingly, as opposed to the wild-type TyrR protein, the mutant TyrR(V67A) protein had a repressive effect on the tyrP promoter in the presence of phenylalanine as the coeffector. PMID:11055921

  13. Activation of R-mediated innate immunity and disease susceptibility is affected by mutations in a cytosolic O-acetylserine (thiol) lyase in Arabidopsis.

    Science.gov (United States)

    Tahir, Jibran; Watanabe, Mutsumi; Jing, Hai-Chun; Hunter, Donald A; Tohge, Takayuki; Nunes-Nesi, Adriano; Brotman, Yariv; Fernie, Alisdair R; Hoefgen, Rainer; Dijkwel, Paul P

    2013-01-01

    O-acetylserine (thiol) lyases (OASTLs) are evolutionarily conserved proteins among many prokaryotes and eukaryotes that perform sulfur acquisition and synthesis of cysteine. A mutation in the cytosolic OASTL-A1 protein ONSET OF LEAF DEATH3 (OLD3) was previously shown to reduce the OASTL activity of the old3-1 protein in vitro and cause auto-necrosis in specific Arabidopsis accessions. Here we investigated why a mutation in this protein causes auto-necrosis in some but not other accessions. The auto-necrosis was found to depend on Recognition of Peronospora Parasitica 1 (RPP1)-like disease resistance R gene(s) from an evolutionarily divergent R gene cluster that is present in Ler-0 but not the reference accession Col-0. RPP1-like gene(s) show a negative epistatic interaction with the old3-1 mutation that is not linked to reduced cysteine biosynthesis. Metabolic profiling and transcriptional analysis further indicate that an effector triggered-like immune response and metabolic disorder are associated with auto-necrosis in old3-1 mutants, probably activated by an RPP1-like gene. However, the old3-1 protein in itself results in largely neutral changes in primary plant metabolism, stress defence and immune responses. Finally, we showed that lack of a functional OASTL-A1 results in enhanced disease susceptibility against infection with virulent and non-virulent Pseudomonas syringae pv. tomato DC3000 strains. These results reveal an interaction between the cytosolic OASTL and components of plant immunity. PMID:22974487

  14. Sex-specific dysregulation of cysteine oxidation and the methionine and folate cycles in female cystathionine gamma-lyase null mice: a serendipitous model of the methylfolate trap

    Directory of Open Access Journals (Sweden)

    Hua Jiang

    2015-09-01

    Full Text Available In addition to its role in the endogenous synthesis of cysteine, cystathionine gamma-lyase (CGL is a major physiological source of the vasorelaxant hydrogen sulfide. Cgl null mice are potentially useful for studying the influence of this compound upon vascular tone and endothelial function. Here, we confirm a previous report that female Cgl null mice exhibit an approximate 45-fold increase in plasma total homocysteine compared to wild type controls. This level of homocysteine is approximately 3.5-fold higher than that observed in male Cgl null mice and is essentially equivalent to that observed in mouse models of cystathionine beta synthase deficient homocystinuria. Cgl null mice of both sexes exhibited decreased expression of methylenetetrahydrofolate reductase and cysteinesulfinate decarboxylase compared to WT controls. Female Cgl null mice exhibited a sex-specific induction of betaine homocysteine S-methyltransferase and methionine adenosyltransferase 1, alpha and a 70% decrease in methionine synthase expression accompanied by significantly decreased plasma methionine. Decreased plasma cysteine levels in female Cgl null mice were associated with sex-specific dysregulation of cysteine dioxygenase expression. Comparative histological assessment between cystathionine beta-synthase and Cgl null mice indicated that the therapeutic potential of cystathionine against liver injury merits possible further investigation. Collectively, our data demonstrates the importance of considering sex when investigating mouse models of inborn errors of metabolism and indicate that while female Cgl null mice are of questionable utility for studying the physiological role of hydrogen sulfide, they could serve as a useful model for studying the consequences of methionine synthase deficiency and the methylfolate trap.

  15. Radiative muon capture on carbon, oxygen and calcium

    International Nuclear Information System (INIS)

    The photon energy spectra from radiative muon capture on 12C, 16O and 40Ca have been measured using a time projection chamber as a pair spectrometer. The branching ratio for radiative muon capture is sensitive to gp, the induced pseudoscalar coupling constant of the weak interaction. Expressed in terms of the axial-vector weak coupling constant ga, values of gp/ga = 5.7 ± 0.8 and gp/ga = 7.3 ± 0.9 are obtained for 40Ca and 16O respectively, from comparison with phenomenological calculations of the nuclear response. From comparison with microscopic calculations, values of gp/ga = 4.6 ± 1.8, 13.6 +1.6-1.9 and 16.2 +1.3-0.7 for 40Ca, 16O and 12C, respectively, are obtained. The microscopic results are suggestive of a renormalization of the nucleonic form factors within the nucleus. (Author) (78 refs., 14 tabs, 22 figs.)

  16. An interpretation of the carbon-oxygen to iron ratio

    Science.gov (United States)

    Balasubrahmanyan, V. K.; Ormes, J. F.; Ramaty, R.

    1974-01-01

    Data from recent measurements on the composition of primary cosmic rays above 1 GeV/nucleon are critically reviewed for information pertaining to selection of suitable energy-dependent composition models. Areas where crucial experimental information may result in selection of a suitable model are pointed out. In particular, the study of the energy distribution of VVH nuclei, and the derivation of more accurate energy spectra of 10 less than or equal to Z less than or equal to 14, 15 less than or equal to Z less than or equal to 23 and Z greater than 24 would contribute to discriminate between models which rely on interstellar propagation and those that ascribe a different source mechanism for Fe group nuclei.

  17. Substrate specificity of three cytochrome c haem lyase isoenzymes from Wolinella succinogenes: unconventional haem c binding motifs are not sufficient for haem c attachment by NrfI and CcsA1

    OpenAIRE

    Kern, Melanie; Eisel, Florian; Scheithauer, Juliane; Kranz, Robert G.; Simon, Jörg

    2009-01-01

    Bacterial c-type cytochrome maturation is dependent on a complex enzymic machinery. The key reaction is catalysed by cytochrome c haem lyase (CCHL) that usually forms two thioether bonds to attach haem b to the cysteine residues of a haem c binding motif (HBM) which is, in most cases, a CX2CH sequence. Here, the HBM specificity of three distinct CCHL isoenzymes (NrfI, CcsA1 and CcsA2) from the Epsilonproteobacterium Wolinella succinogenes was investigated using either W. succinogenes or Esche...

  18. Crystal Structures of Two Bacterial 3-Hydroxy-3-methylglutaryl-CoA Lyases Suggest a Common Catalytic Mechanism among a Family of TIM Barrel Metalloenzymes Cleaving Carbon-Carbon Bonds

    Energy Technology Data Exchange (ETDEWEB)

    Forouhar,F.; Hussain, M.; Farid, R.; Benach, J.; Abashidze, M.; Edstrom, W.; Vorobiev, S.; Montelione, G.; Hunt, J.; et al.

    2006-01-01

    The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase catalyzes the terminal steps in ketone body generation and leucine degradation. Mutations in this enzyme cause a human autosomal recessive disorder called primary metabolic aciduria, which typically kills victims because of an inability to tolerate hypoglycemia. Here we present crystal structures of the HMG-CoA lyases from Bacillus subtilis and Brucella melitensis at 2.7 and 2.3 {angstrom} resolution, respectively. These enzymes share greater than 45% sequence identity with the human orthologue. Although the enzyme has the anticipated triose-phosphate isomerase (TIM) barrel fold, the catalytic center contains a divalent cation-binding site formed by a cluster of invariant residues that cap the core of the barrel, contrary to the predictions of homology models. Surprisingly, the residues forming this cation-binding site and most of their interaction partners are shared with three other TIM barrel enzymes that catalyze diverse carbon-carbon bond cleavage reactions believed to proceed through enolate intermediates (4-hydroxy-2-ketovalerate aldolase, 2-isopropylmalate synthase, and transcarboxylase 5S). We propose the name 'DRE-TIM metallolyases' for this newly identified enzyme family likely to employ a common catalytic reaction mechanism involving an invariant Asp-Arg-Glu (DRE) triplet. The Asp ligates the divalent cation, while the Arg probably stabilizes charge accumulation in the enolate intermediate, and the Glu maintains the precise structural alignment of the Asp and Arg. We propose a detailed model for the catalytic reaction mechanism of HMG-CoA lyase based on the examination of previously reported product complexes of other DRE-TIM metallolyases and induced fit substrate docking studies conducted using the crystal structure of human HMG-CoA lyase (reported in the accompanying paper by Fu, et al. (2006) J. Biol. Chem. 281, 7526-7532). Our model is consistent with extensive mutagenesis

  19. Phosphoenolpyruvate carboxykinase, pyruvate orthophosphate dikinase and isocitrate lyase in both tomato fruits and leaves, and in the flesh of peach and some other fruits.

    Science.gov (United States)

    Famiani, Franco; Paoletti, Andrea; Battistelli, Alberto; Moscatello, Stefano; Chen, Zhi-Hui; Leegood, Richard C; Walker, Robert P

    2016-09-01

    In this study the occurrence of a number of enzymes involved in gluconeogenesis was investigated in both tomato fruits and leaves during their development and senescence and in some other fruits. The enzymes studied were phosphoenolpyruvate carboxykinase (PEPCK), pyruvate orthophosphate dikinase (PPDK) and glyoxysomal isocitrate lyase (ICL). PPDK was detected in the ripe flesh of tomato, and much smaller amounts were detected in the flesh of both peach and pepper, whereas it was not detected (not present or at very low abundance) in the other fruits which were investigated (apricot, aubergine, blackberry, blueberry, cherry, grape, plum, raspberry and red current). By contrast PEPCK was present in the flesh of all the fruits investigated. Very small amounts of ICL were detected in ripe tomato flesh. PEPCK was present in the skin, flesh, locular gel and columella of tomato fruit, and in these its abundance increased greatly during ripening. PPDK showed a similar distribution, however, its abundance did not increase during ripening. PEPCK was not detected in tomato leaves at any stage of their development or senescence. The content of PPDK g(-1) fresh weight (FW) increased in tomato leaves as they matured, however, it declined during their senescence. In tomato leaves the content of ICL g(-1) FW increased until the mid-stage of development, then decreased as the leaf matured, and then increased during the latter stages of senescence. In the flesh of tomato fruits the contents of PPDK and PEPCK g(-1) FW decreased during senescence. The results suggest that in fruits other than tomato the bulk of any gluconeogenic flux proceeds via PEPCK, whereas in tomato both PEPCK and PPDK could potentially be utilised. Further, the results indicate that the conversion of pyruvate/acetyl-CoA to malate by the glyoxylate cycle, for which ICL is necessary, is not a major pathway utilised by gluconeogenesis in fruits under normal conditions of growth. Finally, the results contribute to

  20. Subcellular localisation of Medicago truncatula 9/13-hydroperoxide lyase reveals a new localisation pattern and activation mechanism for CYP74C enzymes

    Directory of Open Access Journals (Sweden)

    Hughes Richard K

    2007-11-01

    Full Text Available Abstract Background Hydroperoxide lyase (HPL is a key enzyme in plant oxylipin metabolism that catalyses the cleavage of polyunsaturated fatty acid hydroperoxides produced by the action of lipoxygenase (LOX to volatile aldehydes and oxo acids. The synthesis of these volatile aldehydes is rapidly induced in plant tissues upon mechanical wounding and insect or pathogen attack. Together with their direct defence role towards different pathogens, these compounds are believed to play an important role in signalling within and between plants, and in the molecular cross-talk between plants and other organisms surrounding them. We have recently described the targeting of a seed 9-HPL to microsomes and putative lipid bodies and were interested to compare the localisation patterns of both a 13-HPL and a 9/13-HPL from Medicago truncatula, which were known to be expressed in leaves and roots, respectively. Results To study the subcellular localisation of plant 9/13-HPLs, a set of YFP-tagged chimeric constructs were prepared using two M. truncatula HPL cDNAs and the localisation of the corresponding chimeras were verified by confocal microscopy in tobacco protoplasts and leaves. Results reported here indicated a distribution of M.truncatula 9/13-HPL (HPLF between cytosol and lipid droplets (LD whereas, as expected, M.truncatula 13-HPL (HPLE was targeted to plastids. Notably, such endocellular localisation has not yet been reported previously for any 9/13-HPL. To verify a possible physiological significance of such association, purified recombinant HPLF was used in activation experiments with purified seed lipid bodies. Our results showed that lipid bodies can fully activate HPLF. Conclusion We provide evidence for the first CYP74C enzyme, to be targeted to cytosol and LD. We also showed by sedimentation and kinetic analyses that the association with LD or lipid bodies can result in the protein conformational changes required for full activation of the enzyme

  1. A mutation in the cytosolic O-acetylserine (thiol lyase induces a genome-dependent early leaf death phenotype in Arabidopsis

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    Schippers Jos HM

    2010-04-01

    Full Text Available Abstract Background Cysteine is a component in organic compounds including glutathione that have been implicated in the adaptation of plants to stresses. O-acetylserine (thiol lyase (OAS-TL catalyses the final step of cysteine biosynthesis. OAS-TL enzyme isoforms are localised in the cytoplasm, the plastids and mitochondria but the contribution of individual OAS-TL isoforms to plant sulphur metabolism has not yet been fully clarified. Results The seedling lethal phenotype of the Arabidopsis onset of leaf death3-1 (old3-1 mutant is due to a point mutation in the OAS-A1 gene, encoding the cytosolic OAS-TL. The mutation causes a single amino acid substitution from Gly162 to Glu162, abolishing old3-1 OAS-TL activity in vitro. The old3-1 mutation segregates as a monogenic semi-dominant trait when backcrossed to its wild type accession Landsberg erecta (Ler-0 and the Di-2 accession. Consistent with its semi-dominant behaviour, wild type Ler-0 plants transformed with the mutated old3-1 gene, displayed the early leaf death phenotype. However, the old3-1 mutation segregates in an 11:4:1 (wild type: semi-dominant: mutant ratio when backcrossed to the Colombia-0 and Wassilewskija accessions. Thus, the early leaf death phenotype depends on two semi-dominant loci. The second locus that determines the old3-1 early leaf death phenotype is referred to as odd-ler (for old3 determinant in the Ler accession and is located on chromosome 3. The early leaf death phenotype is temperature dependent and is associated with increased expression of defence-response and oxidative-stress marker genes. Independent of the presence of the odd-ler gene, OAS-A1 is involved in maintaining sulphur and thiol levels and is required for resistance against cadmium stress. Conclusions The cytosolic OAS-TL is involved in maintaining organic sulphur levels. The old3-1 mutation causes genome-dependent and independent phenotypes and uncovers a novel function for the mutated OAS-TL in cell

  2. Phenolics and Flavonoids Compounds, Phenylanine Ammonia Lyase and Antioxidant Activity Responses to Elevated CO2 in Labisia pumila (Myrisinaceae

    Directory of Open Access Journals (Sweden)

    Hawa Z.E. Jaafar

    2012-05-01

    Full Text Available A split plot 3 × 3 experiment was designed to examine the impact of three concentrations of CO2 (400, 800 and 1,200 µmol·mol−1 on the phenolic and flavonoid compound profiles, phenylalanine ammonia lyase (PAL and antioxidant activity in three varieties of Labisia pumila Benth. (var. alata, pumila and lanceolata after 15 weeks of exposure. HPLC analysis revealed a strong influence of increased CO2 concentration on the modification of phenolic and flavonoid profiles, whose intensity depended on the interaction between CO2 levels and L. pumila varieties. Gallic acid and quercetin were the most abundant phenolics and flavonoids commonly present in all the varieties. With elevated CO2 (1,200 µmol·mol−1 exposure, gallic acid increased tremendously, especially in var. alata and pumila (101–111%, whilst a large quercetin increase was noted in var. lanceolata (260%, followed closely by alata (201%. Kaempferol, although detected under ambient CO2 conditions, was undetected in all varieties after exposure. Instead, caffeic acid was enhanced tremendously in var. alata (338~1,100% and pumila (298~433%. Meanwhile, pyragallol and rutin were only seen in var. alata (810 µg·g−1 DW and pumila (25 µg·g−1 DW, respectively, under ambient conditions; but the former compound went undetected in all varieties while rutin continued to increase by 262% after CO2 enrichment. Interestingly, naringenin that was present in all varieties under ambient conditions went undetected under enrichment, except for var. pumila where it was enhanced by 1,100%. PAL activity, DPPH and FRAP also increased with increasing CO2 levels implying the possible improvement of health-promoting quality of Malaysian L. pumila

  3. Increased renal methylglyoxal formation with down-regulation of PGC-1α-FBPase pathway in cystathionine γ-lyase knockout mice.

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    Ashley A Untereiner

    Full Text Available We have previously reported that hydrogen sulfide (H(2S, a gasotransmitter and vasodilator has cytoprotective properties against methylglyoxal (MG, a reactive glucose metabolite associated with diabetes and hypertension. Recently, H(2S was shown to up-regulate peroxisome proliferator-activated receptor-γ coactivator (PGC-1α, a key gluconeogenic regulator that enhances the gene expression of the rate-limiting gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase. Thus, we sought to determine whether MG levels and gluconeogenic enzymes are altered in kidneys of 6-22 week-old cystathionine γ-lyase knockout (CSE(-/-; H(2S-producing enzyme male mice. MG levels were determined by HPLC. Plasma glucose levels were measured by an assay kit. Q-PCR was used to measure mRNA levels of PGC-1α and FBPase-1 and -2. Coupled-enzymatic assays were used to determine FBPase activity, or triosephosphate levels. Experimental controls were either age-matched wild type mice or untreated rat A-10 cells. Interestingly, we observed a significant decrease in plasma glucose levels along with a significant increase in plasma MG levels in all three age groups (6-8, 14-16, and 20-22 week-old of the CSE(-/- mice. Indeed, renal MG and triosephosphates were increased, whereas renal FBPase activity, along with its mRNA levels, were decreased in the CSE(-/- mice. The decreased FBPase activity was accompanied by lower levels of its product, fructose-6-phosphate, and higher levels of its substrate, fructose-1,6-bisphosphate in renal extracts from the CSE(-/- mice. In agreement, PGC-1α mRNA levels were also significantly down-regulated in 6-22 week-old CSE(-/- mice. Furthermore, FBPase-1 and -2 mRNA levels were reduced in aorta tissues from CSE(-/- mice. Administration of NaHS, a H(2S donor, increased the gene expression of PGC-1α and FBPase-1 and -2 in cultured rat A-10 cells. In conclusion, overproduction of MG in CSE(-/- mice is due to a H(2S-mediated down-regulation of

  4. Phenylalanin Ammonia-lyase Activity,Total Phenolics and Flavonoids Contents in Flowers,Leaves ,Hulls and Kernels of Three Pistachio(Pistacia vera L.) Cultivars%Phenylalanin Ammonia-lyase Activity,Total Phenolics and Flavonoids Contents in Flowers,Leaves,Hulls and Kernels of Three Pistachio(Pistacia vera L.) Cultivars

    Institute of Scientific and Technical Information of China (English)

    Nadernejad Nazi; Ahmadimoghadam Ali; Hosseinifard Javad; Pourseyedi Shahram

    2012-01-01

    Phenylalanin ammonia-lyase (PAL) plays a pivotal role in the production of phenolic compounds,which are responsible for the success of the defense strategies in harsh environments in response to different stimuli.Measurements of the PAL activity,total phenolics,total flavonoids and anthocyanin contents were performed in flowers,leaves and fruits of three pistachio cultivars "Ahmadaghaii","Ohadi" and "Kallehghuchi".The results showed that PAL activity was different in cultivars and in plant organs of pistachio trees (flowers,leaves and fruits).The highest activity rate of their compounds was observed in Ahmadaghaii cultivar.A positive correlation was observed between PAL activity,total phenolics and total flavonoids in leaves,and a negative correlation between PAL activity and anthocyanin contents in leaves and flowers of Ahmadaghaii cultivar.PAL activity and total phenolics in fruits of pistachio suffered a decrease when the maturation processes began.It is suggested that the hulls of the pistachio fruits,containing high level of phenolic compounds ( especially in Ahmadaghaii cultivar),may function as a protective layer of defense chemicals againstultraviolet radiation and pathogens.The final concentration of phenolic compounds,flavonoids and antocyanins in the kernel depend on PAL activity in the kernel' s cultivar.The results led to the conclusion that increase in PAL activity,phenolic compounds and flavonoids in Ahmadaghaii can help the plant to cope with the stresses better than the other cultivars.Since phenolic compounds are antioxidant and scavenge free oxygen,it is postulated that Ahmadaghaii is the most resistant cultivar to the environmental stresses.

  5. Construction of Wine Yeast for Improving Cystathionine β-Lyase Activity%具有高胱硫醚β-裂解酶活性的葡萄酒酵母工程菌株的构建

    Institute of Scientific and Technical Information of China (English)

    马捷; 刘延琳

    2012-01-01

    [Objective] The wine yeast which exhibited higher cystathionine p-Lyase activity was contracted. [Method] Plasmid pAUR123 was used to express the tnaA gene of Escherichia coli in Saccharomyces cerevisiae strain LFP525 which is a indigenous wine yeast selected from China. Then the cystathionine P-Lyase activity of yeast transformants was detected. After that, model grape juice fermentation and white wine fermentation were carried out to evaluate the tenological properties and thiol-producing characteristics of them. [Result] Two engineering wine yeasts, TH1 and TH2, were obtained in this study. Compared with the host strain, their cystathionine p-Lyase activities were increased by 32.25% to 59.44%. The result of model grape juice fermentation showed that the fermentation duration of engineering yeast strains was one-day longer, the residual sugars and acetic acid were higher than their host strain, while the production of thiols increased obviously, which were 2.8-4.3 folds than that in the host strain. In Sauvignon Blanc wine making test, the technological characteristics of engineering yeast strains showed no significant difference with the host strain, whereas the production of thiols was 1.22 times higher than that of the host strains. [Conclusion] The cystathionine β-Lyase activity of wine yeast was enhanced successfully by using the gene modification method in this study. It will have great interests for improving flavor characteristics of wine in the future.%[目的]构建具有较高胱硫醚β-裂解酶活性的葡萄酒酵母工程菌株,用于提高葡萄酒的香气品质.[方法]利用质粒pAUR123将大肠杆菌tnaA基因在中国本土酿酒酵母菌株LFP525中进行表达,得到的转化子进行酶活测定,并通过模拟汁和干白的发酵,评价其酿酒特性和产生硫醇类物质的能力.[结果]成功获得酵母工程菌株TH1和TH2,其胱硫醚β-裂解酶活性与受体菌比提高了32.25%-59.44%.模拟葡萄汁发酵显示,工

  6. Activation of the jasmonic acid pathway by depletion of the hydroperoxide lyase OsHPL3 reveals crosstalk between the HPL and AOS branches of the oxylipin pathway in rice.

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    Xiaoqiang Liu

    Full Text Available The allene oxide synthase (AOS and hydroperoxide lyase (HPL branches of the oxylipin pathway, which underlie the production of jasmonates and aldehydes, respectively, function in plant responses to a range of stresses. Regulatory crosstalk has been proposed to exist between these two signaling branches; however, there is no direct evidence of this. Here, we identified and characterized a jasmonic acid (JA overproduction mutant, cea62, by screening a rice T-DNA insertion mutant library for lineages that constitutively express the AOS gene. Map-based cloning was used to identify the underlying gene as hydroperoxide lyase OsHPL3. HPL3 expression and the enzyme activity of its product, (E-2-hexenal, were depleted in the cea62 mutant, which resulted in the dramatic overproduction of JA, the activation of JA signaling, and the emergence of the lesion mimic phenotype. A time-course analysis of lesion formation and of the induction of defense responsive genes in the cea62 mutant revealed that the activation of JA biosynthesis and signaling in cea62 was regulated in a developmental manner, as was OsHPL3 activity in the wild-type plant. Microarray analysis showed that the JA-governed defense response was greatly activated in cea62 and this plant exhibited enhanced resistance to the T1 strain of the bacterial blight pathogen Xanthomonasoryzaepvoryzae (Xoo. The wounding response was attenuated in cea62 plants during the early stages of development, but partially recovered when JA levels were elevated during the later stages. In contrast, the wounding response was not altered during the different developmental stages of wild-type plants. These findings suggest that these two branches of the oxylipin pathway exhibit crosstalk with regards to biosynthesis and signaling and cooperate with each other to function in diverse stress responses.

  7. Integration of apo-α-phycocyanin into phycobilisomes and its association with FNRL in the absence of the phycocyanin α-subunit lyase (CpcF in Synechocystis sp. PCC 6803.

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    Pengpeng Zhang

    Full Text Available Phycocyanin is an important component of the phycobilisome, which is the principal light-harvesting complex in cyanobacteria. The covalent attachment of the phycocyanobilin chromophore to phycocyanin is catalyzed by the enzyme phycocyanin lyase. The photosynthetic properties and phycobilisome assembly state were characterized in wild type and two mutants which lack holo-α-phycocyanin. Insertional inactivation of the phycocyanin α-subunit lyase (ΔcpcF mutant prevents the ligation of phycocyanobilin to α-phycocyanin (CpcA, while disruption of the cpcB/A/C2/C1 operon in the CK mutant prevents synthesis of both apo-α-phycocyanin (apo-CpcA and apo-β-phycocyanin (apo-CpcB. Both mutants exhibited similar light saturation curves under white actinic light illumination conditions, indicating the phycobilisomes in the ΔcpcF mutant are not fully functional in excitation energy transfer. Under red actinic light illumination, wild type and both phycocyanin mutant strains exhibited similar light saturation characteristics. This indicates that all three strains contain functional allophycocyanin cores associated with their phycobilisomes. Analysis of the phycobilisome content of these strains indicated that, as expected, wild type exhibited normal phycobilisome assembly and the CK mutant assembled only the allophycocyanin core. However, the ΔcpcF mutant assembled phycobilisomes which, while much larger than the allophycocyanin core observed in the CK mutant, were significantly smaller than phycobilisomes observed in wild type. Interestingly, the phycobilisomes from the ΔcpcF mutant contained holo-CpcB and apo-CpcA. Additionally, we found that the large form of FNR (FNR(L accumulated to normal levels in wild type and the ΔcpcF mutant. In the CK mutant, however, significantly less FNR(L accumulated. FNRL has been reported to associate with the phycocyanin rods in phycobilisomes via its N-terminal domain, which shares sequence homology with a phycocyanin

  8. Exposure of E. coli to DNA-methylating agents impairs biofilm formation and invasion of eukaryotic cells via down regulation of the N-acetylneuraminate lyase NanA

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    Pamela eDi Pasquale

    2016-02-01

    Full Text Available DNA methylation damage can be induced by endogenous and exogenous chemical agents, which has led every living organism to develop suitable response strategies. We investigated protein expression profiles of Escherichia coli upon exposure to the alkylating agent methyl-methane sulfonate (MMS by differential proteomics. Quantitative proteomic data showed a massive downregulation of enzymes belonging to the glycolytic pathway and fatty acids degradation, strongly suggesting a decrease of energy production. A strong reduction in the expression of the N-acetylneuraminate lyases (NanA involved in the sialic acid metabolism was also observed. Using a null NanA mutant and DANA, a substrate analogue acting as competitive inhibitor, we demonstrated that down regulation of NanA affects biofilm formation and adhesion properties of E. coli MV1161. Exposure to alkylating agents also decreased biofilm formation and bacterial adhesion to Caco-2 eukaryotic cell line by the adherent invasive E. coli (AIEC strain LF82. Our data showed that methylation stress impairs E. coli adhesion properties and suggest a possible role of NanA in biofilm formation and bacteria host interactions.

  9. Induction of a Proinflammatory Response in Cortical Astrocytes by the Major Metabolites Accumulating in HMG-CoA Lyase Deficiency: the Role of ERK Signaling Pathway in Cytokine Release.

    Science.gov (United States)

    Fernandes, Carolina Gonçalves; Rodrigues, Marília Danyelle Nunes; Seminotti, Bianca; Colín-González, Ana Laura; Santamaria, Abel; Quincozes-Santos, André; Wajner, Moacir

    2016-08-01

    3-Hydroxy-3-methylglutaric aciduria (HMGA) is an inherited metabolic disorder caused by 3-hydroxy-3-methylglutaryl-CoA lyase deficiency. It is biochemically characterized by predominant tissue accumulation and high urinary excretion of 3-hydroxy-3-methylglutarate (HMG) and 3-methylglutarate (MGA). Affected patients commonly present acute symptoms during metabolic decompensation, including vomiting, seizures, and lethargy/coma accompanied by metabolic acidosis and hypoketotic hypoglycemia. Although neurological manifestations are common, the pathogenesis of brain injury in this disease is poorly known. Astrocytes are important for neuronal protection and are susceptible to damage by neurotoxins. In the present study, we investigated the effects of HMG and MGA on important parameters of redox homeostasis and cytokine production in cortical cultured astrocytes. The role of the metabolites on astrocyte mitochondrial function (thiazolyl blue tetrazolium bromide (MTT) reduction) and viability (propidium iodide incorporation) was also studied. Both organic acids decreased astrocytic mitochondrial function and the concentrations of reduced glutathione without altering cell viability. In contrast, they increased reactive species formation (2'-7'-dichlorofluorescein diacetate (DCFHDA) oxidation), as well as IL-1β, IL-6, and TNF α release through the ERK signaling pathway. Taken together, the data indicate that the principal compounds accumulating in HMGA induce a proinflammatory response in cultured astrocytes that may possibly be involved in the neuropathology of this disease. PMID:26099308

  10. AMP-activated protein kinase and ATP-citrate lyase are two distinct molecular targets for ETC-1002, a novel small molecule regulator of lipid and carbohydrate metabolism[S

    Science.gov (United States)

    Pinkosky, Stephen L.; Filippov, Sergey; Srivastava, Rai Ajit K.; Hanselman, Jeffrey C.; Bradshaw, Cheryl D.; Hurley, Timothy R.; Cramer, Clay T.; Spahr, Mark A.; Brant, Ashley F.; Houghton, Jacob L.; Baker, Chris; Naples, Mark; Adeli, Khosrow; Newton, Roger S.

    2013-01-01

    ETC-1002 (8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel investigational drug being developed for the treatment of dyslipidemia and other cardio-metabolic risk factors. The hypolipidemic, anti-atherosclerotic, anti-obesity, and glucose-lowering properties of ETC-1002, characterized in preclinical disease models, are believed to be due to dual inhibition of sterol and fatty acid synthesis and enhanced mitochondrial long-chain fatty acid β-oxidation. However, the molecular mechanism(s) mediating these activities remained undefined. Studies described here show that ETC-1002 free acid activates AMP-activated protein kinase in a Ca2+/calmodulin-dependent kinase β-independent and liver kinase β 1-dependent manner, without detectable changes in adenylate energy charge. Furthermore, ETC-1002 is shown to rapidly form a CoA thioester in liver, which directly inhibits ATP-citrate lyase. These distinct molecular mechanisms are complementary in their beneficial effects on lipid and carbohydrate metabolism in vitro and in vivo. Consistent with these mechanisms, ETC-1002 treatment reduced circulating proatherogenic lipoproteins, hepatic lipids, and body weight in a hamster model of hyperlipidemia, and it reduced body weight and improved glycemic control in a mouse model of diet-induced obesity. ETC-1002 offers promise as a novel therapeutic approach to improve multiple risk factors associated with metabolic syndrome and benefit patients with cardiovascular disease. PMID:23118444

  11. Synthesis of novel 17-(4'-formyl)pyrazolylandrosta-5,16-dienes and their derivatives as potent 17α-hydroxylase/C17,20-lyase inhibitors or antiproliferative agents depending on the substitution pattern of the heteroring.

    Science.gov (United States)

    Kovács, Dóra; Wölfling, János; Szabó, Nikoletta; Szécsi, Mihály; Schelz, Zsuzsanna; Zupkó, István; Frank, Éva

    2016-09-14

    A series of novel 17-(4'-formyl)pyrazolylandrosta-5,16-dienes were efficiently synthesized in two steps from pregnadienolone acetate with monosubstituted hydrazines via the cyclization/formylation sequence of the primarily formed hydrazones on treatment with the Vilsmeier-Haack reagent. The products were further transformed by deacetylation and subsequent reduction in order to enlarge the compound library available for pharmacological studies. Moreover, 4'-formylpyrazoles containing H or Me on the heteroring-N were subjected to oxime formation and Ac2O-induced dehydration to furnish the corresponding 4'-cyano derivatives in good yields. The antiproliferative activities of the structurally related steroidal 17-exo-pyrazole derivatives were tested in vitro on four human adherent breast cancer cell lines (MCF7, T47D, MDA-MB-231 and MDA-MB-361): the microculture tetrazolium assay revealed that seven compounds exerted better cell growth-inhibitory effects on some or all these cell lines than those of the reference cisplatin. With regard to the well-known structural features that a potent C17,20-lyase inhibitor should possess, some relevant derivatives were tested in vitro from the aspects of their inhibitory effects on rat testicular enzyme, and one of them proved to exert noteworthy enzyme-inhibitory action, with an IC50 (26 nM) of the same order of magnitude as that of abiraterone. PMID:27209562

  12. Hydrogen Peroxide Treatment and the Phenylpropanoid Pathway Precursors Feeding Improve Phenolics and Antioxidant Capacity of Quinoa Sprouts via an Induction of L-Tyrosine and L-Phenylalanine Ammonia-Lyases Activities

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    Michał Świeca

    2016-01-01

    Full Text Available Hydrogen peroxide treatment and the phenylpropanoid pathway precursors feeding affected the antioxidant capacity of quinoa sprouts. Compared to the control, total phenolics content was significantly increased by treatment of control sprouts with 50 mM and 200 mM H2O2—an elevation of about 24% and 28%, respectively. The highest increase of flavonoids content was found for the sprouts treated with 200 mM H2O2 obtained from seeds fed with shikimic acid. All the studied modifications increased the antioxidant potential of sprouts (at least by 50% compared to control. The highest reducing power was found for the sprouts treated with 200 mM H2O2 obtained by phenylalanine feeding (5.03 mg TE/g DW and those obtained from the seeds fed with tyrosine (5.26 mg TE/g DW. The activities of L-tyrosine (TAL and L-phenylalanine (PAL ammonia-lyases were strongly affected by germination time as well as the applied modification of sprouting. On the 3rd day the highest PAL activity was determined for both untreated and induced with 50 mM H2O2 sprouts obtained by phenylalanine feeding. H2O2 induced TAL activity; the highest TAL activity was determined for 3-day-old sprouts induced with 200 mM H2O2 obtained from seeds fed with phenylalanine.

  13. 3-Hydroxy-3-methylglutaryl CoA lyase (HL): Mouse and human HL gene (HMGCL) cloning and detection of large gene deletions in two unrelated HL-deficient patients

    Energy Technology Data Exchange (ETDEWEB)

    Wang, S.P.; Robert, M.F.; Mitchell, G.A. [Hopital Sainte-Justine, Quebec (Canada)] [and others

    1996-04-01

    3-hydroxy-3-methylglutaryl CoA lyase (HL, EC 4.1.3.4) catalyzes the cleavage of 3-hydroxy-3-methylglutaryl CoA to acetoacetic acid and acetyl CoA, the final reaction of both ketogenesis and leucine catabolism. Autosomal-recessive HL deficiency in humans results in episodes of hypoketotic hypoglycemia and coma. Using a mouse HL cDNA as a probe, we isolated a clone containing the full-length mouse HL gene that spans about 15 kb of mouse chromosome 4 and contains nine exons. The promoter region of the mouse HL gene contains elements characteristic of a housekeeping gene: a CpG island containing multiple Sp1 binding sites surrounds exon 1, and neither a TATA nor a CAAT box are present. We identified multiple transcription start sites in the mouse HL gene, 35 to 9 bases upstream of the translation start codon. We also isolated two human HL genomic clones that include HL exons 2 to 9 within 18 kb. The mouse and human HL genes (HGMW-approved symbol HMGCL) are highly homologous, with identical locations of intron-exon junctions. By genomic Southern blot analysis and exonic PCR, was found 2 of 33 HL-deficient probands to be homozygous for large deletions in the HL gene. 26 refs., 4 figs., 2 tabs.

  14. 4种竹子木质素合成酶PAL的基因克隆和序列分析%Gene Cloning and Sequence Analysis of Phenylalanine Ammonia-Lyase in Bamboo Lignin Synthase

    Institute of Scientific and Technical Information of China (English)

    李潞滨; 刘蕾; 张淑萍; 何聪芬; 胡桃; 庄彩云

    2009-01-01

    苯丙氨酸解氨酶(Phenylalanine Ammonia-Lyase,PAL;EC 4.3.1.5)是木质素生物合成过程的关键酶和限速酶.应用RACE(Rapid-Amplification of cDNA Ends)方法,获得了白哺鸡竹(Phyllostachys dulcis)、高节竹(Phyllostachys prominens)、龙鳞竹(Phyllostachys pubescens heterocycla)、丽水苦竹(Pleioblastus maculosoides Wen)等4种竹子PAL基因的全长序列,并进行了生物信息学分析.结果表明,PAL基因的开放读码框长度为2 136 bp,共编码712个氨基酸,具有2个外显子和1个内含子.其中高节竹、龙鳞竹、丽水苦竹PAL基因内含子长度为121 bp,白哺鸡竹PLA基因内含子长度为84 bp.推测其氨基酸序列,并分析PAL蛋白单体的三维结构,结果显示PAL蛋白均含有大量的α螺旋和β折叠结构.基于邻接法的进化树对31个物种的PAL基因的氨基酸序列分析表明,竹类植物PAL基因的保守性较高,与禾本科植物玉米、甘蔗的亲缘关系较近,与双子叶植物辣椒等的亲缘关系较远.%Phenylalanine Ammonia-Lyase (PAL EC 4.3.1.5) is one of the key enzymes in the process of lignin synthesis.In this paper,PAL gene sequences from four varieties of bamboo:Phyllostachys dulcis,Phyllostachys prominens,Phyllostachys pubescens heterocycla and Pleioblastus maculosoides Wen,were obtained by Rapid-Amplification of cDNA Ends (RACE).The results show that each PAL gene has two exons and an intron and every Open Reading Frame (ORF) is 2 136 bp in length,encoding 712 amino acids.The bamboo PAL amino acid sequences and threedimensional structures were then analyzed and inferred.The results indicate that all of the PAL proteins contain a large number of α-helix and β plated sheet structure.The neighbor-joining phylogenetic tree based on PAL sequences of bamboo and other 27 plants indicates that PAL gene sequence in bamboo is relatively conservative,and bamboo has a very close relationship with that of Zea may and Saccharum officinarum,and a remote relationship with

  15. Activities of lipoxygenase and phenylalanine ammonia lyase in poplar leaves induced by insect herbivory and volatiles%昆虫取食和挥发物诱导的杨树叶片中LOX和PAL活性变化

    Institute of Scientific and Technical Information of China (English)

    胡增辉; 张雯; 沈应柏; 付怀军; 苏晓华

    2009-01-01

    为了探索昆虫取食诱导的木本植物体内所产生的防御反应,以合作杨(Populus simonii×P.pyramidalis,'Opera 8277')扦插苗为实验材料,经杨扇舟蛾(Clostera anachoreta)幼虫取食后,检测叶片中茉莉酸(jasmonate,JA)途径中的关键酶--脂氧合酶(lipoxygenas,LOX)及苯基丙酸类合成途径中的限速酶--苯丙氨酸解氨酶(phenylalanine ammonia lyase,PAL)的活性变化.结果显示,LOX和PAL的活性不仅在虫咬叶片中出现增加,在虫咬叶片上部的系统叶片中也有显著升高,表明茉莉酸途径和苯基丙酸类合成途径被激活,而且防御反应被系统性诱导.并且,与虫咬植株邻近的健康杨树叶片中LOX和PAL的增加表明,杨树间存在由昆虫取食诱导挥发物介导的信息传递.熏蒸实验也证明,茉莉酸甲酯(methyl jasmonate,MeJA)能够作为气体信号诱导合作杨植株产生防御反应.

  16. Synthesis and in vitro activity of some epimeric 20 alpha-hydroxy, 20-oxime and aziridine pregnene derivatives as inhibitors of human 17 alpha-hydroxylase/C17,20-lyase and 5 alpha-reductase.

    Science.gov (United States)

    Ling, Y Z; Li, J S; Kato, K; Liu, Y; Wang, X; Klus, G T; Marat, K; Nnane, I P; Brodie, A M

    1998-10-01

    Some epimeric 20-hydroxy, 20-oxime, 16 alpha, 17 alpha-, 17,20- and 20,21-aziridine derivatives of progesterone were synthesized and evaluated as inhibitors of human 17 alpha-hydroxylase/C17,20-lyase (P450(17) alpha) and 5 alpha-reductase (5 alpha-R). The reduction of 16-dehydropregenolone acetate (3a) was reinvestigated. NaBH4 in the presence of CeCl3 gave better stereo-selectivity for 20 beta-ol [20 alpha/20 beta-OH (4 alpha/4 beta) = 1/2.7] than LTBAH or the Meerwein-Pondroff method reported; reduction with Zn in HOAc formed exclusively 20 alpha-ol (4 alpha b). The 20 alpha- and 20 beta-hydroxy-4,16-pregnadien-3-one (9 alpha) and (9 beta) were synthesized from the alcohols 4 alpha b and 4 beta b. Several 20-oxime pregnadienes and 16 alpha, 17 alpha-, 17,20- and 20,21-aziridinyl-5-pregnene derivatives were also synthesized. LiAlH4 reduction of the 16-en-20-oxime (12b) yielded 20 (R)-(13a) and 20(S)-17 alpha,20-aziridine (13b) and 20(R)-17 beta,20-aziridine (14a). Several compounds inhibited the human P450(17) alpha with greater potency than ketoconzole. The 5 alpha-R enzyme assay showed that while (9 alpha) did not have any activity, (9 beta) and (3b) were potent 5 alpha-reductase (IC50 = 21 and 31 nM) inhibitors with activities similar to finasteride. The 20-oximes (17a) and (17b) were potent dual inhibitors for both 5 alpha-R (IC50 = 63 and 115 nM, compared to 33 nM for finasteride) and P450(17) alpha (IC50 = 43 and 25 nM, compared to 78 nM for ketoconazole). PMID:9839000

  17. Cystathionine γ lyase-hydrogen sulfide increases peroxisome proliferator-activated receptor γ activity by sulfhydration at C139 site thereby promoting glucose uptake and lipid storage in adipocytes.

    Science.gov (United States)

    Cai, Junyan; Shi, Xiaoqin; Wang, Huamin; Fan, Jinghui; Feng, Yongliang; Lin, Xianjuan; Yang, Jichun; Cui, Qinghua; Tang, Chaoshu; Xu, Guoheng; Geng, Bin

    2016-05-01

    Adipocytes express the cystathionine γ lyase (CSE)-hydrogen sulfide (H2S) system. CSE-H2S promotes adipogenesis but ameliorates adipocyte insulin resistance. We investigated the mechanism of how CSE-H2S induces these paradoxical effects. First, we confirmed that an H2S donor or CSE overexpression promoted adipocyte differentiation. Second, we found that H2S donor inhibited but CSE inhibition increased phosphodiesterase (PDE) activity. H2S replacing isobutylmethylxanthine in the differentiation program induced adipocyte differentiation in part. Inhibiting PDE activity by H2S induced peroxisome proliferator activated receptor γ (PPARγ) protein and mRNA expression. Of note, H2S directly sulfhydrated PPARγ protein. Sulfhydrated PPARγ increased its nuclear accumulation, DNA binding activity and adipogenesis gene expression, thereby increasing glucose uptake and lipid storage, which were blocked by the desulfhydration reagent DTT. H2S induced PPARγ sulfhydration, which was blocked by mutation of the C139 site of PPARγ. In mice fed a high-fat diet (HFD) for 4weeks, the CSE inhibitor decreased but H2S donor increased adipocyte numbers. In obese mice fed an HFD for 13weeks, H2S treatment increased PPARγ sulfhydration in adipose tissues and attenuated insulin resistance but did not increase obesity. In conclusion, CSE-H2S increased PPARγ activity by direct sulfhydration at the C139 site, thereby changing glucose into triglyceride storage in adipocytes. CSE-H2S-mediated PPARγ activation might be a new therapeutic target for diabetes associated with obesity. PMID:26946260

  18. Impact of Soil Field Water Capacity on Secondary Metabolites, Phenylalanine Ammonia-lyase (PAL, Maliondialdehyde (MDA and Photosynthetic Responses of Malaysian Kacip Fatimah (Labisia pumila Benth

    Directory of Open Access Journals (Sweden)

    Hawa Z. E. Jaafar

    2012-06-01

    Full Text Available A randomized complete block design 2 × 4 experiment was designed and conducted for 15 weeks to characterize the relationships between production of total phenolics, flavonoid, anthocyanin, leaf gas exchange, total chlorophyll, phenylalanine ammonia-lyase (PAL and malondialdehyde (MDA activity in two varieties of Labisia pumila Benth, namely the var. alata and pumila, under four levels of evapotranspiration replacement (ER (100%; well watered, (75%, moderate water stress, (50%; high water stress and (25%; severe water stress. The production of total phenolics, flavonoids, anthocyanin, soluble sugar and relative leaf water content was affected by the interaction between varieties and SWC. As the ER levels decreased from 100% to 25%, the production of PAL and MDA activity increased steadily. At the highest (100% ER L. pumila exhibited significantly higher net photosynthesis, apparent quantum yield, maximum efficiency of photosystem II (fv/fm and lower dark respiration rates compared to the other treatment. The production of total phenolics, flavonoids and anthocyanin was also found to be higher under high water stress (50% ER replacement compared to severe water stress (25% ER. From this study, it was observed that as net photosynthesis, apparent quantum yield and chlorophyll content were downregulated under high water stress the production of total phenolics, flavonoids and anthocyanin were upregulated implying that the imposition of high water stress can enhance the medicinal properties of L. pumila Benth.

  19. Construction of Vector Harboring Pectin Lyase C Gene and Its Expression in E. coli%果胶裂解酶基因PelC表达载体的构建及原核表达分析

    Institute of Scientific and Technical Information of China (English)

    邓伟科; 郭安平; 刘恩平; 王炎松; 郭运玲; 孔华; 阳辛凤; 贺立卡

    2009-01-01

    A complete open reading frame of pectin lyase C (PelC) cloned from a pectinase-producing strain BTC105 isolated and collected in the laboratory was constructed on a plasmid pET28a and transferred into E. coli BL21 (DE3) to carry out fuse expression; and flask shaking fermented in LB (Luria-Bertani) , induced with 1 mmol/L IPTG (iso-propyl β-D-1-thiogalactopyranoside). The results showed that the recombinant plasmid pET28a-pelC was constructed successfully and PelC has mainly expressed in E. coli BL21 (DE3). The optimal pH of the enzyme was 5.4, optimal temperature at 50℃, Ca~(2+) stimulated strongly on the enzyme activity, however, Cu~(2+) completely inhibited the activity.%从实验室分离保存的1株产果胶酶的菌株(BTC105)中克隆果胶裂解酶基因(PelC)完整开放阅读框,通过载体构建,将目的基因连接到表达载体pET28a上,转化大肠埃希菌BL21(DE3)进行融合表达,在LB(Luria-Bertani)中进行摇瓶发酵,1 mmol/L IPTG(异丙基-β-D-硫代半乳糖苷)诱导.结果表明,构建了表达载体pET28a-pelC,果胶裂解酶主要在胞内表达,酶活最适pH为5.4,最适温度为50℃,Ca~(2+)对酶活促进作用最为明显,Cu~(2+)完全抑制了酶的活性.

  20. Substrate specificity of three cytochrome c haem lyase isoenzymes from Wolinella succinogenes: unconventional haem c binding motifs are not sufficient for haem c attachment by NrfI and CcsA1

    Science.gov (United States)

    Kern, Melanie; Eisel, Florian; Scheithauer, Juliane; Kranz, Robert G.; Simon, Jörg

    2012-01-01

    Summary Bacterial c-type cytochrome maturation is dependent on a complex enzymic machinery. The key reaction is catalysed by cytochrome c haem lyase (CCHL) that usually forms two thioether bonds to attach haem b to the cysteine residues of a haem c binding motif (HBM) which is, in most cases, a CX2CH sequence. Here, the HBM specificity of three distinct CCHL isoenzymes (NrfI, CcsA1 and CcsA2) from the Epsilonproteobacterium Wolinella succinogenes was investigated using either W. succinogenes or Escherichia coli as host organism. Several reporter c-type cytochromes were employed including cytochrome c nitrite reductases (NrfA) from E. coli and Campylobacter jejuni that differ in their active site HBMs (CX2CK or CX2CH). W. succinogenes CcsA2 was found to attach haem to standard CX2CH motifs in various cytochromes whereas other HBMs were not recognized. NrfI was able to attach haem c to the active site CX2CK motif of both W. succinogenes and E. coli NrfA, but not to NrfA from C. jejuni. Different apo-cytochrome variants carrying the CX15CH motif, assumed to be recognized by CcsA1 during maturation of the octahaem cytochrome MccA, were not processed by CcsA1 in either W. succinogenes or E. coli. It is concluded that the dedicated CCHLs NrfI and CcsA1 attach haem to non-standard HBMs only in the presence of further, as yet uncharacterised structural features. Interestingly, it proved impossible to delete the ccsA2 gene from the W. succinogenes genome; a finding that is discussed in the light of the available genomic, proteomic and functional data on W. succinogenes c-type cytochromes. PMID:19919672

  1. HMG-CoA lyase (HL) gene: Cloning and characterization of the 5{prime} end of the mouse gene, gene targeting in ES cells, and demonstration of large deletions in three HL-deficient patients

    Energy Technology Data Exchange (ETDEWEB)

    Wang, S.; Robert, M.F.; Mitchell, G.A. [Hopital Sainte-Justine, Quebec (Canada)] [and others

    1994-09-01

    3-hydroxy-3-methylglutaryl CoA lyase (HL) is a mitochondrial matrix enzyme which catalyzes the last step of leucine catabolism and of ketogenesis. Autosomal recessive HL deficiency in humans results in episodes of hypoglycemia and coma. We are interested in the pathophysiology of HL deficiency as a model for both amino acid and fatty acid inborn errors. We have cloned the human and mouse HL genes. In order to analyze the 5{prime} nontranslated region of mouse HL gene, we cloned and sequenced a 1.8 kb fragment containing the 5{prime} extremity including exon 1 and about 1.6 kb of 5{prime} nontranslated sequence. The region surrounding exon 1 is CpG-rich (66.4%). Using the criteria of West, the Observed/Expected ratio for CpG dinucleotides is 0.7 ({ge}0.6 is consistent with a CpG island). We are carrying out primer extension and RNase protection experiments to determine the transcription initiation site. We constructed a gene targeting vector by introducing the neomycin resistance gene into exon 2 of a 7.5 kb genomic subclone of the mouse HL gene. Targeting was performed by electroporating 10 mg linearized vector into 10{sup 7} ES cells and selecting for 12 days with G418. 5/228 colonies (2.2%) had homologous recombination as shown by PCR screening and Southern analysis. We are microinjecting the 5 targeted clones into blastocysts to create an HL-deficient mouse. To date we have obtained two chimeras with contributions of 95% and 55% from 129, by coat color estimates. Three of 27 (11%) of the HL-deficient patients studied were suggested by genomic Southern analysis to be homozygous for large intragenic deletions. We confirmed this and defined the boundaries using exonic PCR.

  2. Androgen receptor inactivation contributes to antitumor efficacy of 17{alpha}-hydroxylase/17,20-lyase inhibitor 3beta-hydroxy-17-(1H-benzimidazole-1-yl)androsta-5,16-diene in prostate cancer.

    Science.gov (United States)

    Vasaitis, Tadas; Belosay, Aashvini; Schayowitz, Adam; Khandelwal, Aakanksha; Chopra, Pankaj; Gediya, Lalji K; Guo, Zhiyong; Fang, Hong-Bin; Njar, Vincent C O; Brodie, Angela M H

    2008-08-01

    We previously reported that our novel compound 3beta-hydroxy-17-(1H-benzimidazole-1-yl)androsta-5,16-diene (VN/124-1) is a potent 17alpha-hydroxylase/17,20-lyase (CYP17) inhibitor/antiandrogen and strongly inhibits the formation and proliferation of human prostate cancer LAPC4 tumor xenografts in severe combined immunodeficient mice. In this study, we report that VN/124-1 and other novel CYP17 inhibitors also cause down-regulation of androgen receptor (AR) protein expression in vitro and in vivo. This mechanism of action seems to contribute to their antitumor efficacy. We compared the in vivo antitumor efficacy of VN/124-1 with that of castration and a clinically used antiandrogen, Casodex, and show that VN/124-1 is more potent than castration in the LAPC4 xenograft model. Treatment with VN/124-1 (0.13 mmol/kg twice daily) was also very effective in preventing the formation of LAPC4 tumors (6.94 versus 2410.28 mm(3) in control group). VN/124-1 (0.13 mmol/kg twice daily) and VN/124-1 (0.13 mmol/kg twice daily) + castration induced regression of LAPC4 tumor xenografts by 26.55% and 60.67%, respectively. Treatments with Casodex (0.13 mmol/kg twice daily) or castration caused significant tumor suppression compared with control. Furthermore, treatment with VN/124-1 caused marked down-regulation of AR protein expression, in contrast to treatments with Casodex or castration that caused significant up-regulation of AR protein expression. The results suggest that VN/124-1 acts by several mechanisms (CYP17 inhibition, competitive inhibition, and down-regulation of the AR). These actions contribute to inhibition of the formation of LAPC4 tumors and cause regression of growth of established tumors. VN/124-1 is more efficacious than castration in the LAPC4 xenograft model, suggesting that the compound has potential for the treatment of prostate cancer. PMID:18723482

  3. Phycocyanin alpha-subunit phycocyanobilin lyase.

    OpenAIRE

    Fairchild, C D; Zhao, J.(Central China Normal University (HZNU), Wuhan, 430079, China); Zhou, J.; Colson, S E; Bryant, D A; Glazer, A N

    1992-01-01

    Phycobiliproteins, unlike other light-harvesting proteins involved in photosynthesis, bear covalently attached chromophores. The bilin chromophores are attached through thioether bonds to cysteine residues. The cyanobacterium Synechococcus sp. PCC 7002 has eight distinct bilin attachment sites on seven polypeptides, all of which carry the same chromophore, phycocyanobilin. When two genes in the phycocyanin operon of this organism, cpcE and cpcF, are inactivated by insertion, together or separ...

  4. Genetics Home Reference: adenylosuccinate lyase deficiency

    Science.gov (United States)

    ... Biochemistry. 2009 Jun 16;48(23):5291-302. doi: 10.1021/bi802321m. Citation on PubMed or Free article on ... Biochemistry. 2012 Aug 21;51(33):6701-13. doi: 10.1021/bi300796y. Epub 2012 Aug 7. Citation on PubMed ...

  5. Molecular Cloning and Expression Analysis of a Phenylalanne Ammonial-lyase Gene from Prunella vulgaris%夏枯草苯丙氨酸解氨酶基因的克隆与表达分析

    Institute of Scientific and Technical Information of China (English)

    许锋; 曹腾; 宁迎晶; 蒋丽阳; 张威威; 程水源

    2012-01-01

    苯丙氨酸解氨酶(PAL)是迷迭香酸合成途径中的关键酶之一,根据其他植物PAL基因的保守区域设计特异引物,利用3′-RACE-PCR技术,本研究首次从夏枯草中克隆得到了PAL基因的cDNA片段序列,命名为PvPAL,GenBank登录号为JN65446.PvPAL基因cDNA片段长1 306 bp,其中编码区域为1 047 bp,编码349个氨基酸.蛋白质序列多重比较结果显示,PvPAL蛋白质序列与丹参、地黄、黄芩、藿香等植物的PAL蛋白质高度同源.PAL系统进化树分析结果表明,PvPAL与唇形科植物的PAL基因亲缘关系最近.组织表达分析结果显示,PvPAL基因在根、茎、叶中均表达,其中根中表达量最高.PvPAL基因的克隆为进一步研究夏枯草迷迭香酸合成的分子机制奠定了基础.%Phenylalanne ammonial-lyase( PAL) is one of the key enzymes involved in rosmarinic acid biosyn-thetic pathway. In this study,a PAL gene,named PvPAL,was cloned from Prunella vulgaris at the first time by 3'-RACE-PCR and using the specific primer,which was designed according to the homologus sequences of PAL genes from other plants. The GenBank accession number of PvPAL is JN65446. The length of PvPAL cDNA fragment is 1 306 bp,including a 1 047 bp-length coding sequence,which encoded a 349-amino-acid protein. Sequence multiple-alignment revealed that PvPAL protein had extensive homology with those of other plants as Salvia miltiorrhiza, Rehmannia glutinosa, Scutellaria baicalensis and Agastache rugosa. Phylogenetic tree analysis showed that PvPAL had closest relationship with PALs from Lamiaceae plants than from other plants. Tissue expression analysis indicated that PvPAL expressed in all tissues examined,but highest in roots. The isolation of PvPAL provided basis for further studying the molecular mechanism of rosmarinic acid biosynthesis in P. vulgaris.

  6. Expression of Astragalus membranaceus phenylalanine ammonia-lyase gene in Pichia pastoris%膜荚黄芪苯丙氨酸解氨酶基因在毕赤酵母中的分泌表达

    Institute of Scientific and Technical Information of China (English)

    张健慧; 王首锋

    2014-01-01

    L-phenylalanine , as an essential amino acid for human nutrition , is widely used in pharmaceutical and food industries . Using phenylalanine ammonia-lyase ( PAL ,EC 4 .3 .1 .5) to produce L-phenylalanine is one of the major routes . However , most commercial enzymes are extracted from Rhodotorula glutinis , which is time-consuming and over-priced . Therefore , how to efficiently construct the genetic engineering strain to produce PAL is the hot topic . Pichia pastoris is popular in expressing heterologous proteins due to the advantages of low nutritional demands , excellent genetic stability and high-density fermentation . Inserting the heterologous gene into pPIC 9K vectortoachievesecretedexpressionin P.pastorishasbeenreported.However,unlikeothervectors,pPIC9Khas few desirable restriction enzyme cutting sites , which reduces vector construction efficiency when the classical method of digestion and then ligation is adopted . Under this condition , an efficient cloning strategy , independent of digestion and ligation , is required . Homologous recombination in vitro between pPIC9K and gene can settle this problem .Now ,we intend to employ homologous recombination in vitro cloning method to insert the PAL gene into pPIC9K vector to obtain secreted expression in P . pastoris in order to lay the basis for industrial fermentation . First , total RNA extracted from Astragalus membranaceus was used as template for isolating cDNA . Open reading frame ( ORF) of PA L gene was amplified by PCR from cDNA with a pair of primers designed according to the sequence of PA L gene published in the GenBank . Then , ORF was cloned into vector pUCm-T . The transformant was selected to sequence for further analysis of the PA L gene sequence with the help of bioinformatics tools . After that , pPIC9K-PA L was constructed by homologous recombination in vitro . Similarly , the transformant was selected to sequence to investigate the base mutation caused by PCR . Linearized pPIC 9K-PA L by

  7. Critical review of carbon monoxide pressure measurements in the uranium-carbon-oxygen ternary system

    International Nuclear Information System (INIS)

    For high temperature reactors (HTR), the high level of fuel operating temperature in normal and accidental conditions requires to predict the possible chemical interactions between the fuel components. Among the concerns of the TRISO fuel particle thermomechanical behavior, it is necessary to better understand the carbon monoxide formation due to chemical interactions at the UO2 kernel and graphite buffer's interface. In a first step, the thermodynamic properties of the U-C-O system have to be assessed. The experimental data from literature on the equilibrium CO gas pressure measurements in the UO2-UC2-C ternary section of the U-C-O system are critically reviewed. Discrepancies between the different determinations can be explained - (i) by the different gaseous flow regimes in the experiments and - (ii) by the location of the measuring pressure gauge away from the reaction site. Experimental values are corrected - (i) from the gaseous flow type (molecular, transition or viscous) defined by the Knudsen number and - (ii) from the thermomolecular effect due to the temperature gradient inside the experimental vessels. Taking account of the selected and corrected values improves greatly the consistency of the original set of measurements

  8. Carbon, oxygen and nitrogen dynamics in a soil profile: Model development and application

    Science.gov (United States)

    Batlle-Aguilar, J.; Brovelli, A.; Porporato, A. M.; Barry, D. A.

    2009-12-01

    In order to meet demands for crops, pasture and firewood, the rate of land use change from forested to agricultural uses steadily increased over several decades, resulting in an increased release of nutrients towards groundwater and surface water bodies. In parallel, the degradation of riparian zones has diminished their capacity to provide critical ecosystem functions, such as the ability to control and buffer nutrient cycles. In recent years, however, the key environmental importance of natural, healthy ecosystems has been progressively recognized and restoration of degraded lands towards their former natural state has become an area of active research worldwide. Land use changes and restoration practices are known to affect both soil nutrient dynamics and their transport to neighboring areas. To this end, in order to interpret field experiments and elucidate the different mechanisms taking place, numerical tools are beneficial. Microbial decomposition is the main driving force on biogeochemical transformations of soil organic matter and soil nutrients. The activity of the soil biota is primarily controlled by water availability and by the pore-solution oxygen concentrations, which ultimately depend to a large extent on meteorological conditions, e.g., precipitation. In this work a model is presented that simulates carbon and nitrogen turnover and transport in a 1D profile under variably-saturated conditions. The model is based on the mechanistic batch model of Porporato et al. (Adv. Water Res., 26: 45-58, 2003), but extends its capabilities to simulate the vertical transport of the mobile components. Furthermore, oxygen dynamics are included such that the pore-water concentration is dependent on microbial degradation rates and soil moisture level. The model was applied to simulate the effect of land use change from forested to agricultural soils on pedo-fauna activity and nutrient distribution and abundance across the vertical profile. External forcing, i.e., precipitation time series, were generated stochastically. Modeling results showed that soil tillage practices, which modify soil structure and thus soil aeration, were responsible for higher decomposition and mineralization rates in agricultural soils, thus for the lower soil carbon and nitrogen concentrations. Furthermore, higher plant uptake rates and leaching of agricultural soils contributed also to lower nitrate concentrations in such soils.

  9. Reversible CO2 Capture by Conjugated Ionic Liquids through Dynamic Covalent Carbon-Oxygen Bonds.

    Science.gov (United States)

    Pan, Mingguang; Cao, Ningning; Lin, Wenjun; Luo, Xiaoyan; Chen, Kaihong; Che, Siying; Li, Haoran; Wang, Congmin

    2016-09-01

    The strong chemisorption of CO2 is always accompanied by a high absorption enthalpy, and traditional methods to reduce the absorption enthalpy lead to decreased CO2 capacities. Through the introduction of a large π-conjugated structure into the anion, a dual-tuning approach for the improvement of CO2 capture by anion-functionalized ionic liquids (ILs) resulted in a high capacity of up to 0.96 molCO2  mol-1IL and excellent reversibility. The increased capacity and improved desorption were supported by quantum chemical calculations, spectroscopic investigations, and thermogravimetric analysis. The increased capacity may be a result of the strengthened dynamic covalent bonds in these π-electron-conjugated structures through anion aggregation upon the uptake of CO2 , and the improved desorption originates from the charge dispersion of interaction sites through the large π-electron delocalization. These results provide important insights into effective strategies for CO2 capture. PMID:27458723

  10. Type Ia Supernova Explosions from Hybrid Carbon-Oxygen-Neon White Dwarf Progenitors

    CERN Document Server

    Willcox, Donald E; Calder, Alan C; Denissenkov, Pavel A; Herwig, Falk

    2016-01-01

    Motivated by recent results in stellar evolution that predict the existence of hybrid white dwarf (WD) stars with a C-O core inside an O-Ne shell, we simulate thermonuclear (Type Ia) supernovae from these hybrid progenitors. We use the FLASH code to perform multidimensional simulations in the deflagration to detonation transition (DDT) explosion paradigm. Our hybrid progenitor models were produced with the MESA stellar evolution code and include the effects of the Urca process, and we map the progenitor model to the FLASH grid. We performed a suite of DDT simulations over a range of ignition conditions consistent with the progenitor's thermal and convective structure assuming multiple ignition points. To compare the results from these hybrid WD stars to previous results from C-O white dwarfs, we construct a set of C-O WD models with similar properties and similarly simulate a suite of explosions. We find that despite significant variability within each suite, trends distinguishing the explosions are apparent ...

  11. Carbon, oxygen and sulfur isotope studies of hydrothermal mineralization in the Gavnunim valley, Makhtesh Ramon, Israel

    International Nuclear Information System (INIS)

    Quartz and sulfide mineral from polymetallic vein-type mineralization within the quartz syenites of the Gavnunim valley, Makhtesh Ramon, exhibit isotopic composition quite different from the hypothermal ore occurrence. Quartz samples have δ18O in the range 19.2 to 25.3 per mille and the δ34S of sulfides varies from 17.5 to -30.6 per mille. At mineral deposition temperatures of 200-350 degree Celsius this compositional ranges are strongly indicative of isotopic buffering of the fluids by sediments in contact with the intrusive rocks. The distribution of δ13C and δ18O in secondary carbonates is consistent with epigenetic remobilization. The mineralization phenomena in the Gavnunim valley are constraint to the later stages of magmatic activity, when circulating hypothermal fluids moving through the sediments infiltrated fractures within the roof of the igneous body and deposited primary sulfide and gangue minerals. (author)

  12. Carbon, oxygen and strontium isotopic systematics of Mediterranean white marbles used in the Antiquity

    Science.gov (United States)

    Zoeldfoeldi, J.; Szekely, B.

    2009-04-01

    Many geological and petrographic systems have been utilized to determine the provenance of classical marble artefacts. Normally the first steps in sourcing a lithic artefact should be the macroscopic and thin section study with the petrographic microscope. Unfortunately, often to take sample, which is large enough to make thin section is not allowed from precious artefacts. A common procedure to analyse powder sample in various ways: The most widely used system today is that of stable δ13C and δ18O isotopic signatures. Previous studies showed that, unfortunately, many quarry fields overlap in values. Because of great advantage of isotopic ratio analysis, principally the need of only small samples and homogeneity over large areas, we decided to include the 87Sr/86Sr isotopic ratios. Therefore we tested the δ13C, δ18O and 87Sr/86Sr isotopic system, based on the data set entry in MissMarble measurement and information system of marble (Z

  13. The ground part of data acquisition system for carbon/oxygen well logging

    International Nuclear Information System (INIS)

    The communication between the ground computer and the pulsed neutron multi-parameter spectrum instrument in well logging, i.e. Atlas-Dress 3700-2727 C/O well logging instrument, is studied. A data acquisition system based on the microprocessor 80C196 is designed. The 80C196 can encode as well as decode the Manchester code by means of software. Furthermore, the system has a diagnostic function for checking the transmission performance

  14. Emission Lines of Boron, Carbon, Oxygen and Iron in Tokamak Plasma

    Institute of Scientific and Technical Information of China (English)

    DI Long; WAN Bao-Nian; ZHAO Gang; ZHANG Jie; SHI Jian-Rong; WANG Shou-Jun; DONG Quan-Li; ZHAO Jing; LI Yu-Tong; FU Jia; WANG Fu-Di; SHI Yue-Jiang

    2011-01-01

    The emission lines of B,C,O and Fe in tokamak plasma are reported. The spectra are compared with those calculated by the CHIANTI code,which is based on the collisional-radiative models with a large amount of accurate atomic data.General agreement is obtained between the results of experiment and computation.Most of the lines in the spectra are identified,and the relative number density ratios orB,C,O and Fe are determined.It is found that the processes of line formation in our experiment are similar to those in the stellar coronae.The line-averaged electron density of the tokamak plasma is measured by the HCN laser,indicating a good agreement with the theoretical prediction by the density-dependent line ratio of Fe XXI.

  15. Carbon, oxygen and intrinsic defect interactions in germanium-doped silicon

    International Nuclear Information System (INIS)

    Production and annealing of oxygen-vacancy (VO) and oxygen-carbon (CiOi, CiOiI) defects in germanium-doped Czochralski-grown silicon (Cz-Si) containing carbon are investigated. All the samples were irradiated with 2 MeV fast electrons. Radiation-produced defects are studied using infrared spectroscopy by monitoring the relevant bands in optical spectra. For the VO defects, it is established that the doping with Ge affects the thermal stability of VO (830 cm−1) defects as well as their fraction converted to VO2 (888 cm−1) defects. In Ge-free samples containing carbon, it was found that carbon impurity atoms do not affect the thermal stability of VO defects, although they affect the fraction of VO defects that is converted to VO2 complexes. Considering the oxygen–carbon complexes, it is established that the annealing of the 862 cm−1 band associated with the CiOi defects is accompanied with the emergence of the 1048 cm−1 band, which has earlier been assigned to the CsO2i center. The evolution of the CiOiI bands is also traced. Ge doping does not seem to affect the thermal stability of the CiOi and CiOiI defects. Density functional theory (DFT) calculations provide insights into the stability of the defect clusters (VO, CiOi, CiOiI) at an atomic level. Both experimental and theoretical results are consistent with the viewpoint that Ge affects the stability of the VO but does not influence the stability of the oxygen–carbon clusters. DFT calculations demonstrate that C attracts both Oi and VO pairs predominately forming next nearest neighbor clusters in contrast to Ge where the interactions with Oi and VO are more energetically favorable at nearest neighbor configurations

  16. Contribution to the study of the system uranium-carbon-oxygen

    International Nuclear Information System (INIS)

    The region U-UC-UO2 of the U-C-O diagram has been investigated by the saturation method. This system is of the ternary eutectic type, the eutectic point lying close to pure uranium. The UO2-UC plane can be considered as pseudo-binary; it involves a eutectic UO2-U (C,O) melting at about 2250 C. The oxycarbides U (C,O) precipitated from the melt can be considered as stoichiometric ((C+O)/U = 1), with a formula close to UC(0.65)O(0.35) for deposition temperatures below 2200 C. (author)

  17. Carbon combustion supernovae - Numerical studies of the final evolution of degenerate carbon-oxygen cores

    Science.gov (United States)

    Mueller, E.; Arnett, W. D.

    1986-01-01

    The evolution of polytropic 1.46-solar-mass 3-Gg/cu cm (4 x 10 to the 8th)-K pure C cores is investigated theoretically by means of model computations starting from the runaway stage. The temperature in central zones is raised to the runaway temperature, and the evolution is followed in a spherical coordinate system. In models of burning without detonation, it is found that the initially spherical burning front is Rayleigh-Taylor unstable, without bipolar jets or equatorial rings; that some C is not burned to Ni; and that the Ni in the 0.8-1.0 solar mass of burned fuel produced can account for observed properties of type I supernovae.

  18. [pi] Backbonding in Carbonyl Complexes and Carbon-Oxygen Stretching Frequencies: A Molecular Modeling Exercise

    Science.gov (United States)

    Montgomery, Craig D.

    2007-01-01

    An exercise is described that has illustrated the effect of various factors on [pi] backbonding to carbonyl ligands, where the students can view the molecular orbitals corresponding to the M-CO [pi] interaction as well as the competing interaction between the metal and co-ligands. The visual and hands-on nature of the modeling exercise has helped…

  19. Climatic forcing of carbon-oxygen isotopic covariance in temperate-region marl lakes

    Science.gov (United States)

    Drummond, C. N.; Patterson, W. P.; Walker, J. C.

    1995-01-01

    Carbon and oxygen stable isotopic compositions of lacustrine carbonate from a southeastern Michigan marl lake display linear covariance over a range of 4.0% Peedee belemnite (PDB) in oxygen and 3.9% (PDB) in carbon. Mechanisms of delta 13 C-delta 18 O coupling conventionally attributed to lake closure in arid-region basins are inapplicable to hydrologically open lake systems. Thus, an alternative explanation of isotopic covariance in temperate region dimictic marl lakes is required. We propose that isotopic covariance is a direct record of change in regional climate. In short-residence-time temperate-region lake basins, summer meteoric precipitation is enriched in 18O relative to winter values, and summer organic productivity enriches epilimnic dissolved inorganic carbon in 13C. Thus, climate change toward longer summers and/or shorter winters could result in greater proportions of warm-month meteoric precipitation, longer durations of warm-month productivity, and net long-term enrichment in carbonate 18O and 13C. Isotopic covariance observed in the Michigan marl lake cores is interpreted to reflect postglacial warming from 10 to 3 ka followed by cooler mean annual temperature, a shift toward greater proportions of seasonal summer precipitation, a shortening of the winter season, or some combination of these three factors.

  20. Interstellar gas phase abundance of carbon, oxygen, nitrogen, copper, gallium, germanium, and krypton toward Zeta Ophiuchi

    Science.gov (United States)

    Cardelli, Jason A.; Savage, Blair D.; Ebbets, Dennis C.

    1991-01-01

    An analysis of weak (less than 10 mA) UV interstellar absorption line data obtained for the line of sight to the O9.5 IV star Zeta Oph is presented. Measurements of weak semiforbidden lines of N I, O I, Cu II, and a new UV detection of Na I are reported along with a small upper limit for C II. Interstellar detections of Ga II, Ge II, and Kr I are also presented. Ga, Ge, and Kr represent the heaviest elements detected in the ISM. A comparison of the derived column densities to cosmic abundances shows Ga to be depleted by about -1.2 dex while Ge is overabundant by +0.2 dex. Assuming Kr to be undepleted, a logarithmic cosmic abundance of Kr/H = 2.95 is obtained on the scale where H = 12.00.

  1. Population synthesis of triple systems in the context of mergers of carbon-oxygen white dwarfs

    OpenAIRE

    Hamers, A.S.; Pols, O. R; Claeys, J. S. W.; Nelemans, G.A.

    2013-01-01

    Hierarchical triple systems are common among field stars yet their long-term evolution is poorly understood theoretically. In such systems Kozai cycles can be induced in the inner binary system during which the inner orbit eccentricity and the inclination between both binary orbits vary periodically. These cycles, combined with tidal friction and gravitational wave emission, can significantly affect the inner binary evolution. To investigate these effects quantitatively we perform a populatio...

  2. Ionic charge state distribution of helium, carbon, oxygen, and iron in an energetic storm particle enhancement

    Science.gov (United States)

    Hovestadt, D.; Klecker, B.; Hoefner, H.; Scholer, M.; Gloeckler, G.; Ipavich, F. M.

    1982-01-01

    An analysis is presented of the ionic charge state distribution of He, C, O and Fe in the energetic storm particle event of September 28-29, 1978. Data were obtained with the ULEZEQ electrostatic analyzer-proportional counter on board the ISEE 3 spacecraft. The He(+)/He(++) ratio between 0.4 and 1 MeV/n is shown to be significantly lower during the energetic storm particle event than during the preceding period of solar flare particle enhancement, with a temporal evolution similar to that of the Fe/He ratio as reported by Klecker et al. (1981). Increases in the mean charge state for oxygen by about 3% and for iron by about 16% are also noted. The temporal variations in charge states are accounted for in terms of first-order Fermi acceleration of the pre-existing solar flare particles by a propagating interplanetary shock wave.

  3. Conservation and Functional Importance of Carbon-Oxygen Hydrogen Bonding in AdoMet-Dependent Methyltransferases

    Energy Technology Data Exchange (ETDEWEB)

    Horowitz, Scott; Dirk, Lynnette M.A.; Yesselman, Joseph D.; Nimtz, Jennifer S.; Adhikari, Upendra; Mehl, Ryan A.; Scheiner, Steve; Houtz, Robert L.; Al-Hashimi, Hashim M.; Trievel, Raymond C. [Oregon State U.; (Michigan); (Utah SU); (HHMI); (Kentucky)

    2013-09-06

    S-Adenosylmethionine (AdoMet)-based methylation is integral to metabolism and signaling. AdoMet-dependent methyltransferases belong to multiple distinct classes and share a catalytic mechanism that arose through convergent evolution; however, fundamental determinants underlying this shared methyl transfer mechanism remain undefined. A survey of high-resolution crystal structures reveals that unconventional carbon–oxygen (CH···O) hydrogen bonds coordinate the AdoMet methyl group in different methyltransferases irrespective of their class, active site structure, or cofactor binding conformation. Corroborating these observations, quantum chemistry calculations demonstrate that these charged interactions formed by the AdoMet sulfonium cation are stronger than typical CH···O hydrogen bonds. Biochemical and structural studies using a model lysine methyltransferase and an active site mutant that abolishes CH···O hydrogen bonding to AdoMet illustrate that these interactions are important for high-affinity AdoMet binding and transition-state stabilization. Further, crystallographic and NMR dynamics experiments of the wild-type enzyme demonstrate that the CH···O hydrogen bonds constrain the motion of the AdoMet methyl group, potentially facilitating its alignment during catalysis. Collectively, the experimental findings with the model methyltransferase and structural survey imply that methyl CH···O hydrogen bonding represents a convergent evolutionary feature of AdoMet-dependent methyltransferases, mediating a universal mechanism for methyl transfer.

  4. Coupling between the nickel-carbon and carbon-oxygen stretch motion in NiCO

    Science.gov (United States)

    Dunlap, B. I.; Yu, H. L.; Antoniewicz, P. R.

    1982-01-01

    Linear-combination-of-Gaussian-type-orbital (LCGTO) X-alpha calculations are performed on NiCO for various linear Ni-CO and NiC-O bond distances. The basis sets used are moderately large, approximately double zeta, and thus are fairly accurate within the X-alpha approximation. The electronic structure and equilibrium bond distances are consistent with recent CI calculations, indicating a much stronger Ni-CO bond than occurs in Ni(CO)4. The calculations agree to within 100/cm with the experimentally observed NiC-O stretch frequency. The Ni-CO stretch is predicted to occur at 656/cm, although at an intensity that is 640 times less. Compared with experimental results for CO adsorbed on single crystal Ni(100), the computed vibrational frequencies and intensities are less appropriate than the relevant experimental values for Ni(CO)4. This is interpreted to mean that an accurate description of the electronic structure of the nickel atom participating directly in the surface bond requires proper accounting of the bonds to its other nearest neighbors.

  5. The carbon/oxygen ratio in high-energy cosmic rays

    Science.gov (United States)

    Juliusson, E.; Meyer, P.

    1973-01-01

    The abundance distribution of cosmic ray nuclei at 0.6 GeV/nucleon, 1.6 GeV/nucleon and between 20 and 100 GeV/nucleon has been measured by balloon-borne instrumentation. We find that the abundance ratio of carbon to oxygen decreases from 1.1 around 1 GeV/nucleon to a value of about 0.8 at energies above about 30 GeV/nucleon. Some implications of this result are discussed.

  6. Carbon, oxygen and hydrogen isotopic intercomparison of fruit and vegetable juices

    International Nuclear Information System (INIS)

    Within the framework of the CEN (European Committee for Standardization) the Working Group 1 of the Technical Committee 174 ''Fruit and Vegetable Juices'' is in charge of developing and validating isotope analytical methods, capable to improve the authentication of fruit juices. Here we report the results of several round robins recently carried out. (author). 4 refs, 1 fig., 8 tabs

  7. Deficiência da 3-OH-3-metil-glutaril-CoA-liase como causa de coma no período neonatal: relato de caso 3-hydroxy-3-methylglutaryl-CoA-lyase deficiency as coma etiology in the neonatal period: case report

    Directory of Open Access Journals (Sweden)

    ERASMO BARBANTE CASELLA

    1998-09-01

    Full Text Available Estudamos um paciente que apresentou dois episódios de coma no primeiro mês de vida, com descompensação metabólica, nos quais se observou hipoglicemia e acidose metabólica acentuada, sem cetonúria. O estudo dos ácidos orgânicos urinários demonstrou elevação acentuada de 3-OH-3-metil-glutárico, 3-metil-glutacônico, 3-metil-glutárico e 3-OH-isovalérico. Os sinais e sintomas clínicos associados às alterações metabólicas citadas permitiram o diagnóstico da deficiência da 3-OH-3-metil-glutaril-CoA-liase, entidade de origem autossômica recessiva, passível de ser tratada, como no caso estudado, com dieta hipoproteica, restrita em leucina, hipogordurosa e rica em carboidratos, associada a L-carnitina e evitando-se períodos prolongados de jejum.We report a patient that presented two episodes of coma in the neonatal period, with severe metabolic acidosis and hypoglycemia, without ketosis. The urinary organic acid analysis showed increased amounts of 3-hydroxy-3-methyl-glutaric, 3-methylglutaconic, 3-methylglutaric and 3-hydroxyisovaleric acid. The deficiency of 3-hydroxy-3-methylglutaryl-CoA lyase was diagnosed by the clinical and metabolic features. This disease shows autosomal recessive inheritance and the treatment is done by a diet with restriction of protein (mainly leucine and lipids, high in carbohydrate content, and the avoidance of fasting and carnitine supplementation.

  8. Sediment-water column fluxes of carbon, oxygen and nutrients in Bedford Basin, Nova Scotia, inferred from 224Ra measurements

    Directory of Open Access Journals (Sweden)

    E. Horne

    2012-07-01

    Full Text Available Exchanges between sediment pore waters and the overlying water column play a significant role in the chemical budgets of many important chemical constituents. Quantification of such benthic fluxes requires explicit knowledge of the sediment properties and biogeochemistry. Alternatively, changes in water column properties near the sediment-water column interface can be exploited to gain insight into the sediment biogeochemistry and benthic fluxes. Here, we apply a 1-D diffusive mixing model to near-bottom water column profiles of 224Ra activity in order to yield vertical eddy diffusivities (KZ, based upon which we assess the diffusive exchange of inorganic carbon (DIC, nutrients and oxygen (O2, across the sediment-water interface in a coastal inlet, Bedford Basin, Nova Scotia, Canada. Near-bottom observations of DIC, O2 and nutrients provide flux ratios similar to Redfield values, suggesting that benthic respiration of primarily marine organic matter is the dominant driver. Furthermore, we did not observe any significant release of alkalinity (AT from the sediments to the overlying water column, providing further insight into the dominant reactions taking place within sediments: the respiration of organic matter occurs largely under aerobic conditions or products of anaerobic processes are reoxidized quickly in oxygenated layers of the sediments. Finally, comparison with other carbon sources reveal the observed benthic DIC release as a significant contributor to the Bedford Basin carbon system.

  9. Type Ia supernovae within dense carbon-oxygen rich envelopes: a model for 'Super-Chandrasekhar' explosions?

    CERN Document Server

    Noebauer, U M; Blinnikov, S; Sorokina, E; Hillebrandt, W

    2016-01-01

    We investigate the consequences of fairly normal Type Ia supernovae being embedded in compact and dense envelopes of carbon and oxygen rich circumstellar material by means of detailed radiation hydrodynamic simulations. Our main focus rests on exploring the effects of the interaction between ejecta and circumstellar material on the ejecta evolution and the broad-band light curve. In our calculations, we find that a strong reverse shock efficiently decelerates and compresses the ejecta material. This leads to a significant broadening of the optical light curve, a longer rise to maximum and a slower decline in the tail phase. During the interaction, substantial radiative energy is generated, which mostly emerges in the extreme ultraviolet and X-ray regime. Only if reprocessing due to radiation-matter interactions is very efficient, a significant boost in the optical light curve is observed. We discuss these findings in particular in the context of the super-luminous event SN 2009dc. As our calculations are able...

  10. Disequilibrium Carbon, Oxygen, and Nitrogen Chemistry in the Atmospheres of HD 189733b and HD 209458b

    CERN Document Server

    Moses, Julianne I; Fortney, Jonathan J; Showman, Adam P; Lewis, Nikole K; Griffith, Caitlin A; Shabram, Megan; Friedson, A James; Marley, Mark S; Freedman, Richard S

    2011-01-01

    We have developed 1-D photochemical and thermochemical kinetics and diffusion models for the transiting exoplanets HD 189733b and HD 209458b to study the effects of disequilibrium chemistry on the atmospheric composition of "hot Jupiters." Here we investigate the coupled chemistry of neutral carbon, hydrogen, oxygen, and nitrogen species, and we compare the model results with existing transit and eclipse observations. We find that the vertical profiles of molecular constituents are significantly affected by transport-induced quenching and photochemistry, particularly on cooler HD 189733b; however, the warmer stratospheric temperatures on HD 209458b can help maintain thermochemical equilibrium and reduce the effects of disequilibrium chemistry. For both planets, the methane and ammonia mole fractions are found to be enhanced over their equilibrium values at pressures of a few bar to less than a mbar due to transport-induced quenching, but CH$_4$ and NH$_3$ are photochemically removed at higher altitudes. Atomi...

  11. Carbon, oxygen and their interaction with intrinsic point defects in solar silicon ribbon material: A speculative approach

    Science.gov (United States)

    Goesele, U.; Ast, D. G.

    1983-01-01

    Some background information on intrinsic point defects is provided and on carbon and oxygen in silicon in so far as it may be relevant for the efficiency of solar cells fabricated from EFG ribbon material. The co-precipitation of carbon and oxygen and especially of carbon and silicon self interstitials are discussed. A simple model for the electrical activity of carbon-self-interstitial agglomerates is presented. The self-interstitial content of these agglomerates is assumed to determine their electrical activity and that both compressive stresses (high self-interstitial content) and tensile stresses (low self-interstitial content) give rise to electrical activity of the agglomerates. The self-interstitial content of these carbon-related agglomerates may be reduced by an appropriate high temperature treatment and enhanced by a supersaturation of self-interstitials generated during formation of the p-n junction of solar cells. Oxygen present in supersaturation in carbon-rich silicon may be induced to form SiO, precipitates by self-interstitials generated during phosphorus diffusion. It is proposed that the SiO2-Si interface of the precipates gives rise to a continuum of donor stables and that these interface states are responsible for at least part of the light inhancement effects observed in oxygen containing EFG silicon after phosphorus diffusion.

  12. Sediment-water column fluxes of carbon, oxygen and nutrients in Bedford Basin, Nova Scotia, inferred from 224Ra measurements

    Directory of Open Access Journals (Sweden)

    E. Horne

    2013-01-01

    Full Text Available Exchanges between sediment pore waters and the overlying water column play a significant role in the chemical budgets of many important chemical constituents. Direct quantification of such benthic fluxes requires explicit knowledge of the sediment properties and biogeochemistry. Alternatively, changes in water-column properties near the sediment-water interface can be exploited to gain insight into the sediment biogeochemistry and benthic fluxes. Here, we apply a 1-D diffusive mixing model to near-bottom water-column profiles of 224Ra activity in order to yield vertical eddy diffusivities (KZ, based upon which we assess the diffusive exchange of dissolved inorganic carbon (DIC, nutrients and oxygen (O2, across the sediment-water interface in a coastal inlet, Bedford Basin, Nova Scotia, Canada. Numerical model results are consistent with the assumptions regarding a constant, single benthic source of 224Ra, the lack of mixing by advective processes, and a predominantly benthic source and sink of DIC and O2, respectively, with minimal water-column respiration in the deep waters of Bedford Basin. Near-bottom observations of DIC, O2 and nutrients provide flux ratios similar to Redfield values, suggesting that benthic respiration of primarily marine organic matter is the dominant driver. Furthermore, a relative deficit of nitrate in the observed flux ratios indicates that denitrification also plays a role in the oxidation of organic matter, although its occurrence was not strong enough to allow us to detect the corresponding AT fluxes out of the sediment. Finally, comparison with other carbon sources reveal the observed benthic DIC release as a significant contributor to the Bedford Basin carbon system.

  13. Carbon, oxygen and biological productivity in the Southern Ocean in and out the Kerguelen plume: CARIOCA drifter results

    Directory of Open Access Journals (Sweden)

    L. Merlivat

    2014-12-01

    Full Text Available The Kerguelen Plateau region in the Indian sector of the Southern Ocean supports annually a large-scale phytoplankton bloom which is naturally fertilized with iron. As part of the second Kerguelen Ocean and Plateau compared Study expedition (KEOPS2 in austral spring (October–November 2011, one Carioca buoy was deployed east of the Kerguelen plateau. It drifted eastward downstream in the Kerguelen plume. Hourly surface measurements of pCO2, O2 and ancillary observations were collected between 1 November 2011 to 12 February 2012 with the aim of characterizing the spatial and temporal variability of the biological Net Community Production (NCP downstream the Kerguelen plateau, assess the impact of iron-induced productivity on the biological carbon consumption and consequently on the CO2 flux exchanged at the air–sea interface. The trajectory of the buoy until mid-December was within the longitude range, 72–83° E, close to the polar front and then in the polar frontal zone, PFZ, until 97° E. From 17 November to 16 December, the buoy drifted within the Kerguelen plume following a filament carrying dissolved iron, DFe, for a total distance of 700 km. In the first part of the trajectory, the ocean surface waters are a sink for CO2 and a source for CO2, with fluxes of respective mean values equal to −8 and +38 mmol CO2 m−2 d−1. Eastward, as the buoy escapes the iron enriched filament, the fluxes are in opposite direction, with respective mean values of +5 and −48 mmol O2 m−2 d−1. These numbers clearly indicate the strong impact of biological processes on the biogeochemistry in the surface waters within the Kerguelen plume in November-mid-December, while it is undetectable eastward in the PFZ from mid-December to mid-February. While the buoy follows the Fe enriched filament, simultaneous observations of dissolved inorganic carbon, DIC, and dissolved oxygen, O2, highlight biological events lasting from 2 to 4 days. Stoichiometric ratios, O2/C, between 1.1 and 1.4 are observed indicating new and regenerated production regimes. NCP estimates range from 60 to 140 mmol C m−2 d−1. Based on the relationship between the time a water parcel has left the plateau and its iron content, we have highlighted that the main control on the value of NCP is the availability of iron in the upper water column, with the largest NCP occurring in waters that have recently left the plateau and presented the highest iron concentrations.

  14. Carbon, oxygen and their interaction with intrinsic point defects in solar silicon ribbon material. Annual report, September 1982-September 1983

    Energy Technology Data Exchange (ETDEWEB)

    Goesele, U.; Ast, D.G.

    1983-10-01

    This report first provides some background information on intrinsic point defects, and on carbon and oxygen in silicon in so far as it may be relevant for the efficiency of solar cells fabricated from EFG ribbon material. We discuss the co-precipitation of carbon and oxygen and especially of carbon and silicon self interstitials. A simple model for the electrical activity of carbon-self-interstitial agglomerates is presented. We assume that the self-interstitial content of these agglomerates determines their electrical activity and that both compressive stresses (high self-interstitial content) and tensile stresses (low self-interstitial content) give rise to electrical activity of the agglomerates. The self-interstitial content of these carbon-related agglomerates may be reduced by an appropriate high-temperature treatment and enhanced by a supersaturation of self-interstitials generated during formation of the p-n junction of solar cells. It is suggested that oxygen present in supersaturation in carbon-rich silicon may be induced to form SiO/sub 2/ precipitates by self-interstitials generated during phosphorus diffusion. It is proposed that the SiO/sub 2/-Si interface of the precipates gives rise to a continuum of donor states and that these interface states are responsible for at least part of the light-enhancement effects observed in oxygen containing EFG silicon after phosphorus diffusion.

  15. Facies and carbon/oxygen isotopes of the Calabozo Formation (Middle Jurassic), Arroyo La Vaina, Mendoza, Argentina

    International Nuclear Information System (INIS)

    Facial / microfacial studies and geochemical isotopic analyses on marine jurassic carbonates of the Calabozo Formation (Dessanti, 1973) were carried out to reconstruct the palaeoenvironment and postdepositional history of the unit. This study is part of a project which purpose is the sedimentological and geochemical characterization of the Jurassic carbonate sequences of Cuenca Neuquina, in the southwestern Mendoza, Argentina. A detailed description about this basin can be found in Legarreta and Uliana (1999) and Riccardi et al. (2000). During the Late Bathonian and Early Callovian, the basin showed a reduction of the sedimentation area and a marked marginal facies progradation. West of Malargue, in areas with low detritic contribution, limestones of the Calabozo Formation were deposited. At the end of the Early Callovian, the basin was isolated, prevailing hypersaline conditions which caused the accumulation of the evaporites of the Tabanos Formation (Stipanicic, 1966) (au)

  16. INDUCCIÓN DE FENILALANINA AMONIO LIASA Y VARIACIÓN EN EL CONTENIDO DE COMPUESTOS FENÓLICOS EN FRUTOS DE LULO (Solanum quitoense Lam INFECTADOS CON Colletotrichum acutatum. Induction of phenylalanine ammonia lyase and variation in phenolic compounds content in Lulo fruits (Solanum quitoense Lam infected by Colletotrichum acutatum

    Directory of Open Access Journals (Sweden)

    MAIRA ANDREA ARRIETA-GUEVARA

    Full Text Available Se evaluó la dinámica de la actividad fenilalanina amonio liasa (PAL en corteza de frutos de lulo (Solanum quitoense Lam con el fin de determinar su participación en respuestas bioquímicas hacia Colletotrichum acutatum. Se establecieron como mejores condiciones para la extracción de la enzima, buffer ácido bórico-borato de sodio 0.1M pH 8.8, 1% SDS, 3% PVPP y para medir la actividad, sustrato L-fenilalanina 5 mM , pH 8,0, 20°C , 30 ΜL de extracto y 45 min. Se realizó un ensayo in vivo usando frutos en tres estados de madurez, los cuales fueron inoculados con el patógeno o tratados con agua estéril. A cinco tiempos (hpi = horas post-infección se determinó la actividad PAL y el contenido total de fenoles, encontrándose que hay una respuesta diferencial de la enzima por efecto del patógeno y por el estado de madurez. Para frutos en el estado pintón se obtuvo el mayor aumento de PAL, el que perduró hasta 48 hpi, al compararlo con los controles y con los otros dos estados de madurez. Este aumento mostró relación con un marcado incremento en el contenido total de fenoles y con el desarrollo más tardío de síntomas característicos de antracnosis, observado para los frutos pintones. Estos resultados permiten postular, una posible relación positiva entre inducción de PAL, aumento de fenólicos y respuesta de tolerancia a C. acutatum. Para lulos en estado verde y maduro se observó aumento de PAL a 12 y 24 hpi que coincidió también con incremento en el contenido de fenoles totales, aunque para estos dos últimos estados dicho contenido disminuyó significativamente a tiempos mayores.Phenylalanine ammonia lyase (PAL activity induction was evaluated in lulo fruits to determine the role of this enzyme in biochemical responses towards the pathogen Colletotrichum acutatum. We studied the experimental conditions to obtain the enzyme, using lulo peel, and found that the best conditions for extraction were buffer of boric acid

  17. 葡萄苯丙氨酸解氨酶基因家族的全基因组鉴定及表达分析%Genome-wide characterization and expression analysis of the phenylalanine ammonia-lyase gene family in grapevine (Vitis vinifera L.)

    Institute of Scientific and Technical Information of China (English)

    孙润泽; 张雪; 成果; 李强; 朱燕溶; 陈武; 潘秋红; 段长青; 王军

    2016-01-01

    Phenylalanine ammonia-lyase (PAL), the first enzyme in the phenylpropanoid pathway, plays critical roles in plant growth, development and resistance to biotic/abiotic stresses. In this research, a total of 17 PAL genes were identified from the Vitis vinifera reference genome by the bioinformatics methods. Thirteen of the VviPAL genes are arranged in tandem in two duplication blocks located on chromosomes 11 and 16, the rests are randomly distributed on other chromosomes. Phylogenetic tree analysis revealed that most of the VviPAL members have a closer relationship with PALs from other dicots species, whereas the four VviPALs arranged in tandem on chromosome 11, which classified into neither the dicot nor monocot group, may serve as ancestral PALs in plants. The full-length cDNA of five VviPAL genes were isolated from ‘Cabernet Sauvignon’ according to the reference transcriptome of ‘Cabernet Sauvignon’ grape berries. Sequence analysis of the deduced VviPAL proteins showed that all of them contain conserved functional domains, confirming that these VviPALs may have similar functions with PALs from other plant species. Expression profiling reveals that VviPAL2 and VviPAL15 were strongly expressed in all test organs/tissues, and their expression throughout berry development was positively correlated with the accumulation of phenolic compounds. Furthermore, the transcripts of VviPAL2, VviPAL4 and VviPAL7 in grape berries were induced by cluster sunlight exposure and suppressed by cluster shading, which might contribute to changes in metabolites accumulation.%苯丙氨酸解氨酶(PAL)作为苯丙烷途径的入口酶,与植物生长发育以及生物和非生物胁迫响应等生物过程密切相关。本研究通过生物信息学方法,在已测序的葡萄基因组中预测得到17个PAL基因,其中13个串联分布在第11和16号染色体的2个重复基因群中,4个随机分布于其他染色体。系统发育树分析表明,葡萄大部分PAL基因与

  18. One-week habitation of two humans in an airtight facility with two goats and 23 crops Analysis of carbon, oxygen, and water circulation

    Science.gov (United States)

    Tako, Y.; Tsuga, S.; Tani, T.; Arai, R.; Komatsubara, O.; Shinohara, M.

    Human habitation and animal holding experiments in a closed environment, the Closed Ecology Experiment Facilities (CEEF), were carried out. The CEEF were established for collecting experimental data to estimate carbon transfer in the ecosystem around Rokkasho nuclear fuel reprocessing plant. Circulation of O2 and CO2, and supply of food from crops cultivated in the CEEF were conducted for the first time in the habitation experiments. Two humans known as eco-nauts inhabited the CEEF, living and working in the Plant Module (PM) and the Animal and Habitation Module (AHM), for a week three times in 2005. On a fresh weight basis, 82% of their food was supplied from 23 crops including rice and soybean, cultivated and harvested in the PM, in the 2nd and 3rd experiments. For the goats, the animals held in the experiments, all of their feed, consisting of rice straw, soybean plant leaves, and peanut shells and peanut plant leaves, was produced in the PM in the 2nd and 3rd experiments. The O2 produced in the PM by photosynthesis of the crops was separated by the O2 separator using molecular sheaves, then accumulated, transferred, and supplied to the AHM atmosphere. The CO2 produced in the AHM by respiration of the humans and animals was separated by the CO2 separator using solid amine, then accumulated, transferred, and supplied to the PM atmosphere. The amount of O2 consumed in the AHM was 46 51% of that produced in the PM, and the amount of CO2 produced in the AHM was 43 56% of that consumed in the PM. The surplus of O2 and the shortage of CO2 was a result of the fact that waste of the goats and the crops and part of the human waste were not processed in these habitation experiments. The estimated amount of carbon ingested by the eco-nauts was 64 92% of that in the harvested edible part of the crops. The estimated amount of carbon ingested by the goats was 36 53% of that in the harvested inedible part of the crops. One week was not enough time for determination of gas exchange especially for humans and animals, because fluctuation of their gas exchange was quite high. The amount of transpired water collected as condensate was 818 938 L d-1, and it was recycled as replenishing water compensating transpiration loss of nutrient solution. The amount of waste nutrient solution discharged from the PM was 1421 1644 L d-1. The waste nutrient solutions from rice and other crops were processed through micro filters (MFs) separately. The MF filtrated solutions were processed with reverse osmosis (RO) membrane filter separately and divided into filtrated water and concentrated waste nutrient solution. The concentrated waste nutrient solution from the crops other than rice was processed through an ultra-micro filter (UF) and reused, although that from rice was discharged in 2005. Concentrations of nutritional ions in the UF filtrated solution were determined, the depleted ions were added back, the UF filtrated solution was diluted with the RO membrane filtrated water, and the nutrient solution for the crops other than rice was regenerated. The nutrient solution for rice was newly made each time, using concentrated solution from an external source and the RO membrane filtrated water. Average amounts of water used in the AHM (L d-1) were determined as follows: drinking by humans (filtrated water), 1.5; cooking, etc. (filtrated water other than for drinking), 14.3; drinking by goats, 3.8; showering (hot water), 13.2; showering (cold water), 0.1; washing of hand and face and brushing teeth, 4.1; washing of dishes, dish clothes and towels, 36.4; and washing of animal holding tools, 0.3. The waste water was processed by a RO purification system and recycled for toilet flushing and animal pens washing. A circulation experiment for water was started in 2006 and a circulation experiment for waste materials is planned for 2007. In 2006, a single duration of the air circulation experiments was 2 weeks, although the human habitants were changed after 1 week.

  19. Carbon, Oxygen and Uranium Isotopes in Pedothem Carbonates Reveal Anomalous North American Atmospheric Circulation 70,000 to 55,000 Years Ago

    Science.gov (United States)

    Oerter, E.; Sharp, W. D.; Oster, J. L.; Ebeling, A.; Valley, J. W.; Kozdon, R.; Orland, I. J.; Hellstrom, J. C.; Woodhead, J. D.; Hergt, J.; Chadwick, O.; Amundson, R.

    2015-12-01

    Climate conditions in North America during the past two glacial cycles remain uncertain in part because long, well-dated, continuous paleoclimate records are limited in number and sparsely distributed. Here we present the first continuous, millennial resolution paleoclimate proxy record derived from C, O and U isotopes in mm-thick pedogenic carbonate clast-coatings (pedothems), which are widely distributed in semi-arid to arid regions worldwide. Carbonate 230Th/U ages were obtained from laser ablation ICP-MS spots of 93um diameter size, which also yielded 234U/238Ui proxy values for soil water infiltration and paleoprecipitation. The U-series transects were coupled to δ13C and δ18O values obtained by ion probe on ~10um diameter spots. Modern soil carbonate in the region has δ18O and δ13C values that are similar to the youngest (early Holocene) samples acquired in the laminations, strengthening the interpretation that the carbonate reflects soil isotopic conditions at the time of formation. Our new record from the Wind River Basin in Wyoming spans the last 120,000 years and confirms a previously hypothesized period of increased transport of Gulf of Mexico moisture northward into the continental interior from 70,000 to 55,000 years ago. Such pronounced meridional moisture transport, which contrasts with the dominant zonal transport of Pacific moisture into the North American interior by westerly winds before and after 70,000 to 55,000 years ago, may have resulted from a persistent anticyclone developed above the North American ice sheet during Marine Isotope Stage 4. Subsequently, the carbonate record suggests that climate in mid-latitude central North America became progressively more arid during the onset of the last glaciation (both δ13C and 234U/238Ui values become more positive), culminating in peak aridity during the LGM. We conclude that pedothems, when analyzed using micro-analytical techniques, can provide unique insights into past terrestrial climates.

  20. Purification and characterization of Clostridium sticklandii D-selenocystine alpha, beta-lyase.

    OpenAIRE

    Esaki, N; Seraneeprakarn, V; Tanaka, H.; Soda, K

    1988-01-01

    We have found a novel enzyme that decomposes D-selenocystine into pyruvate, ammonia, and elemental selenium in extracts of Clostridium sticklandii and C. sporogenes. The enzyme of C. sticklandii has been purified to homogeneity. It has a molecular weight of 74,000 and consists of two subunits identical in molecular weight (35,000). Pyridoxal 5'-phosphate is required as a cofactor. In addition to D-selenocystine, D-cystine, D-lanthionine, meso-lanthionine, and D-cysteine serve as substrates. H...

  1. Genetics Home Reference: 3-hydroxy-3-methylglutaryl-CoA lyase deficiency

    Science.gov (United States)

    ... cause the blood to become too acidic (metabolic acidosis). If untreated, the disorder can lead to breathing ... byproducts called organic acids can result in metabolic acidosis. A shortage of ketones often leads to hypoglycemia. ...

  2. Metabolism of the cysteine S-conjugate of busulfan involves a β-lyase reaction

    OpenAIRE

    Cooper, Arthur J.L.; Younis, Islam R.; Niatsetskaya, Zoya V; Krasnikov, Boris F.; Pinto, John T.; Petros, William P.; Callery, Patrick S.

    2008-01-01

    The present work documents the first example of an enzyme-catalyzed β-elimination of a thioether from a sulfonium cysteine S-conjugate. β-(S-Tetrahydrothiophenium)-L-alanine (THT-A) is the cysteine S-conjugate of busulfan. THT-A slowly undergoes a non-enzymatic β-elimination reaction at pH 7.4 and 37°C to yield tetrahydrothiophene, pyruvate and ammonia. This reaction is accelerated by a) rat liver, kidney and brain homogenates, b) isolated rat liver mitochondria, and c) pyridoxal 5′-phosphate...

  3. Densities, cellulases, alginate and pectin lyases of luminous and other heterotrophic bacteria associated with marine algae

    Digital Repository Service at National Institute of Oceanography (India)

    Ramaiah, N.; Chandramohan, D.

    Epiphytic luminous and non-luminous bacteria were determined quantitatively for eight intertidal algal species from rocky beaches of Goa and Lakshadweep coral reef lagoon. Luminous bacteria were present on all eight algal species and contributed 2...

  4. Functions and cellular localization of cysteine desulfurase and selenocysteine lyase in Trypanosoma brucei

    Czech Academy of Sciences Publication Activity Database

    Poliak, Pavel; Van Hoewyk, D.; Oborník, Miroslav; Zíková, Alena; Stuart, K. D.; Tachezy, J.; Pilon, M.; Lukeš, Julius

    2010-01-01

    Roč. 277, č. 2 (2010), s. 383-393. ISSN 1742-464X R&D Projects: GA ČR GA204/09/1667 Institutional research plan: CEZ:AV0Z60220518 Keywords : Fe–S cluster * mitochondrion * RNAi * selenoprotein * Trypanosoma Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.129, year: 2010

  5. S1P lyase regulates DNA damage responses through a novel sphingolipid feedback mechanism

    OpenAIRE

    Kumar, A.; Oskouian, B; Fyrst, H; Zhang, M.; Paris, F; Saba, J D

    2011-01-01

    The injurious consequences of ionizing radiation (IR) to normal human cells and the acquired radioresistance of cancer cells represent limitations to cancer radiotherapy. IR induces DNA damage response pathways that orchestrate cell cycle arrest, DNA repair or apoptosis such that irradiated cells are either repaired or eliminated. Concomitantly and independent of DNA damage, IR activates acid sphingomyelinase (ASMase), which generates ceramide, thereby promoting radiation-induced apoptosis. H...

  6. SULFUR ASSIMILATION IN SOYBEAN: MOLECULAR CLONING AND CHARACTERIZATION OF O-ACETYLSERINE (THIOL) LYASE (CYSTEINE SYNTHASE)

    Science.gov (United States)

    Soybean (Glycine max [L.] Merr.) is a good protein source for both humans and livestock. However, soybean seed proteins are deficient in the sulfur-containing amino acids cysteine and methionine. This deficiency has stimulated efforts to improve the amino acid composition of soybean seed proteins. ...

  7. The Salmonella effector protein SpvC, a phosphothreonine lyase is functional in plant cells

    OpenAIRE

    Neumann, Christina; Fraiture, Malou; Hernàndez-Reyes, Casandra; Akum, Fidele N.; Virlogeux-Payant, Isabelle; Chen, Ying; Pateyron, Stephanie; Colcombet, Jean; Kogel, Karl-Heinz; Hirt, Heribert; Brunner, Frédéric; Schikora, Adam

    2014-01-01

    Salmonella is one of the most prominent causes of food poisoning and growing evidence indicates that contaminated fruits and vegetables are an increasing concern for human health. Successful infection demands the suppression of the host immune system, which is often achieved via injection of bacterial effector proteins into host cells. In this report we present the function of Salmonella effector protein in plant cell, supporting the new concept of trans-kingdom competence of this bacterium. ...

  8. Overexpression of isocitrate lyase-glyoxylate bypass influence on metabolism in Aspergillus niger

    DEFF Research Database (Denmark)

    Meijer, Susan Lisette; Otero, José Manuel; Olivares Hernandez, Roberto; Andersen, Mikael Rørdam; Olsson, Lisbeth; Nielsen, Jens

    2009-01-01

    the cells was investigated. Inhibition of SDH was expected to lead to succinate production, but this was not observed. There was an increase in citrate and oxalate production in the wild-type strain. Furthermore, in the strain with over-expression of icl the organic acid production shifted from...... fumarate towards malate production when malonate was added to the cultivation medium. Overall, the icl over-expression and malonate addition had a significant impact on metabolism and on organic acid production profiles. Although the expected succinate and malate formation was not observed, a distinct and...

  9. A cardioprotective insight of the cystathionine γ-lyase/hydrogen sulfide pathway

    Directory of Open Access Journals (Sweden)

    Steve Huang

    2015-06-01

    However, CSE/H2S pathway's role in inflammation mitigation is still clouded, due to both pro and anti-inflammatory results presented in the literature, depending on the concentration and form of H2S used in specific experiment models.

  10. Evaluation of the Hydroxynitrile Lyase Activity in Cell Cultures of Capulin (Prunus serotina)`

    Institute of Scientific and Technical Information of China (English)

    Liliana Hernáindez; Héctor Luna; Arturo Navarro-Oca(n)a; Ma Teresa de Jesús Olivera-Flores; Ivon Ayala

    2008-01-01

    Enzymatic preparations obtained from young plants and cell cultures of capulin were screened for hydroxynitrile lyaseactivity. The thrceweek old plants, grown under sterile conditions, were used to establish a solid cell culture. Crude preparationsobtained from this plant material were evaluated for the transformation of benzaldehyde to the corresponding eyanohydrin(mandelonitrile). The results show that the crude material from roots, stalks, and leaves of young plants and calli of roots, stalks,internodes and petioles biocatalyzed the addition of hydrogen cyanide (HCN) to benzaidehyde with a modest to excellentenantioselectivity.

  11. Cloning and characterization of alfalfa hydroperoxide lyase : a biocatalyst for the production of green note flavors

    OpenAIRE

    Noordermeer, M.A.

    2001-01-01

    Plants continuously have to defend themselves against life threatening events such as drought, mechanical damage, temperature stress and potential pathogens. A main component of the plant defense mechanism is the lipoxygenase pathway. Products of this pathway are involved in wound healing, pest resistance, signaling, or have antimicrobial and antifungal activity. The first step in the lipoxygenase pathway is the reaction of linoleic or linolenic acids with molecular oxygen, catalyzed by the e...

  12. 陆西地区火山岩中碳酸盐矿物氧碳稳定同位素特征%Characteristic of Carbonate Oxygen-Carbon Stable Isotopes in Volcanic Rocks in Luxi Area,Junggar Basin

    Institute of Scientific and Technical Information of China (English)

    孔玉华; 沈新安; 李桂秋; 单守会; 李刚

    2009-01-01

    准噶尔盆地陆西地区火山岩孔隙中充填的碳酸盐岩对储集层物性产生巨大影响,通过对碳酸盐充填物的氧碳稳定同位素分析表明,δ~(13)C和δ~(18)O值均较轻且Z值较低,反映其形成于淡水环境,碳酸盐结晶温度大致分3种情况:早成岩作用阶段早期占大多数,部分形成于表生风化淋滤阶段,个别形成于热液作用阶段.通过氧同位素与古地温间的关系,推测碳酸盐充填物的结晶时间大致为晚三叠世到早侏罗世.烃源岩低成熟至成熟早期,靠近生烃凹陷的低凸带上,受淡水淋滤作用,火山岩中仍然存在部分未被碳酸盐岩充填的气孔.烃源岩大量排烃期,靠近凹陷区受溶蚀作用较强,可能存在相对优质的火山岩储集层发育区.

  13. Optical spectra of the carbon-oxygen accretion discs in the ultra-compact X-ray binaries 4U 0614+09, 4U 1543-624 and 2S 0918-549

    OpenAIRE

    Nelemans, G.; Jonker, P. G.; Marsh, T. R.; Klis, van der, M.

    2004-01-01

    We present optical spectra in the range 4600 -- 8600 A for three low-mass X-ray binaries which have been suggested to belong to the class of ultra-compact X-ray binaries based on their X-ray spectra. Our spectra show no evidence for hydrogen or helium emission lines, as are seen in classical X-ray binaries. The spectrum of 4U~0614+09 does show emission lines which we identify with carbon and oxygen lines of CII, CIII, OII and OIII. While the spectra of 4U 1543-624 and 2S 0918-549 have a lower...

  14. VLT/UVES observations of extremely strong intervening damped Lyman-alpha systems: Molecular hydrogen and excited carbon, oxygen and silicon at log N(HI)=22.4

    CERN Document Server

    Noterdaeme, P; Rahmani, H; Petitjean, P; Pâris, I; Ledoux, C; Gupta, N; López, S

    2015-01-01

    We present a detailed analysis of three extremely strong intervening DLAs (log N(HI)>=21.7) observed towards quasars with VLT/UVES. We measure overall metallicities of [Zn/H]~-1.2, -1.3 and -0.7 at respectively zabs=2.34 towards SDSS J2140-0321 (log N(HI) = 22.4+/-0.1), zabs=3.35 towards SDSS J1456+1609 (log N(HI) = 21.7+/-0.1) and zabs=2.25 towards SDSS J0154+1935 (log N(HI) = 21.75+/-0.15). We detect H2 towards J2140-0321 (log N(H2) = 20.13+/-0.07) and J1456+1609 (log N(H2) = 17.10+/-0.09) and argue for a tentative detection towards J0154+1935. Absorption from the excited fine-structure levels of OI, CI and SiII are detected in the system towards J2140-0321, that has the largest HI column density detected so far in an intervening DLA. This is the first detection of OI fine-structure lines in a QSO-DLA, that also provides us a rare possibility to study the chemical abundances of less abundant atoms like Co and Ge. Simple single phase photo-ionisation models fail to reproduce all the observed quantities. Inst...

  15. Bacterial Conversion of Hydroxylamino Aromatic Compounds by both Lyase and Mutase Enzymes Involves Intramolecular Transfer of Hydroxyl Groups

    OpenAIRE

    Nadeau, Lloyd J.; He, Zhongqi; Spain, Jim C.

    2003-01-01

    Hydroxylamino aromatic compounds are converted to either the corresponding aminophenols or protocatechuate during the bacterial degradation of nitroaromatic compounds. The origin of the hydroxyl group of the products could be the substrate itself (intramolecular transfer mechanism) or the solvent water (intermolecular transfer mechanism). The conversion of hydroxylaminobenzene to 2-aminophenol catalyzed by a mutase from Pseudomonas pseudoalcaligenes JS45 proceeds by an intramolecular hydroxyl...

  16. Dissimilation of cysteate via 3-sulfolactate sulfo-lyase and a sulfate exporter in Paracoccus pantotrophus NKNCYSA

    OpenAIRE

    Rein, Ulrike; Gueta, Ronnie; Denger, Karin; Ruff, Jürgen; Hollemeyer, Klaus; Cook, Alasdair M.

    2005-01-01

    Paracoccus pantotrophus NKNCYSA utilizes (R)-cysteate (2-amino-3-sulfopropionate) as a sole source of carbon and energy for growth, with either nitrate or molecular oxygen as terminal electron acceptor, and the specific utilization rate of cysteate is about 2 mkat (kg protein)(-1). The initial degradative reaction is catalysed by an (R)-cysteate : 2-oxoglutarate aminotransferase, which yields 3-sulfopyruvate. The latter was reduced to 3-sulfolactate by an NAD-linked sulfolactate dehydrogenase...

  17. THE THREE DIMENSIONAL STRUCTURE OF ASPERGILLUS NIGER PECTIN LYASE AT 1.7-A RESOLUTION. (U915444)

    Science.gov (United States)

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  18. Gamma irradiation induced enhancement of phenylalanine ammonia-lyase (PAL) and antioxidant activity in peach (Prunus persica Bausch, Cv. Elberta)

    Energy Technology Data Exchange (ETDEWEB)

    Hussain, Peerzada R., E-mail: hussainpr@rediffmail.co [Nuclear Research Laboratory, Bhabha Atomic Research Centre, Zakura, Srinagar 190006, Kashmir (India); Wani, Ali M.; Meena, Raghuveer S.; Dar, Mohd A. [Nuclear Research Laboratory, Bhabha Atomic Research Centre, Zakura, Srinagar 190006, Kashmir (India)

    2010-09-15

    Effect of medium dose gamma irradiation on PAL and antioxidant activity of peach fruit was investigated. Peach fruit after harvest at commercial maturity was irradiated in the dose range 1.0-2.0 kGy, stored under refrigerated conditions (3{+-}1 {sup o}C, RH 80%) and evaluated at intervals of 7 days. The antioxidant activity as determined by DPPH and FRAP methods revealed significant (p{<=}0.05) increase particularly in the dose range 1.6-2.0 kGy. During storage, maximum increase in both PAL and antioxidant activity was observed after 21 days. Positive correlation (r=0.75) existed between antioxidant activity and total phenols. EC{sub 50} values as obtained from DPPH and FRAP experiments were significantly (p{<=}0.05) lower in irradiated fruits compared to control.

  19. SpvC is a Salmonella effector with phosphothreonine lyase activity on host mitogen-activated protein kinases

    OpenAIRE

    Mazurkiewicz, Piotr; Thomas, Jerry; Thompson, Jessica A.; Liu, Mei; Arbibe, Laurence; Sansonetti, Philippe; Holden, David W.

    2008-01-01

    SpvC is encoded by the Salmonella virulence plasmid. We have investigated the biochemical function of SpvC and the mechanism by which it is secreted by bacteria and translocated into infected macrophages. We constructed a strain carrying a deletion in spvC and showed that the strain is attenuated for systemic virulence in mice. SpvC can be secreted in vitro by either the SPI-1 or SPI-2 type III secretion systems. Cell biological and genetic experiments showed that translocation of the protein...

  20. MalY of Escherichia coli is an enzyme with the activity of a beta C-S lyase (cystathionase).

    OpenAIRE

    Zdych, E; Peist, R; Reidl, J.; Boos, W

    1995-01-01

    The Escherichia coli maltose system consists of a number of genes whose products are involved in the uptake and metabolism of maltose and maltodextrins. MalT is the central positive gene activator of the regulon and is, together with the cyclic AMP-catabolite gene activator protein system, necessary for the expression of the maltose genes. Expression of malY, a MalT-independent gene, leads to the repression of all MalT-dependent genes. We have purified MalY to homogeneity and found it to be a...

  1. Fatty acid hydroperoxide lyase : a plant cytochrome P450 enzyme involved in wound healing and pest resistance

    OpenAIRE

    Vliegenthart, J. F. G.; Noordermeer, M.A.; Veldink, G.A.

    2001-01-01

    Plants continuously have to defend themselves against life-threatening events such as drought, mechanical damage, temperature stress, and potential pathogens. Nowadays, more and more similarities between the defense mechanism of plants and that of animals are being discovered. In both cases, the lipoxygenase pathway plays an important role. In plants, products of this pathway are involved in wound healing, pest resistance, and signaling, or they have antimicrobial and antifungal activity. The...

  2. Enzymatic modification of bacterial exopolysaccharides; xanthan lyase as a tool for structural and functional modification of xanthan

    OpenAIRE

    Ruijssenaars, H.J.

    2001-01-01

    Bacterial extracellular polysaccharides (EPSs) can be applied, e.g., in foods, as a thickener or stabilizer. The functional properties that make a polysaccharide suitable for such applications are largely determined by the primary structure, i.e., the sugar composition, the linkage types between the sugar units, and the presence of side chains and non-sugar substituents. The aim of this research was to obtain EPS-modifying enzymes that could be used as tools both for studying structure-functi...

  3. Gamma irradiation induced enhancement of phenylalanine ammonia-lyase (PAL) and antioxidant activity in peach (Prunus persica Bausch, Cv. Elberta)

    International Nuclear Information System (INIS)

    Effect of medium dose gamma irradiation on PAL and antioxidant activity of peach fruit was investigated. Peach fruit after harvest at commercial maturity was irradiated in the dose range 1.0-2.0 kGy, stored under refrigerated conditions (3±1 oC, RH 80%) and evaluated at intervals of 7 days. The antioxidant activity as determined by DPPH and FRAP methods revealed significant (p≤0.05) increase particularly in the dose range 1.6-2.0 kGy. During storage, maximum increase in both PAL and antioxidant activity was observed after 21 days. Positive correlation (r=0.75) existed between antioxidant activity and total phenols. EC50 values as obtained from DPPH and FRAP experiments were significantly (p≤0.05) lower in irradiated fruits compared to control.

  4. Myosin-cross-reactive antigen (MCRA) protein from Bifidobacterium breve is a FAD-dependent fatty acid hydratase which has a function in stress protection

    LENUS (Irish Health Repository)

    Rosberg-Cody, Eva

    2011-02-17

    Abstract Background The aim of this study was to determine the catalytic activity and physiological role of myosin-cross-reactive antigen (MCRA) from Bifidobacterium breve NCIMB 702258. MCRA from B. breve NCIMB 702258 was cloned, sequenced and expressed in heterologous hosts (Lactococcus and Corynebacterium) and the recombinant proteins assessed for enzymatic activity against fatty acid substrates. Results MCRA catalysed the conversion of palmitoleic, oleic and linoleic acids to the corresponding 10-hydroxy fatty acids, but shorter chain fatty acids were not used as substrates, while the presence of trans-double bonds and double bonds beyond the position C12 abolished hydratase activity. The hydroxy fatty acids produced were not metabolised further. We also found that heterologous Lactococcus and Corynebacterium expressing MCRA accumulated increasing amounts of 10-HOA and 10-HOE in the culture medium. Furthermore, the heterologous cultures exhibited less sensitivity to heat and solvent stresses compared to corresponding controls. Conclusions MCRA protein in B. breve can be classified as a FAD-containing double bond hydratase, within the carbon-oxygen lyase family, which may be catalysing the first step in conjugated linoleic acid (CLA) production, and this protein has an additional function in bacterial stress protection.

  5. SwissProt search result: AK101382 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK101382 J033036E19 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 1e-69 ...

  6. SwissProt search result: AK103461 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103461 J033129M07 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 1e-48 ...

  7. SwissProt search result: AK242427 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242427 J080088O13 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 4e-35 ...

  8. SwissProt search result: AK067493 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK067493 J013111L08 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 1e-73 ...

  9. SwissProt search result: AK067912 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK067912 J013128F10 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 3e-12 ...

  10. SwissProt search result: AK066243 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK066243 J013050J23 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 2e-81 ...

  11. SwissProt search result: AK121476 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK121476 J023149B05 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 2e-23 ...

  12. SwissProt search result: AK104431 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK104431 006-206-H12 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 3e-75 ...

  13. SwissProt search result: AK073320 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK073320 J033029C16 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 4e-98 ...

  14. SwissProt search result: AK111801 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK111801 J013059J16 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 3e-82 ...

  15. SwissProt search result: AK104807 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK104807 001-040-E08 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 1e-27 ...

  16. SwissProt search result: AK106800 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK106800 002-116-B10 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 3e-78 ...

  17. SwissProt search result: AK067247 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK067247 J013098N22 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 2e-81 ...

  18. SwissProt search result: AK064814 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK064814 J013000E09 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 2e-20 ...

  19. SwissProt search result: AK064874 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK064874 J013000J10 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 1e-38 ...

  20. SwissProt search result: AK107270 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK107270 002-125-H11 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 1e-102 ...

  1. SwissProt search result: AK072954 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072954 J023149K09 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 1e-27 ...

  2. SwissProt search result: AK061078 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061078 006-206-E09 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 4e-15 ...

  3. SwissProt search result: AK105523 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK105523 001-127-F12 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 5e-55 ...

  4. SwissProt search result: AK099397 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK099397 J013000M18 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 3e-37 ...

  5. SwissProt search result: AK111818 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK111818 J023009B06 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 7e-82 ...

  6. SwissProt search result: AK071183 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK071183 J023086P08 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 1e-101 ...

  7. SwissProt search result: AK066477 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK066477 J013070H07 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 5e-24 ...

  8. SwissProt search result: AK106667 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK106667 002-113-H08 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 1e-21 ...

  9. SwissProt search result: AK107218 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK107218 002-125-C12 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 4e-72 ...

  10. SwissProt search result: AK069959 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK069959 J023038G19 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 2e-75 ...

  11. SwissProt search result: AK072798 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072798 J023140F24 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 8e-34 ...

  12. SwissProt search result: AK241380 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241380 J065155L15 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 4e-49 ...

  13. SwissProt search result: AK073508 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK073508 J033044O22 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 4e-15 ...

  14. SwissProt search result: AK070195 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK070195 J023043E04 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 2e-44 ...

  15. SwissProt search result: AK106123 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK106123 001-207-F01 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 2e-23 ...

  16. SwissProt search result: AK066902 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK066902 J013092B20 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 4e-26 ...

  17. SwissProt search result: AK241620 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241620 J065186H23 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 3e-12 ...

  18. SwissProt search result: AK111439 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK111439 002-183-A05 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 9e-15 ...

  19. SwissProt search result: AK064747 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK064747 002-120-E08 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 4e-55 ...

  20. SwissProt search result: AK068732 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK068732 J013159H01 (P52708) P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitrile... lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile lyase chain B] (Fragment) HNLS_SORBI 4e-37 ...

  1. Protein (Cyanobacteria): 155688 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available orismate lyase Microcystis aeruginosa PCC 9701 MYWYNGELIENERISLGIDDIGLLYGATLFTTLRIYQQSLDHPLTHWQEHLERLHSSLQVF...RWSTPDWRRIRQGAEELSLFYPVLRVTIFPDGREWIKGRFLPEDLNIRQERGIIGWVTDNPAWQRVLGEHKTGNYLTPWLASQTAQKKGAKEAILVDRVGNWLETSTGNLWGWKDNCWYSPLLTADILPGI...GRSALIGWLKKQNISLQENLWTPEFVGTLQGIAYSNSLVEIIPFNSILGWDIDINTAIYRQVLPDLQQYFSSQLENK ...

  2. Arabidopsis CDS blastp result: AK119521 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK119521 001-202-D09 At3g57050.2 cystathionine beta-lyase, chloroplast / beta-cystathionase...thionase) (Cysteine lyase) {Arabidopsis thaliana} 1e-173 ... ... / cysteine lyase (CBL) identical to SP|P53780 Cystathionine beta-lyase, chloroplast precursor (EC 4.4.1.8) (CBL) (Beta-cysta

  3. Arabidopsis CDS blastp result: AK108403 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK108403 002-142-G06 At3g57050.2 cystathionine beta-lyase, chloroplast / beta-cystathionase...thionase) (Cysteine lyase) {Arabidopsis thaliana} 5e-36 ... ... / cysteine lyase (CBL) identical to SP|P53780 Cystathionine beta-lyase, chloroplast precursor (EC 4.4.1.8) (CBL) (Beta-cysta

  4. 17-Imidazolyl, pyrazolyl, and isoxazolyl androstene derivatives. Novel steroidal inhibitors of human cytochrome C17,20-lyase (P450(17 alpha).

    Science.gov (United States)

    Ling, Y Z; Li, J S; Liu, Y; Kato, K; Klus, G T; Brodie, A

    1997-09-26

    We recently described a number of inhibitors of P450(17 alpha), the key enzyme of androgen biosynthesis. Here, we report the synthesis and activity of novel 17-imidazolyl, pyrazolyl, and isoxazolyl androstene derivatives as potential agents for the treatment of prostatic cancer. A number of 17-(4'-Imidazolyl) derivatives were prepared by condensing the corresponding 17-ketol acetate side chain with aldehyde and ammonium hydroxide. The 17 beta-(4'imidazolyl) derivatives (2a, 2e, 4a, 4c) were found to be potent inhibitors of human testicular P450(17 alpha), with greater activity than ketoconazole. The juxtaposition between the imidazole ring and the steroid D ring appears to be important in contributing inhibitory properties, Compounds having a 17 beta-(2'-imidazolyl) ring (9a, 10) or a 20 beta-(2'-imidazolyl) ring (12), instead of the 17 beta-(4'-imidazolyl) ring (2a, 4a), are weak inhibitors. Among the 17-(4'-imidazolyl) derivatives, introduction of the 17 alpha-hydroxy group (4b) and 16 alpha,17 alpha-epoxide group (2d) diminished potency (2a-->2d; lC50 66-->430 nM; 4a-->4b; lC50 58-->1200 nM), while the 16,17 double bond increased the inhibitory activity by almost three times in the 5-en-3 beta-ol inhibitors (2a-->2e; lC50 60-->24 nM). There was virtually no difference in the inhibitory activity in the 4-en-3-one inhibitors (4a-->4c; IC50 58-->50 nM). The introduction of a methyl (2b) or phenyl group (2c) on the 2'-position of 4'-imidazolyl ring caused a dramatic decrease in the potency. As to modification of the A,B rings, the 3-acetate (2f, 2g) decreased the potency almost 3-fold compared with the 3-alcohol (2e-->2f, IC50 24-->75 nM; 2a-->2g, 66-->199 nM) and the conversion from the 5-en-3 beta-ol into the 4-en-3-one hardly affected the potency. As expected, 4c was more potent than 2e for the rat p450(17 alpha). 17-(3'Pyrazolyl)-(14b) and 17-(5'-isoxazolyl)-androsta-5,16-dien-3 beta-ol (15b) were also potent inhibitors of P450(17 alpha), whereas the 17-(2'-imidazolyl) compound (9b) was one of the most potent inhibitor in this series. However, their 16-saturated counterparts (9a, 14a, 15a) were weak inhibitors. The 17 beta-(3'-isoxazolyl)- (16) and 17 beta-(5'-methyl-3'-oxazolyl)androst-5-en-3 beta-ol (18) were also inactive. The introduction of a methyl of phenyl group on the nitrogen of the pyrazolyl ring of 14b [see 14c, 14d, and 14e] also caused some loss of inhibition for P450(17 alpha). Compounds 2e, 4a, 4c, 9b, 14d, 17a, and 17b are among the most potent inhibitors of human P450(17 alpha) so far reported. PMID:9379450

  5. The level of pyruvate-formate lyase controls the shift from homolactic to mixed-acid product formation in Lactococcus lactis

    DEFF Research Database (Denmark)

    Melchiorsen, C.R.; Jokumsen, K.V.; Villadsen, John;

    2002-01-01

    promoters in L. lactis MG1363 and in the PFL-deficient strain CRM40. Strains with five different PFL levels were obtained. Variation in the PFL level markedly affected the resulting end-product formation in these strains. During growth on galactose, the flux towards mixed-acid products was to a great extent...

  6. Complementation of a phycocyanin-bilin lyase from Synechocystis sp. PCC 6803 with a nucleomorph-encoded open reading frame from the cryptophyte Guillardia theta

    OpenAIRE

    Nyalwidhe Julius; Gruenheit Nicole; Prechtl Julia; Kawach Oliver; Bolte Kathrin; Maier Uwe-G

    2008-01-01

    Abstract Background Cryptophytes are highly compartmentalized organisms, expressing a secondary minimized eukaryotic genome in the nucleomorph and its surrounding remnant cytoplasm, in addition to the cell nucleus, the mitochondrion and the plastid. Because the members of the nucleomorph-encoded proteome may contribute to essential cellular pathways, elucidating nucleomorph-encoded functions is of utmost interest. Unfortunately, cryptophytes are inaccessible for genetic transformations thus f...

  7. The Chemolithoautotroph Acidithiobacillus ferrooxidans Can Survive under Phosphate-Limiting Conditions by Expressing a C-P Lyase Operon That Allows It To Grow on Phosphonates▿ †

    OpenAIRE

    Vera, Mario; Pagliai, Fernando; Guiliani, Nicolas; Jerez, Carlos A.

    2008-01-01

    The chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans is of great importance in biomining operations. During the bioleaching of ores, microorganisms are subjected to a variety of environmental stresses and to the limitations of some nutrients, such as inorganic phosphate (Pi), which is an essential component for all living cells. Although the primary source of phosphorus for microorganisms is Pi, some bacteria are also able to metabolize Pi esters (with a C-O-P bond) and phosphon...

  8. Expression of Cystathionine β-synthase and Cystathionine γ-lyase in Human Pregnant Myometrium and Their Roles in the Control of Uterine Contractility

    OpenAIRE

    You, Xing-Ji; Xu, Chen; Lu, Jian-Qiang; Zhu, Xiao-Yan; Gao, Lu; Cui, Xiao-Rui; Li, Yuan; Gu, Hang; Ni, Xin

    2011-01-01

    Background Human uterus undergoes distinct molecular and functional changes during pregnancy and parturition. Hydrogen sulfide (H2S) has recently been shown to play a key role in the control of smooth muscle tension. The role of endogenous H2S produced locally in the control of uterine contractility during labour is unknown. Methodology/Principal Findings Human myometrium biopsies were obtained from pregnant women undergoing cesarean section at term. Immunohistochemistry analysis showed that ...

  9. Biodegradation of All Stereoisomers of the EDTA Substitute Iminodisuccinate by Agrobacterium tumefaciens BY6 Requires an Epimerase and a Stereoselective C-N Lyase

    OpenAIRE

    Cokesa, Z̆eljko; Knackmuss, Hans-Joachim; Rieger, Paul-Gerhard

    2004-01-01

    Biodegradation tests according to Organization for Economic Cooperation and Development standard 301F (manometric respirometry test) with technical iminodisuccinate (IDS) revealed ready biodegradability for all stereoisomers of IDS. The IDS-degrading strain Agrobacterium tumefaciens BY6 was isolated from activated sludge. The strain was able to grow on each IDS isomer as well as on Fe2+-, Mg2+-, and Ca2+-IDS complexes as the sole carbon, nitrogen, and energy source. In contrast, biodegradatio...

  10. Apurinic/apyrimidinic (AP) site recognition by the 5′-dRP/AP lyase in poly(ADP-ribose) polymerase-1 (PARP-1)

    OpenAIRE

    Khodyreva, S. N.; Prasad, R; Ilina, E. S.; Sukhanova, M. V.; Kutuzov, M. M.; Liu, Y.; Hou, E. W.; Wilson, S H; Lavrik, O. I.

    2010-01-01

    The capacity of human poly(ADP-ribose) polymerase-1 (PARP-1) to interact with intact apurinic/apyrimidinic (AP) sites in DNA has been demonstrated. In cell extracts, sodium borohydride reduction of the PARP-1/AP site DNA complex resulted in covalent cross-linking of PARP-1 to DNA; the identity of cross-linked PARP-1 was confirmed by mass spectrometry. Using purified human PARP-1, the specificity of PARP-1 binding to AP site-containing DNA was confirmed in competition binding experiments. PARP...

  11. HMG CoA Lyase (HL): Mutation detection and development of a bacterial expression system for screening the activity of mutant alleles from HL-deficient patients

    Energy Technology Data Exchange (ETDEWEB)

    Robert, M.F.; Ashmarina, L.; Poitier, E. [Hospital Ste-Justine, Montreal (Canada)] [and others

    1994-09-01

    HL catalyzes the last step of ketogenesis, and autosomal recessive HL deficiency in humans can cause episodes of hypoglycemia and coma. Structurally, HL is a dimer of identical 325-residue peptides which requires a reducing environment to maintain activity. We cloned the human and mouse HL cDNAs and genes and have performed mutation analysis on cells from 30 HL-deficient probands. Using SSCP and also genomic Southern analysis we have identified putative mutations on 53/60 alleles of these patients (88%). To date, we have found 20 mutations: 3 large deletions, 4 termination mutations, 5 frameshift mutations, and 8 missense mutations which we suspect to be pathogenic based on evolutionary conservation and/or our previous studies on purified HL protein. We have also identified 3 polymorphic variants. In order to directly test the activity of the missense mutations, we established a pGEX-based system, using a glutathione S transferase (GST)-HL fusion protein. Expressed wild-type GST-HL was insoluble. We previously located a reactive Cys at the C-terminus of chicken HL which is conserved in human HL. We produced a mutant HL peptide, C323S, which replaced Cys323 with Ser. Purified C323S is soluble and has similar kinetics to wild-type HL. C323S-containing GST-HL is soluble and enzymatically active. We are cloning and expressing the 8 missense mutations.

  12. Carbon, oxygen, strontium isotope characteristics and cause analysis of Carboniferous carbonate rocks in the eastern Sichuan Basin%川东地区石炭系碳酸盐岩碳、氧、锶同位素特征及其成因分析

    Institute of Scientific and Technical Information of China (English)

    王坤; 李伟; 陆进; 张朝军

    2011-01-01

    利用微量元素和碳、氧同位素特征对样品有效性作出了检验.在证明样品未受明显蚀变的前提下对川东地区石炭系碳酸盐岩碳、氧、锶同位素进行了分析.在层序地层格架中对比不同体系域、不同岩性的碳、氧同位素特征,87Sr/86Sr比值特征,Z值与古温度特征,分析了碳酸盐岩的成岩环境.低位体系域以膏盐湖及萨巴哈环境为主,炎热干旱,陆源淡水对成岩作用影响有限,去膏化、去白云岩化作用导致次生灰岩和“鸡笼铁丝”构造的发育.海侵体系域以半局限—局限陆棚环境为主,锶同位素特征表明陆源淡水对成岩作用影响增强,广泛沉积白云岩.高位体系域仍以陆棚环境为主,沉积海相灰岩为主,幔源锶含量增加,表明海平面上升,陆源锶的注入明显降低.白云岩碳、氧、锶同位素值差异较大,主要存在4种沉积环境:(1)Z值较低的淡水沉积环境;(2)海相环境下的中—浅埋藏环境;(3)海相环境,温度较低的蒸发潟湖环境;(4)温度最高的高温环境.结合87Sr/86Sr比值特征与岩石学特征相,认为川东地区主要存在淡水、埋藏、准同生、热液等4种白云岩化作用.%The validity of the samples was tested using trace elements and carbon and oxygen isotopes. Carbon, O and Sr isotope data of least altered Carboniferous carbonate rock in the eastern Sichuan Basin were processed. The C, O, and 87Sr/86Sr isotope characteristics, and paleosalinity and paleotemperature of different stratigraphic framework and different lithology were compared, aiming at better constraints on the diagenetic environment of carbonate rocks. The results suggest that it is mainly gypsic saline lake and sabkha environment when lowstand system tract (LST) deposited. The climate was hot and dry, terrestrial source fresh water was limited on impacting the diagenesis, anti-gypsumization and anti-dolomitization cause secondary limestone, with "wire cages" structures developed. When transgressive system tract (TST) deposited, it was mainly semi-limitated or limitated shelf environment. 87Sr/86Sr value suggests that the impact on diagenesis from terrestrial source freshwater was enhanced, with dolomite well developed. It was mainly shelf environment when highstand system tract (HST) deposited, sediments were marine limestone, with mantle source Sr increased, indicating sea level rise, with significantly reduced terrestrial source Sr. Dolomite C, O, and Sr isotopes show big differences indicating four kinds of sedimentary environments: (1) freshwater sedimentary environment with low salinity; (2) intermediate-shallow burial marine environment; (3) marine and low temperature evaporation lagoon environment; (4) high-temperature environment with the highest temperature. Combined with 87Sr/86Sr and petrological characteristics, it is concluded that there are four kinds of dolomitizations: freshwater, buried, penecon-temporaneous, and hydrothermal dolomitizations in the eastern Sichuan Basin.

  13. Carbon, Oxygen Isotope and Trace element Characteristics of Carbonate Rocks in Changxin-Feixianguan Formation of Puguang Gas Pool and Its Palaeoenvironment Significance%普光气藏长兴-飞仙关组碳酸盐岩C、O同位素、微量元素分析及古环境意义

    Institute of Scientific and Technical Information of China (English)

    徐立恒; 陈践发; 李玲; 马广宇; 刘钰丹

    2009-01-01

    以普光气藏普光2、6井为研究对象,取其长兴-飞仙关组116个白云岩样品,分析C、O同位素及微量元素特征,判断古海水盐度,并与沉积特征相结合,研究其沉积环境.分析得δ13C与盐度Z呈正相关关系.普光2井飞仙关组一段、二段-飞仙关组三段,盐度及δ13C、Sr、P、Ti、Mn值呈逐渐增大的趋势,沉积相经边缘浅滩-潮坪的演化进程.飞仙关组一段、二段时期,其δ13C及Sr、P、Ti、Mn值较低、δ13C波动小,反映海平面变化不大,且遭到淡水的冲淋,导致盐度降低;飞仙关组三段时期,处于潮坪环境,气候干燥炎热,淡水不断地被大量蒸发,其结果导致盐度增大,δ13C及Sr、P、Ti、Mn值增高,在此期间海平面变化较明显,造成δ13C波动较大.碳同位素值在长兴组末期发生突降,其原因可能是在二叠纪末,大量狭盐生物死亡而造成的.

  14. 西藏申扎地区下二叠统昂杰组C-O同位素地球化学特征%Carbon-oxygen isotope and stratigraphic study of the Early Permian Angjie For-mation in Xainza area, Tibet

    Institute of Scientific and Technical Information of China (English)

    安显银; 张予杰; 朱同兴; 张以春

    2015-01-01

    根据岩石地层及生物地层研究,西藏申扎地区下二叠统昂杰组可分为下段的砂质灰岩、微晶灰岩、生物碎屑灰岩段和上段的灰黑色薄层状泥岩、页岩夹钙质粉细砂岩段,时代为早二叠世亚丁斯克期—空谷期。系统采集了24件昂杰组灰岩段C-O同位素样品,测试结果表明,昂杰组样品δ13C最大值为6.312‰,最小值为4.032‰,平均值为5.794‰;δ18O最大值为-1.785‰,最小值为-7.476‰,平均值为-4.494‰。无机碳同位素的变化存在一次缓慢的正向偏移,这与早二叠世中晚期冈瓦纳北缘冰川作用及陆生植物大规模繁衍有很大的相关性。%The Permian Angjie Formation is deposited in Xainza area, Tibet. The upper part is composed of sandstone and mudstone, whereas the lower part is sandstone intercalated with limestone. The age of the Angjie Formation is considered to be from Artinskian to Kungurian. 24 carbonate samples were collected in accordance with the carbon and oxygen isotope analysis, and the dating results yielded the maximum value of 6.312‰, the minimum value of 4.032‰, and the average carbon isotope value of 5.794‰. The maxi⁃mum value of oxygen isotope is-1.785‰, the minimum value is-7.476‰, and the average value is-4.494‰. Theδ13C has a posi⁃tive shift, which is obvious and slow. It might have been influenced by the glaciation of the northern margin of Gondwana in the middle-late Early Permian period and the large-scale terrestrial plant reproduction.

  15. GenBank blastx search result: AK287650 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287650 J065092E02 AY187726.1 AY187726 Spirulina platensis phycocyanin ... alpha subunit phycocyano ... bilin lyase (cpcE), phycocyanin ... alpha subunit phycocyanobilin lyase (cpcF), and DN ...

  16. Main: 1N8P [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available cule: Cystathionine Gamma-Lyase; Chain: A, B, C, D; Synonym: Gamma-Cystathionase; Engineered: Yes Lyase 4.4....Length: 393 AA, Molecular weight: 42411 Da TLQESDKFATKAIHAGEHVDVHGSVIEPISLSTTFKQSSPANPIGTYEYSRSQNPNRENLERAVA...PTNPTLKVTDIQKVADLIKKHAAGQDVILVVDNTFLSPYISNPLNFGADIVVHSATKYINGHSDVVLGVLATNNKPLYERLQFLQNAIGAIPSPFDAWLTHRGLKTLH

  17. Sequence Classification: 400116 [

    Lifescience Database Archive (English)

    Full Text Available BLE ENDONUCLEASE III NTH (DNA-(APURINIC OR APYRIMIDINIC SITE)LYASE) (AP LYASE) (AP ENDONUCLEASE CLASS I) (ENDODEOXYRIBONUCLEASE (APU...RINIC OR APYRIMIDINIC)) (DEOXYRIBONUCLEASE (APURINIC OR APYRIMIDINIC)) || http://www.ncbi.nlm.nih.gov/protein/57117142 ...

  18. Urease Inhibitor Drug Treatment for Urea Cycle Disorders

    Science.gov (United States)

    2016-01-28

    Ornithine Transcarbamylase Deficiency; Argininosuccinate Synthetase Deficiency (Citrullinemia); Argininosuccinic Acid Lyase Deficiency (Argininosuccinic Aciduria); Carbamyl-Phosphate Synthase I Deficiency

  19. Disease: H00197 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available osuccinate lyase [HSA:158] [KO:K01756] Succinyladenosine [CPD:C03794] Succinylaminoimidazole carboxamide ribotid...e metabolism hsa00250(158) Alanine, aspartate and glutamate metabolism ADSL; adenyl...H00197 Adenylosuccinate lyase deficiency Adenylosuccinate lyase deficiency is an au... Spiegel EK, Colman RF, Patterson D Adenylosuccinate lyase deficiency. Mol Genet ...Metab 89:19-31 (2006) PMID:11392513 Ciardo F, Salerno C, Curatolo P Neurologic aspects of adenylosuccinate l

  20. UniProt search blastx result: AK287750 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287750 J065155L15 P52708|HNLS_SORBI P-(S)-hydroxymandelonitrile lyase precursor (EC 4.1.2.11) (Hydroxynitr...ile lyase) (HNL) [Contains: P-(S)-hydroxymandelonitrile lyase chain A; P-(S)-hydroxymandelonitrile

  1. Disease: H00179 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available H00179 3-Hydroxy-3-methylglutaryl-CoA lyase deficiency 3-Hydroxy-3-methylglutaryl-CoA lyase (HL) ... Dincer A, Gunduz E, Ficicioglu C, Kocer N, Aydin A MRI ... and MRS in HMG-CoA lyase deficiency. Pediatr Neuro ...

  2. A novel CYP17A1 deletion causes a functional knockout of the steroid enzyme 17-hydroxylase and 17,20-lyase in a Turkish family and illustrates the precise role of the CYP17A1 gene.

    Science.gov (United States)

    Camats, Núria; Üstyol, Ala; Atabek, Mehmet Emre; Dick, Bernhard; Flück, Christa E

    2015-10-01

    A novel homozygous long-range deletion of the CYP17A1 gene abolished protein expression and caused the severest form of 17-hydroxylase deficiency in one kindred of a Turkish family. The affected subjects presented with 46,XY sex reversal and 46,XX lack of pubertal development as well as severe hypertension. PMID:26509008

  3. 褐藻胶裂解酶产生菌的分离鉴定及产酶发酵优化%Identification of an Alginate Lyase Producing Strain Halomonas sp.WF6 and Fermentation Optimization

    Institute of Scientific and Technical Information of China (English)

    李恒; 朱思婷; 刘旭梅; 龚劲松; 蒋敏; 许正宏; 史劲松

    2014-01-01

    对一株从腐烂海带中筛选得到的产褐藻胶裂解酶的菌株进行鉴定,并对其产酶条件进行发酵优化.经形态学、生理生化特征和分子生物学鉴定,将其鉴定为盐单胞菌属,并命名为Halomonas sp.WF6.通过在摇瓶培养水平上进行单因素和多因素正交试验,确定褐藻胶裂解酶产生菌WF6的最适产酶培养基为:褐藻酸钠6.0 g/L,蛋白胨5.0 g/L,酵母粉2.5 g/L,NaC1 30 g/L,K+5 mmol/L.进而采用最适培养基进行产酶条件的优化,优化后的发酵产酶条件为:初始pH8.0,培养温度25℃,接种量为2%,摇瓶装液量30 ml/250 ml,培养时间39 h.优化后的褐藻胶裂解酶酶活达117.66 U/ml,是优化前的2.1倍.该酶对褐藻酸钠的酶解产物主要由聚合度为二和三的褐藻寡糖组成.

  4. Cloning and Sequence Analysis of Phenylalanine Ammonia-lyase(PAL)Gene from Fagopyrum esculentum%甜荞苯丙氨酸解氨酶基因PAL的克隆及序列分析

    Institute of Scientific and Technical Information of China (English)

    李成磊; 蒙华; 张晓伟; 陈惠; 邵继荣; 吴琦

    2011-01-01

    利用RT-PCR技术,首次从甜荞(Fagopyrum esculentum)中克隆得到苯丙氨酸解氨酶基因(PAL)的cDNA ORF序列,命名为FePAL.该序列长2169bp,编码722个氨基酸,与其他植物PAL基因同源性较高,为80%~97%,其推导的氨基酸序列含有PAL酶活性中心特征序列GTITASGDLVPLSYIA和多个脱氨基、催化活性位点.系统发育树表明,甜养PAL基因与苦荞PAL基因聚类关系最近.

  5. Cloning and expression of Bacillus subtilis alkaline pectate lyase gene in Escherichia coli%Bacillus subtilis碱性果胶酶基因在大肠杆菌中的克隆和表达

    Institute of Scientific and Technical Information of China (English)

    肖静; 路福平; 杨晓杰; 张东向

    2011-01-01

    针对实验室筛选获得的Bacillus subtilis,设计2对引物扩增出编码碱性果胶酶基因pelG1和pelG2,酶切后分别连接到大肠杆菌分泌表达载体pET-22b(+)多克隆位点上得到重组载体,在BL21( DE3)中表达并进行了分析.

  6. 银杏叶苯丙氨酸解氨酶分离纯化条件的研究%Study on the Conditions of Isolating and Purifying L-phenylalanine Ammonia-lyase (PAL) in Ginkgo biloba Leaves

    Institute of Scientific and Technical Information of China (English)

    刘卫红; 王燕; 杜何为; 程水源

    2004-01-01

    对银杏(Ginkgo biloba)叶中苯丙氨酸解氨酶(PAL)的分离纯化条件进行了研究.离子交换条件优化的研究结果是最适洗脱缓冲液的pH8.9,最佳洗脱离子浓度(NaCl)为0.3 mol*L-1.经过一系列的分离纯化,蛋白质得率为0.35%,酶的得率为1.17%,纯化倍数为3.32.

  7. Cloning and expression analysis about fruit coloring related Phenylalanine Ammonia-lyase gene fragment from Pingguoli%苹果梨果实着色相关PAL基因片段克隆及表达分析

    Institute of Scientific and Technical Information of China (English)

    孙百灵; 王旭; 曲柏宏

    2012-01-01

    Make the Pingguoli peel as the material,coloring related PAL(fragment) was cloned,accession number:JN120855.The results showed that the fragment includes 407 basepair,the consistency between fragment and Yali pear PAL gene is 97%,and the amino acid sequence homology is the highest,restriction analysis showed that there was a commonly used restriction enzyme site for AccI.The semi-quantitative RT-PCR technique showed that as fruit mature,expression of PAL gene is increasing constantly about bagged fruit and no bagged fruit,but expression of the bagged fruit is obviously higher than no bagged fruit in the fruit mature.%以苹果梨果皮为试材,克隆了苹果梨果实着色相关PAL基因片段,GenBank登录号为JN120855。结果表明:该基因片段长度为407bp,与鸭梨PAL基因序列一致性达97%,与鸭梨氨基酸序列同源性最高,酶切位点分析表明,该PAL基因序列含有常用的限制性内切酶AccI的识别位点。半定量RT-PCR分析表明,随着果实的成熟,套袋果与未套袋果PAL基因的表达量都不断增加,但套袋果在果实成熟期的表达量明显高于未套袋果。

  8. Characterisation of bovine testicular hyaluronidase and a hyaluronate lyase from Streptococcus agalactiae. Investigations on the effect of pH on hyaluronan degradation and preclinical studies on the adjuvant administration of the enzymes in cancer chemotherapy

    OpenAIRE

    Hoechstetter, Julia

    2005-01-01

    Pharmaceutical preparations of bovine testicular hyaluronidase (BTH) have been therapeutically applied in several medical fields for many years. In the context with the risk of BSE, companies stopped the production of BTH preparations resulting in a shortage of this drug, which is the only highly effective antidote in the treatment of extravasations of vinca alkaloids. To find a substitute for the poorly characterised BTH preparations, two BTH preparations (Neopermease®; Hylase® �Dessau�)...

  9. AcEST: DK957009 [AcEST

    Lifescience Database Archive (English)

    Full Text Available 0. 5' end sequence. DK957009 CL1105Contig1 Show DK957009 Clone id TST39A01NGRL0027_D20 Library TST39 Length 552 Definition Adian...Q9SS45|PAL4_ARATH Phenylalanine ammonia-lyase 4 OS=Arabidopsi... 77 7e-14 sp|P25872|PAL1_TOBAC Phenylalanine ammonia...a-lyase 2 OS=Arabidopsi... 74 4e-13 sp|P45733|PAL3_TOBAC Phenylalanine ammonia-lyase OS=Nicotian...-lyase 3 OS=Arabidopsi... 73 8e-13 sp|P35513|PAL2_TOBAC Phenylalanine ammonia-lyase OS=Nicotian... sp|P52777|PALY_PINTA Phenylalanine ammonia-lyase OS=Pinus taeda Align length 91 Scor

  10. Conversion of Methionine to Thiols by Lactococci, Lactobacilli, and Brevibacteria†

    OpenAIRE

    Dias, Benjamin; Weimer, Bart

    1998-01-01

    Methanethiol has been strongly associated with desirable Cheddar cheese flavor and can be formed from the degradation of methionine (Met) via a number of microbial enzymes. Methionine γ-lyase is thought to play a major role in the catabolism of Met and generation of methanethiol in several species of bacteria. Other enzymes that have been reported to be capable of producing methanethiol from Met in lactic acid bacteria include cystathionine β-lyase and cystathionine γ-lyase. The objective of ...

  11. Genome-Wide Search for Eliminylating Domains Reveals Novel Function for BLES03-Like Proteins

    OpenAIRE

    Khater, Shradha; Mohanty, Debasisa

    2014-01-01

    Bacterial phosphothreonine lyases catalyze a novel posttranslational modification involving formation of dehydrobutyrine/dehyroalanine by β elimination of the phosphate group of phosphothreonine or phosphoserine residues in their substrate proteins. Though there is experimental evidence for presence of dehydro amino acids in human proteins, no eukaryotic homologs of these lyases have been identified as of today. A comprehensive genome-wide search for identifying phosphothreonine lyase homolog...

  12. NCBI nr-aa BLAST: CBRC-EEUR-01-1199 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-EEUR-01-1199 ref|NP_484576.1| phycocyanobilin lyase alpha subunit [Nostoc sp. PCC 7120] sp| ... 25|CPCE_ANASP Phycocyanobilin lyase subunit alpha (Phycocyanin ... operon protein cpcE) pir||E29674 probable phycocya ...

  13. Anticoagulant substance released from human lung mast cells by stimulation with anti-IgE or Ca-ionophore A23187.

    OpenAIRE

    Hayashi, Hisatomo; TSUDA, Takashi; Tsurumi, Naokazu; Takai,Yutaka; Maeda, Masanori; Takahashi,Kiyoshi; Kimura,Ikuro

    1987-01-01

    A significant amount of anticoagulant substance was released along with histamine, when human lung mast cells were stimulated with anti-IgE and Ca-ionophore A23187. Its activity was lost by heparinase, not by chondroitin-ABC lyase or chondroitin-AC lyase, and also inhibited by Polybrene, suggesting it would be heparin.

  14. Anticoagulant substance released from human lung mast cells by stimulation with anti-IgE or Ca-ionophore A23187.

    Directory of Open Access Journals (Sweden)

    Hayashi,Hisatomo

    1987-04-01

    Full Text Available A significant amount of anticoagulant substance was released along with histamine, when human lung mast cells were stimulated with anti-IgE and Ca-ionophore A23187. Its activity was lost by heparinase, not by chondroitin-ABC lyase or chondroitin-AC lyase, and also inhibited by Polybrene, suggesting it would be heparin.

  15. GenBank blastx search result: AK240729 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK240729 J043030H19 AJ421809.1 PFL421809 Pseudomonas fluorescens hutH-2 gene (partial) for putat ... ive histidine ... ammonia lyase, hutH-1 gene for putative histidine ... ammonia lyase and hutT gene (partial) for putative histidine ... transport protein. BCT 2e-27 1 ...

  16. GenBank blastx search result: AK061959 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061959 001-042-G01 AJ421809.1 Pseudomonas fluorescens hutH-2 gene (partial) for putative histidine ... dine ammonia lyase, hutH-1 gene for putative histidine ... ammonia lyase and hutT gene (partial) for putative ... histidine ... transport protein.|BCT BCT 2e-17 +1 ...

  17. 78 FR 48692 - Government-Owned Inventions; Availability for Licensing

    Science.gov (United States)

    2013-08-09

    ... tumors, Alzheimer's, Parkinson's, and multiple sclerosis. Competitive Advantages: Quickly achieves a high... applications. Rabbit Antibody to Mouse Sphingosine-1-phosphate (S1P) Lyase Description of Technology: The... at NIH injected rabbits with the C-terminal peptide of the mouse S1P lyase--551-...

  18. Oxygen neutronization in accreting white dwarfs

    OpenAIRE

    Bravo Guil, Eduardo; Isern Vilaboy, Jordi; Labay, Javier; Canal Masgoret, Ramon

    1983-01-01

    Solid carbon-oxygen white dwarf cores have been shown to be likely initial configurations for collapse to neutron star densities. Solidification seems to entail carbon/oxygen separation, with oxygen settling at the star's center and carbon being confined to more external, lower-density layers. Electron captures on 16O are then the triggering mechanism for collapse. The authors elucidate the outcome of the complete reaction network started by those captures and derive simple expressions for ac...

  19. 芽孢杆菌(Bacillus subtilis No.16A)苎麻脱胶聚半乳糖醛酸裂解酶的纯化及酶学性质%Purification and properties of Polygalacturonic acid lyase from ramie degumming Bacillus subtilis No.16A

    Institute of Scientific and Technical Information of China (English)

    颜涛; 苏静; 李德舜

    2006-01-01

    通过丙酮沉淀,阳离子交换层析,凝胶过滤,从苎麻脱胶菌Bacillus subtilis No.16A发酵液中纯化了聚半乳糖醛酸酶(Pgase).结果表明该酶分子量为30.2ku,最适作用pH为9.0,最适作用温度为50℃,在55℃以下、中性pH(6.0~8.0)范围内稳定, Ca2+、Mg2+对该酶有激活作用, Cu2+、Mn2+、Co2+、Hg2+有抑制作用,酶解产物在235 nm处有强吸收峰,说明该酶是聚半乳糖醛酸裂解酶.

  20. Human Cytochrome P450c17: Single Step Purification and Phosphorylation of Serine 258 by Protein Kinase A

    OpenAIRE

    Wang, Yue-Hao; Tee, Meng Kian; Miller, Walter L.

    2010-01-01

    Cytochrome P450c17 (P450c17) is the single microsomal enzyme that catalyzes steroid 17α-hydroxylase and 17,20 lyase activities. The ratio of lyase to hydroxylase activity of human P450c17 determines whether steroidogenesis leads to the synthesis of cortisol or sex steroids. This ratio is regulated posttranslationally by factors that influence the efficiency of electron transfer from P450 oxidoreductase to P450c17. One factor favoring more efficient electron transfer and 17,20 lyase activity i...