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Sample records for lyases revealed conserved

  1. 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 fro...... structural homology to non-catalytic domains from other carbohydrate active enzymes.......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...

  2. 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

    2010-12-01

    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 CX(2)CH (by CcsA2), CX(2)CK (by NrfI) and CX(15)CH (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.

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

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    Hongnan Cao

    2016-05-01

    Full Text Available 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-morpholinoethanesulfonic 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.

  4. 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.

  5. Structural Snapshots of an Engineered Cystathionine-γ-lyase Reveal the Critical Role of Electrostatic Interactions in the Active Site

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Wupeng; Stone, Everett; Zhang, Yan Jessie

    2017-02-01

    Enzyme therapeutics that can degrade l-methionine (l-Met) are of great interest as numerous malignancies are exquisitely sensitive to l-Met depletion. To exhaust the pool of methionine in human serum, we previously engineered an l-Met-degrading enzyme based on the human cystathionine-γ-lyase scaffold (hCGL-NLV) to circumvent immunogenicity and stability issues observed in the preclinical application of bacterially derived methionine-γ-lyases. To gain further insights into the structure–activity relationships governing the chemistry of the hCGL-NLV lead molecule, we undertook a biophysical characterization campaign that captured crystal structures (2.2 Å) of hCGL-NLV with distinct reaction intermediates, including internal aldimine, substrate-bound, gem-diamine, and external aldimine forms. Curiously, an alternate form of hCGL-NLV that crystallized under higher-salt conditions revealed a locally unfolded active site, correlating with inhibition of activity as a function of ionic strength. Subsequent mutational and kinetic experiments pinpointed that a salt bridge between the phosphate of the essential cofactor pyridoxal 5'-phosphate (PLP) and residue R62 plays an important role in catalyzing β- and γ-eliminations. Our study suggests that solvent ions such as NaCl disrupt electrostatic interactions between R62 and PLP, decreasing catalytic efficiency.

  6. Kinetic characterization of the human O-phosphoethanolamine phospho-lyase reveals unconventional features of this specialized pyridoxal phosphate-dependent lyase.

    Science.gov (United States)

    Schiroli, Davide; Ronda, Luca; Peracchi, Alessio

    2015-01-01

    Human O-phosphoethanolamine (PEA) phospho-lyase is a pyridoxal 5'-phosphate (PLP) dependent enzyme that catalyzes the degradation of PEA to acetaldehyde, phosphate and ammonia. Physiologically, the enzyme is involved in phospholipid metabolism and is expressed mainly in the brain, where its expression becomes dysregulated in the course of neuropsychiatric diseases. Mechanistically, PEA phospho-lyase shows a remarkable substrate selectivity, strongly discriminating against other amino compounds structurally similar to PEA. Herein, we studied the enzyme under steady-state and pre-steady-state conditions, analyzing its kinetic features and getting insights into the factors that contribute to its specificity. The pH dependence of the catalytic parameters and the pattern of inhibition by the product phosphate and by other anionic compounds suggest that the active site of PEA phospho-lyase is optimized to bind dianionic groups and that this is a prime determinant of the enzyme specificity towards PEA. Single- and multiple-wavelength stopped-flow studies show that upon reaction with PEA the main absorption band of PLP (λmax  = 412 nm) rapidly blue-shifts to ~ 400 nm. Further experiments suggest that the newly formed and rather stable 400-nm species most probably represents a Michaelis (noncovalent) complex of PEA with the enzyme. Accumulation of such an early intermediate during turnover is unusual for PLP-dependent enzymes and appears counterproductive for absolute catalytic performance, but it can contribute to optimize substrate specificity. PEA phospho-lyase may hence represent a case of selectivity-efficiency tradeoff. In turn, the strict specificity of the enzyme seems important to prevent inactivation by other amines, structurally resembling PEA, that occur in the brain. © 2014 FEBS.

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

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    Taraka R. Donti

    2016-09-01

    Full Text Available 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.

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

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    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.

  9. Pseudomonas aeruginosa 4-amino-4-deoxychorismate lyase: spatial conservation of an active site tyrosine and classification of two types of enzyme.

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    Patrick E F O'Rourke

    Full Text Available 4-Amino-4-deoxychorismate lyase (PabC catalyzes the formation of 4-aminobenzoate, and release of pyruvate, during folate biosynthesis. This is an essential activity for the growth of gram-negative bacteria, including important pathogens such as Pseudomonas aeruginosa. A high-resolution (1.75 Å crystal structure of PabC from P. aeruginosa has been determined, and sequence-structure comparisons with orthologous structures are reported. Residues around the pyridoxal 5'-phosphate cofactor are highly conserved adding support to aspects of a mechanism generic for enzymes carrying that cofactor. However, we suggest that PabC can be classified into two groups depending upon whether an active site and structurally conserved tyrosine is provided from the polypeptide that mainly forms an active site or from the partner subunit in the dimeric assembly. We considered that the conserved tyrosine might indicate a direct role in catalysis: that of providing a proton to reduce the olefin moiety of substrate as pyruvate is released. A threonine had previously been suggested to fulfill such a role prior to our observation of the structurally conserved tyrosine. We have been unable to elucidate an experimentally determined structure of PabC in complex with ligands to inform on mechanism and substrate specificity. Therefore we constructed a computational model of the catalytic intermediate docked into the enzyme active site. The model suggests that the conserved tyrosine helps to create a hydrophobic wall on one side of the active site that provides important interactions to bind the catalytic intermediate. However, this residue does not appear to participate in interactions with the C atom that undergoes an sp(2 to sp(3 conversion as pyruvate is produced. The model and our comparisons rather support the hypothesis that an active site threonine hydroxyl contributes a proton used in the reduction of the substrate methylene to pyruvate methyl in the final stage of

  10. Characterisation of the willow phenylalanine ammonia-lyase (PAL) gene family reveals expression differences compared with poplar.

    Science.gov (United States)

    de Jong, Femke; Hanley, Steven J; Beale, Michael H; Karp, Angela

    2015-09-01

    Willow is an important biomass crop for the bioenergy industry, and therefore optimal growth with minimal effects of biotic and abiotic stress is essential. The phenylpropanoid pathway is responsible for the biosynthesis of not only lignin but also of flavonoids, condensed tannins, benzenoids and phenolic glycosides which all have a role in protecting the plant against biotic and abiotic stress. All products of the phenylpropanoid pathway are important for the healthy growth of short rotation cropping species such as willow. However, the phenylpropanoid pathway in willow remains largely uncharacterised. In the current study we identified and characterised five willow phenylalanine ammonia-lyase (PAL) genes, which encode enzymes that catalyse the deamination of l-phenylalanine to form trans-cinnamic acid, the entry point into the phenylpropanoid pathway. Willow PAL1, PAL2, PAL3 and PAL4 genes were orthologous to the poplar genes. However no orthologue of PAL5 appears to be present in willow. Moreover, two tandemly repeated PAL2 orthologues were identified in a single contig. Willow PALs show similar sub-cellular localisation to the poplar genes. However, the enzyme kinetics and gene expression of the willow PAL genes differed slightly, with willow PAL2 being more widely expressed than its poplar orthologues implying a wider role for PALs in the production of flavonoids, condensed tannins, benzenoids, and phenolic glycosides, in willow.

  11. Characterisation of the willow phenylalanine ammonia-lyase (PAL) gene family reveals expression differences compared with poplar

    Science.gov (United States)

    de Jong, Femke; Hanley, Steven J.; Beale, Michael H.; Karp, Angela

    2015-01-01

    Willow is an important biomass crop for the bioenergy industry, and therefore optimal growth with minimal effects of biotic and abiotic stress is essential. The phenylpropanoid pathway is responsible for the biosynthesis of not only lignin but also of flavonoids, condensed tannins, benzenoids and phenolic glycosides which all have a role in protecting the plant against biotic and abiotic stress. All products of the phenylpropanoid pathway are important for the healthy growth of short rotation cropping species such as willow. However, the phenylpropanoid pathway in willow remains largely uncharacterised. In the current study we identified and characterised five willow phenylalanine ammonia-lyase (PAL) genes, which encode enzymes that catalyse the deamination of l-phenylalanine to form trans-cinnamic acid, the entry point into the phenylpropanoid pathway. Willow PAL1, PAL2, PAL3 and PAL4 genes were orthologous to the poplar genes. However no orthologue of PAL5 appears to be present in willow. Moreover, two tandemly repeated PAL2 orthologues were identified in a single contig. Willow PALs show similar sub-cellular localisation to the poplar genes. However, the enzyme kinetics and gene expression of the willow PAL genes differed slightly, with willow PAL2 being more widely expressed than its poplar orthologues implying a wider role for PALs in the production of flavonoids, condensed tannins, benzenoids, and phenolic glycosides, in willow. PMID:26070140

  12. Metabolic profiling reveals sphingosine-1-phosphate kinase 2 and lyase as key targets of (phyto- estrogen action in the breast cancer cell line MCF-7 and not in MCF-12A.

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    Nadja Engel

    Full Text Available To search for new targets of anticancer therapies using phytoestrogens we performed comparative metabolic profiling of the breast cancer cell line MCF-7 and the non-tumorigenic breast cell line MCF-12A. Application of gas chromatography-mass spectrometry (GC-MS revealed significant differences in the metabolic levels after exposure with 17ß-estradiol, genistein or a composition of phytoestrogens within a native root flax extract. We observed the metabolites 3-(4-hydroxyphenyl-lactic acid, cis-aconitic acid, 11-beta-hydroxy-progesterone, chenodeoxycholic acid and triacontanoic acid with elevated levels due to estrogen action. Particularly highlighted were metabolites of the sphingolipid metabolism. Sphingosine and its dihydro derivate as well as ethanolaminephosphate were significantly altered after exposure with 1 nM 17ß-estradiol in the cell line MCF-7, while MCF-12A was not affected. Treatment with genistein and the flax extract normalized the sphingosine concentrations to the basic levels found in MCF-12A cells. We could further demonstrate that the expression levels of the sphingosine metabolizing enzymes: sphingosine-1-phosphate kinase (Sphk and lyase (S1P lyase were significantly influenced by estrogens as well as phytoestrogens. The isoform Sphk2 was overexpressed in the tumorigenic cell line MCF-7, while S1P lyase was predominantly expressed in the non-tumorigenic cell line MCF-12A. Importantly, in MCF-7 the weak S1P lyase expression could be significantly increased after exposure with 10 µM genistein and 1 µg/ml root flax extract. Here, we present, for the first time, an analysis of metabolic response of phytoestrogens to breast cancer cell lines. The contrasting regulation of sphingolipid enzymes in MCF-7 and MCF-12A render them as preferred targets for future anticancer strategies.

  13. Gnotobiotic zebrafish reveal evolutionarily conserved responses to the gut microbiota

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    Rawls, John F.; Samuel, Buck S.; Gordon, Jeffrey I.

    2004-01-01

    Animals have developed the means for supporting complex and dynamic consortia of microorganisms during their life cycle. A transcendent view of vertebrate biology therefore requires an understanding of the contributions of these indigenous microbial communities to host development and adult physiology. These contributions are most obvious in the gut, where studies of gnotobiotic mice have disclosed that the microbiota affects a wide range of biological processes, including nutrient processing and absorption, development of the mucosal immune system, angiogenesis, and epithelial renewal. The zebrafish (Danio rerio) provides an opportunity to investigate the molecular mechanisms underlying these interactions through genetic and chemical screens that take advantage of its transparency during larval and juvenile stages. Therefore, we developed methods for producing and rearing germ-free zebrafish through late juvenile stages. DNA microarray comparisons of gene expression in the digestive tracts of 6 days post fertilization germ-free, conventionalized, and conventionally raised zebrafish revealed 212 genes regulated by the microbiota, and 59 responses that are conserved in the mouse intestine, including those involved in stimulation of epithelial proliferation, promotion of nutrient metabolism, and innate immune responses. The microbial ecology of the digestive tracts of conventionally raised and conventionalized zebrafish was characterized by sequencing libraries of bacterial 16S rDNA amplicons. Colonization of germ-free zebrafish with individual members of its microbiota revealed the bacterial species specificity of selected host responses. Together, these studies establish gnotobiotic zebrafish as a useful model for dissecting the molecular foundations of host-microbial interactions in the vertebrate digestive tract. PMID:15070763

  14. Studies on pectin lyase

    NARCIS (Netherlands)

    Houdenhoven, van F.E.A.

    1975-01-01

    The pectin lyase activity in the commercial enzyme preparation Ultrazym originates from more then one type of enzyme; two of them, accounting for 95 % of the total activity, have been completely purified. As purity criteria specific activity, polyacrylamide disc gel electrophoresis and SDS

  15. Studies on pectin lyase

    NARCIS (Netherlands)

    Houdenhoven, van F.E.A.

    1975-01-01

    The pectin lyase activity in the commercial enzyme preparation Ultrazym originates from more then one type of enzyme; two of them, accounting for 95 % of the total activity, have been completely purified. As purity criteria specific activity, polyacrylamide disc gel electrophoresis and SDS electroph

  16. Comparison of protein interaction networks reveals species conservation and divergence

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    Teng Maikun

    2006-10-01

    Full Text Available Abstract Background Recent progresses in high-throughput proteomics have provided us with a first chance to characterize protein interaction networks (PINs, but also raised new challenges in interpreting the accumulating data. Results Motivated by the need of analyzing and interpreting the fast-growing data in the field of proteomics, we propose a comparative strategy to carry out global analysis of PINs. We compare two PINs by combining interaction topology and sequence similarity to identify conserved network substructures (CoNSs. Using this approach we perform twenty-one pairwise comparisons among the seven recently available PINs of E.coli, H.pylori, S.cerevisiae, C.elegans, D.melanogaster, M.musculus and H.sapiens. In spite of the incompleteness of data, PIN comparison discloses species conservation at the network level and the identified CoNSs are also functionally conserved and involve in basic cellular functions. We investigate the yeast CoNSs and find that many of them correspond to known complexes. We also find that different species harbor many conserved interaction regions that are topologically identical and these regions can constitute larger interaction regions that are topologically different but similar in framework. Based on the species-to-species difference in CoNSs, we infer potential species divergence. It seems that different species organize orthologs in similar but not necessarily the same topology to achieve similar or the same function. This attributes much to duplication and divergence of genes and their associated interactions. Finally, as the application of CoNSs, we predict 101 protein-protein interactions (PPIs, annotate 339 new protein functions and deduce 170 pairs of orthologs. Conclusion Our result demonstrates that the cross-species comparison strategy we adopt is powerful for the exploration of biological problems from the perspective of networks.

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

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    Lara-Márquez, Alicia; Zavala-Páramo, María G; López-Romero, Everardo; Calderón-Cortés, Nancy; López-Gómez, Rodolfo; Conejo-Saucedo, Ulises; Cano-Camacho, Horacio

    2011-12-09

    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. 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. The Clpnl2 gene of C. lindemuthianum shares the characteristic elements of genes coding for pectin lyases. A time-course analysis

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

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    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

  19. IMMUNOHISTOCHEMICAL APPROACH REVEALS LOCALIZATION OF CYSTATHIONINE-?-LYASE AND CYSTATHIONINE-ß-SYNTHETASE IN ETHANOL-INDUCED GASTRIC MUCOSA DAMAGE IN MICE

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    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.

  20. How Stakeholder Co-management Reproduces Conservation Conflicts: Revealing Rationality Problems in Swedish Wolf Conservation

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    Erica von Essen

    2015-01-01

    Full Text Available 'Stakeholder' has become the primary category of political actor in decision-making, not least within nature conservation. Drawing from Habermas' theory on communicative action, this article argues that there are democratic deficits to the stakeholder model that promote citizens to remain locked in predetermined, polarized positions. It contends that the stakeholder model must, hence, be scrutinized with respect to its potential role in perpetuating conservation conflicts in modernity. Using the case study of stakeholder-based game management delegations (GMDs in Sweden, our research identifies four barriers, which tie to the instrumental basis and liberal democratic legacy of the stakeholder approach: 1 strong sense of accountability; 2 overly purposive atmosphere; 3 overemphasis on decision as final outcome; and 4 perceived inability on the part of the delegates to influence science-led decision-making. The article suggests that these democratic deficits preclude the deliberation and contestation necessary to legitimate conservation policy. Indeed, stakeholder rationality causes citizens to become inert, instrumental agents who approach discussion with strategic rather than communicative rationality. We conclude that the deficits of the stakeholder model currently: 1 restrict democratic freedom for citizens; 2 engender a crisis of legitimacy of management; and 3 reproduce the conflict, which in Sweden relates to the conservation of wolves.

  1. 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.

  2. Genome-wide identification of citrus ATP-citrate lyase genes and their transcript analysis in fruits reveals their possible role in citrate utilization.

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    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.

  3. Advancing Eucalyptus Genomics: Cytogenomics Reveals Conservation of Eucalyptus Genomes

    Science.gov (United States)

    Ribeiro, Teresa; Barrela, Ricardo M.; Bergès, Hélène; Marques, Cristina; Loureiro, João; Morais-Cecílio, Leonor; Paiva, Jorge A. P.

    2016-01-01

    The genus Eucalyptus encloses several species with high ecological and economic value, being the subgenus Symphyomyrtus one of the most important. Species such as E. grandis and E. globulus are well characterized at the molecular level but knowledge regarding genome and chromosome organization is very scarce. Here we characterized and compared the karyotypes of three economically important species, E. grandis, E. globulus, and E. calmadulensis, and three with ecological relevance, E. pulverulenta, E. cornuta, and E. occidentalis, through an integrative approach including genome size estimation, fluorochrome banding, rDNA FISH, and BAC landing comprising genes involved in lignin biosynthesis. All karyotypes show a high degree of conservation with pericentromeric 35S and 5S rDNA loci in the first and third pairs, respectively. GC-rich heterochromatin was restricted to the 35S rDNA locus while the AT-rich heterochromatin pattern was species-specific. The slight differences in karyotype formulas and distribution of AT-rich heterochromatin, along with genome sizes estimations, support the idea of Eucalyptus genome evolution by local expansions of heterochromatin clusters. The unusual co-localization of both rDNA with AT-rich heterochromatin was attributed mainly to the presence of silent transposable elements in those loci. The cinnamoyl CoA reductase gene (CCR1) previously assessed to linkage group 10 (LG10) was clearly localized distally at the long arm of chromosome 9 establishing an unexpected correlation between the cytogenetic chromosome 9 and the LG10. Our work is novel and contributes to the understanding of Eucalyptus genome organization which is essential to develop successful advanced breeding strategies for this genus. PMID:27148332

  4. Advancing Eucalyptus genomics: cytogenomics reveals conservation of Eucalyptus genomes

    Directory of Open Access Journals (Sweden)

    Teresa Mousinho Resina Ribeiro

    2016-04-01

    Full Text Available The genus Eucalyptus encloses several species with high ecological and economic value, being the subgenus Symphyomyrtus one of the most important. Species such as E. grandis and E. globulus are well characterized at the molecular level but knowledge regarding genome and chromosome organization is very scarce. Here we characterized and compared the karyotypes of three economically important species, E. grandis, E. globulus and E. calmadulensis, and three with ecological relevance, E. pulverulenta, E. cornuta and E. occidentalis, through an integrative approach including genome size estimation, fluorochrome banding, rDNA FISH and BAC landing comprising genes involved in lignin biosynthesis. All karyotypes show a high degree of conservation with pericentromeric 35S and 5S rDNA loci in the first and third pairs, respectively. GC-rich heterochromatin was restricted to the 35S locus while the AT-rich het pattern was species-specific. The slight differences in karyotype formulas and distribution of AT-rich het, along with genome sizes estimations, supports the idea of Eucalyptus genome evolution by local expansions of heterochromatin clusters. The unusual co-localization of both rDNA with AT-rich het was attributed mainly to the presence of silent transposable elements in those loci. The cinnamoyl CoA reductase gene (CCR1 previously assessed to linkage group 10 (LG10 was clearly localized distally at the long arm of chromosome 9 establishing an unexpected correlation between the cytogenetic chromosome 9 and the LG10. Our work is novel and contributes to the understanding of Eucalyptus genome organization which is essential to develop successful advanced breeding strategies for this genus.

  5. Subcellular localisation of Medicago truncatula 9/13-hydroperoxide lyase reveals a new localisation pattern and activation mechanism for CYP74C enzymes

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    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

  6. 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.

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

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    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.

  8. Phycobilin:cystein-84 biliprotein lyase, a near-universal lyase for cysteine-84-binding sites in cyanobacterial phycobiliproteins.

    Science.gov (United States)

    Zhao, Kai-Hong; Su, Ping; Tu, Jun-Ming; Wang, Xing; Liu, Hui; Plöscher, Matthias; Eichacker, Lutz; Yang, Bei; Zhou, Ming; Scheer, Hugo

    2007-09-04

    Phycobilisomes, the light-harvesting complexes of cyanobacteria and red algae, contain two to four types of chromophores that are attached covalently to seven or more members of a family of homologous proteins, each carrying one to four binding sites. Chromophore binding to apoproteins is catalyzed by lyases, of which only few have been characterized in detail. The situation is complicated by nonenzymatic background binding to some apoproteins. Using a modular multiplasmidic expression-reconstitution assay in Escherichia coli with low background binding, phycobilin:cystein-84 biliprotein lyase (CpeS1) from Anabaena PCC7120, has been characterized as a nearly universal lyase for the cysteine-84-binding site that is conserved in all biliproteins. It catalyzes covalent attachment of phycocyanobilin to all allophycocyanin subunits and to cysteine-84 in the beta-subunits of C-phycocyanin and phycoerythrocyanin. Together with the known lyases, it can thereby account for chromophore binding to all binding sites of the phycobiliproteins of Anabaena PCC7120. Moreover, it catalyzes the attachment of phycoerythrobilin to cysteine-84 of both subunits of C-phycoerythrin. The only exceptions not served by CpeS1 among the cysteine-84 sites are the alpha-subunits from phycocyanin and phycoerythrocyanin, which, by sequence analyses, have been defined as members of a subclass that is served by the more specialized E/F type lyases.

  9. Molecular characterization of a Penicillium chrysogenum exo-rhamnogalacturonan lyase that is structurally distinct from other polysaccharide lyase family proteins.

    Science.gov (United States)

    Iwai, Marin; Kawakami, Takuya; Ikemoto, Takeshi; Fujiwara, Daisuke; Takenaka, Shigeo; Nakazawa, Masami; Ueda, Mitsuhiro; Sakamoto, Tatsuji

    2015-10-01

    We previously described an endo-acting rhamnogalacturonan (RG) lyase, termed PcRGL4A, of Penicillium chrysogenum 31B. Here, we describe a second RG lyase, called PcRGLX. We determined the cDNA sequence of the Pcrglx gene, which encodes PcRGLX. Based on analyses using a BLAST search and a conserved domain search, PcRGLX was found to be structurally distinct from known RG lyases and might belong to a new polysaccharide lyase family together with uncharacterized fungal proteins of Nectria haematococca, Aspergillus oryzae, and Fusarium oxysporum. The Pcrglx cDNA gene product (rPcRGLX) expressed in Escherichia coli demonstrated specific activity against RG but not against homogalacturonan. Divalent cations were not essential for the enzymatic activity of rPcRGLX. rPcRGLX mainly released unsaturated galacturonosyl rhamnose (ΔGR) from RG backbones used as the substrate from the initial stage of the reaction, indicating that the enzyme can be classified as an exo-acting RG lyase (EC 4.2.2.24). This is the first report of an RG lyase with this mode of action in Eukaryota. rPcRGLX acted synergistically with PcRGL4A to degrade soybean RG and released ΔGR. This ΔGR was partially decorated with galactose (Gal) residues, indicating that rPcRGLX preferred oligomeric RGs to polymeric RGs, that the enzyme did not require Gal decoration of RG backbones for degradation, and that the enzyme bypassed the Gal side chains of RG backbones. These characteristics of rPcRGLX might be useful in the determination of complex structures of pectins.

  10. Conservation and Rewiring of Functional Modules Revealed by an Epistasis Map in Fission Yeast

    Science.gov (United States)

    Roguev, Assen; Bandyopadhyay, Sourav; Zofall, Martin; Zhang, Ke; Fischer, Tamas; Collins, Sean R.; Qu, Hongjing; Shales, Michael; Park, Han-Oh; Hayles, Jacqueline; Hoe, Kwang-Lae; Kim, Dong-Uk; Ideker, Trey; Grewal, Shiv I.; Weissman, Jonathan S.; Krogan, Nevan J.

    2009-01-01

    An epistasis map (E-MAP) was constructed in the fission yeast, Schizosaccharomyces pombe, by systematically measuring the phenotypes associated with pairs of mutations. This high-density, quantitative genetic interaction map focused on various aspects of chromosome function, including transcription regulation and DNA repair/replication. The E-MAP uncovered a previously unidentified component of the RNA interference (RNAi) machinery (rsh1) and linked the RNAi pathway to several other biological processes. Comparison of the S. pombe E-MAP to an analogous genetic map from the budding yeast revealed that, whereas negative interactions were conserved between genes involved in similar biological processes, positive interactions and overall genetic profiles between pairs of genes coding for physically associated proteins were even more conserved. Hence, conservation occurs at the level of the functional module (protein complex), but the genetic cross talk between modules can differ substantially. PMID:18818364

  11. Evolutionary conservation of codon optimality reveals hidden signatures of cotranslational folding.

    Science.gov (United States)

    Pechmann, Sebastian; Frydman, Judith

    2013-02-01

    The choice of codons can influence local translation kinetics during protein synthesis. Whether codon preference is linked to cotranslational regulation of polypeptide folding remains unclear. Here, we derive a revised translational efficiency scale that incorporates the competition between tRNA supply and demand. Applying this scale to ten closely related yeast species, we uncover the evolutionary conservation of codon optimality in eukaryotes. This analysis reveals universal patterns of conserved optimal and nonoptimal codons, often in clusters, which associate with the secondary structure of the translated polypeptides independent of the levels of expression. Our analysis suggests an evolved function for codon optimality in regulating the rhythm of elongation to facilitate cotranslational polypeptide folding, beyond its previously proposed role of adapting to the cost of expression. These findings establish how mRNA sequences are generally under selection to optimize the cotranslational folding of corresponding polypeptides.

  12. 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.

  13. Managing shifting species: Ancient DNA reveals conservation conundrums in a dynamic world.

    Science.gov (United States)

    Waters, Jonathan M; Grosser, Stefanie

    2016-11-01

    The spread of exotic species represents a major driver of biological change across the planet. While dispersal and colonization are natural biological processes, we suggest that the failure to recognize increasing rates of human-facilitated self-introductions may represent a threat to native lineages. Notably, recent biogeographic analyses have revealed numerous cases of biological range shifts in response to anthropogenic impacts and climate change. In particular, ancient DNA analyses have revealed several cases in which lineages traditionally thought to be long-established "natives" are in fact recent colonizers. Such range expansion events have apparently occurred in response to human-mediated native biodiversity declines and ecosystem change, particularly in recently colonized, isolated ecosystems such as New Zealand. While such events can potentially boost local biodiversity, the spread of exotic lineages may also hasten the decline of indigenous species, so it is essential that conservation managers recognize these rapid biotic shifts.​. © 2016 WILEY Periodicals, Inc.

  14. Comparative genome analysis reveals a conserved family of actin-like proteins in apicomplexan parasites

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    Sibley L David

    2005-12-01

    Full Text Available Abstract Background The phylum Apicomplexa is an early-branching eukaryotic lineage that contains a number of important human and animal pathogens. Their complex life cycles and unique cytoskeletal features distinguish them from other model eukaryotes. Apicomplexans rely on actin-based motility for cell invasion, yet the regulation of this system remains largely unknown. Consequently, we focused our efforts on identifying actin-related proteins in the recently completed genomes of Toxoplasma gondii, Plasmodium spp., Cryptosporidium spp., and Theileria spp. Results Comparative genomic and phylogenetic studies of apicomplexan genomes reveals that most contain only a single conventional actin and yet they each have 8–10 additional actin-related proteins. Among these are a highly conserved Arp1 protein (likely part of a conserved dynactin complex, and Arp4 and Arp6 homologues (subunits of the chromatin-remodeling machinery. In contrast, apicomplexans lack canonical Arp2 or Arp3 proteins, suggesting they lost the Arp2/3 actin polymerization complex on their evolutionary path towards intracellular parasitism. Seven of these actin-like proteins (ALPs are novel to apicomplexans. They show no phylogenetic associations to the known Arp groups and likely serve functions specific to this important group of intracellular parasites. Conclusion The large diversity of actin-like proteins in apicomplexans suggests that the actin protein family has diverged to fulfill various roles in the unique biology of intracellular parasites. Conserved Arps likely participate in vesicular transport and gene expression, while apicomplexan-specific ALPs may control unique biological traits such as actin-based gliding motility.

  15. Genetic diversity and population structure of endangered Aquilaria malaccensis revealed potential for future conservation.

    Science.gov (United States)

    Singh, Pradeep; Nag, Akshay; Parmar, Rajni; Ghosh, Sneha; Bhau, Brijmohan Singh; Sharma, Ram Kumar

    2015-12-01

    The endangered Aquilaria malaccensis,is an important plant with high economic values. Characterization of genetic diversity and population structure is receiving tremendous attention for effective conservation of genetic resources. Considering important repositories of biological diversity, the genetic relationships of 127 A. malaccensis accessions from 10 home gardens of three states of northeast India were assessed using amplified fragment length polymorphism (AFLP). Of the 1153 fragments amplified with four AFLP primer combinations, 916 (79.4%) were found to be polymorphic. Polymorphic information content (PIC) and marker index (MI) of each primer combination correlate significantly with the number of genotypes resolved. Overall, a high genetic diversity (avg. 71.85%) was recorded. Further, high gene flow (Nm: 3.37), low genetic differentiation (FST: 0.069) and high within population genetic variation (93%) suggests that most of the genetic diversity is restricted within population. Neighbour joining (NJ), principal coordinate analysis (PCoA) and Bayesian-based STRUCTURE grouped all the accessions in two clusters with significant intermixing between populations, therefore, revealed that two genetically distinct gene pools are operating in the A. malaccensis populations cultivated in home gardens. Based on the various diversity inferences, five diverse populations (JOH, FN, HLF, DHM and ITN) were identified, which can be potentially exploited to develop conservation strategies for A. malaccensis.

  16. Genetic diversity and population structure of endangered Aquilaria malaccensis revealed potential for future conservation

    Indian Academy of Sciences (India)

    Pradeep Singh; Akshay Nag; Rajni Parmar; Sneha Ghosh; Brijmohan Singh Bhau; Ram Kumar Sharma

    2015-12-01

    The endangered Aquilaria malaccensis, is an important plant with high economic values. Characterization of genetic diversity and population structure is receiving tremendous attention for effective conservation of genetic resources. Considering important repositories of biological diversity, the genetic relationships of 127 A. malaccensis accessions from 10 home gardens of three states of northeast India were assessed using amplified fragment length polymorphism (AFLP). Of the 1153 fragments amplified with four AFLP primer combinations, 916 (79.4%) were found to be polymorphic. Polymorphic information content (PIC) and marker index (MI) of each primer combination correlate significantly with the number of genotypes resolved. Overall, a high genetic diversity (avg. 71.85%) was recorded. Further, high gene flow (m : 3.37), low genetic differentiation (ST : 0.069) and high within population genetic variation (93%) suggests that most of the genetic diversity is restricted within population. Neighbour joining (NJ), principal coordinate analysis (PCoA) and Bayesian-based STRUCTURE grouped all the accessions in two clusters with significant intermixing between populations, therefore, revealed that two genetically distinct gene pools are operating in the A. malaccensis populations cultivated in home gardens. Based on the various diversity inferences, five diverse populations (JOH, FN, HLF, DHM and ITN) were identified, which can be potentially exploited to develop conservation strategies for A. malaccensis.

  17. Intragenic suppressor of Osiaa23 revealed a conserved tryptophan residue crucial for protein-protein interactions.

    Directory of Open Access Journals (Sweden)

    Jun Ni

    Full Text Available The Auxin/Indole-3-Acetic Acid (Aux/IAA and Auxin Response Factor (ARF are two important families that play key roles in auxin signal transduction. Both of the families contain a similar carboxyl-terminal domain (Domain III/IV that facilitates interactions between these two families. In spite of the importance of protein-protein interactions among these transcription factors, the mechanisms involved in these interactions are largely unknown. In this study, we isolated six intragenic suppressors of an auxin insensitive mutant, Osiaa23. Among these suppressors, Osiaa23-R5 successfully rescued all the defects of the mutant. Sequence analysis revealed that an amino acid substitution occurred in the Tryptophan (W residue in Domain IV of Osiaa23. Yeast two-hybrid experiments showed that the mutation in Domain IV prevents the protein-protein interactions between Osiaa23 and OsARFs. Phylogenetic analysis revealed that the W residue is conserved in both OsIAAs and OsARFs. Next, we performed site-specific amino acid substitutions within Domain IV of OsARFs, and the conserved W in Domain IV was exchanged by Serine (S. The mutated OsARF(WSs can be released from the inhibition of Osiaa23 and maintain the transcriptional activities. Expression of OsARF(WSs in Osiaa23 mutant rescued different defects of the mutant. Our results suggest a previously unknown importance of Domain IV in both families and provide an indirect way to investigate functions of OsARFs.

  18. Conserved BK channel-protein interactions reveal signals relevant to cell death and survival.

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    Bernd Sokolowski

    Full Text Available The large-conductance Ca(2+-activated K(+ (BK channel and its β-subunit underlie tuning in non-mammalian sensory or hair cells, whereas in mammals its function is less clear. To gain insights into species differences and to reveal putative BK functions, we undertook a systems analysis of BK and BK-Associated Proteins (BKAPS in the chicken cochlea and compared these results to other species. We identified 110 putative partners from cytoplasmic and membrane/cytoskeletal fractions, using a combination of coimmunoprecipitation, 2-D gel, and LC-MS/MS. Partners included 14-3-3γ, valosin-containing protein (VCP, stathmin (STMN, cortactin (CTTN, and prohibitin (PHB, of which 16 partners were verified by reciprocal coimmunoprecipitation. Bioinformatics revealed binary partners, the resultant interactome, subcellular localization, and cellular processes. The interactome contained 193 proteins involved in 190 binary interactions in subcellular compartments such as the ER, mitochondria, and nucleus. Comparisons with mice showed shared hub proteins that included N-methyl-D-aspartate receptor (NMDAR and ATP-synthase. Ortholog analyses across six species revealed conserved interactions involving apoptosis, Ca(2+ binding, and trafficking, in chicks, mice, and humans. Functional studies using recombinant BK and RNAi in a heterologous expression system revealed that proteins important to cell death/survival, such as annexinA5, γ-actin, lamin, superoxide dismutase, and VCP, caused a decrease in BK expression. This revelation led to an examination of specific kinases and their effectors relevant to cell viability. Sequence analyses of the BK C-terminus across 10 species showed putative binding sites for 14-3-3, RAC-α serine/threonine-protein kinase 1 (Akt, glycogen synthase kinase-3β (GSK3β and phosphoinositide-dependent kinase-1 (PDK1. Knockdown of 14-3-3 and Akt caused an increase in BK expression, whereas silencing of GSK3β and PDK1 had the opposite

  19. Transcriptome profiling of a curdlan-producing Agrobacterium reveals conserved regulatory mechanisms of exopolysaccharide biosynthesis

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    Ruffing Anne M

    2012-02-01

    Full Text Available Abstract Background The ability to synthesize exopolysaccharides (EPS is widespread among microorganisms, and microbial EPS play important roles in biofilm formation, pathogen persistence, and applications in the food and medical industries. Although it is well established that EPS synthesis is invariably in response to environmental cues, it remains largely unknown how various environmental signals trigger activation of the biochemical synthesis machinery. Results We report here the transcriptome profiling of Agrobacterium sp. ATCC 31749, a microorganism that produces large amounts of a glucose polymer known as curdlan under nitrogen starvation. Transcriptome analysis revealed a nearly 100-fold upregulation of the curdlan synthesis operon upon transition to nitrogen starvation, thus establishing the prominent role that transcriptional regulation plays in the EPS synthesis. In addition to known mechanisms of EPS regulation such as activation by c-di-GMP, we identify novel mechanisms of regulation in ATCC 31749, including RpoN-independent NtrC regulation and intracellular pH regulation by acidocalcisomes. Furthermore, we show evidence that curdlan synthesis is also regulated by conserved cell stress responses, including polyphosphate accumulation and the stringent response. In fact, the stringent response signal, pppGpp, appears to be indispensible for transcriptional activation of curdlan biosynthesis. Conclusions This study identifies several mechanisms regulating the synthesis of curdlan, an EPS with numerous applications. These mechanisms are potential metabolic engineering targets for improving the industrial production of curdlan from Agrobacterium sp. ATCC 31749. Furthermore, many of the genes identified in this study are highly conserved across microbial genomes, and we propose that the molecular elements identified in this study may serve as universal regulators of microbial EPS synthesis.

  20. Conserved S-Layer-Associated Proteins Revealed by Exoproteomic Survey of S-Layer-Forming Lactobacilli.

    Science.gov (United States)

    Johnson, Brant R; Hymes, Jeffrey; Sanozky-Dawes, Rosemary; Henriksen, Emily DeCrescenzo; Barrangou, Rodolphe; Klaenhammer, Todd R

    2015-10-16

    The Lactobacillus acidophilus homology group comprises Gram-positive species that include L. acidophilus, L. helveticus, L. crispatus, L. amylovorus, L. gallinarum, L. delbrueckii subsp. bulgaricus, L. gasseri, and L. johnsonii. While these bacteria are closely related, they have varied ecological lifestyles as dairy and food fermenters, allochthonous probiotics, or autochthonous commensals of the host gastrointestinal tract. Bacterial cell surface components play a critical role in the molecular dialogue between bacteria and interaction signaling with the intestinal mucosa. Notably, the L. acidophilus complex is distinguished in two clades by the presence or absence of S-layers, which are semiporous crystalline arrays of self-assembling proteinaceous subunits found as the outermost layer of the bacterial cell wall. In this study, S-layer-associated proteins (SLAPs) in the exoproteomes of various S-layer-forming Lactobacillus species were proteomically identified, genomically compared, and transcriptionally analyzed. Four gene regions encoding six putative SLAPs were conserved in the S-layer-forming Lactobacillus species but not identified in the extracts of the closely related progenitor, L. delbrueckii subsp. bulgaricus, which does not produce an S-layer. Therefore, the presence or absence of an S-layer has a clear impact on the exoproteomic composition of Lactobacillus species. This proteomic complexity and differences in the cell surface properties between S-layer- and non-S-layer-forming lactobacilli reveal the potential for SLAPs to mediate intimate probiotic interactions and signaling with the host intestinal mucosa.

  1. Characterization of the PRMT gene family in rice reveals conservation of arginine methylation.

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    Ayaz Ahmad

    Full Text Available Post-translational methylation of arginine residues profoundly affects the structure and functions of protein and, hence, implicated in a myriad of essential cellular processes such as signal transduction, mRNA splicing and transcriptional regulation. Protein arginine methyltransferases (PRMTs, the enzymes catalyzing arginine methylation have been extensively studied in animals, yeast and, to some extent, in model plant Arabidopsis thaliana. Eight genes coding for the PRMTs were identified in Oryza sativa, previously. Here, we report that these genes show distinct expression patterns in various parts of the plant. In vivo targeting experiment demonstrated that GFP-tagged OsPRMT1, OsPRMT5 and OsPRMT10 were localized to both the cytoplasm and nucleus, whereas OsPRMT6a and OsPRMT6b were predominantly localized to the nucleus. OsPRMT1, OsPRMT4, OsPRMT5, OsPRMT6a, OsPRMT6b and OsPRMT10 exhibited in vitro arginine methyltransferase activity against myelin basic protein, glycine-arginine-rich domain of fibrillarin and calf thymus core histones. Furthermore, they depicted specificities for the arginine residues in histones H3 and H4 and were classified into type I and Type II PRMTs, based on the formation of type of dimethylarginine in the substrate proteins. The two homologs of OsPRMT6 showed direct interaction in vitro and further titrating different amounts of these proteins in the methyltransferase assay revealed that OsPRMT6a inhibits the methyltransferase activity of OsPRMT6b, probably, by the formation of heterodimer. The identification and characterization of PRMTs in rice suggests the conservation of arginine methylation in monocots and hold promise for gaining further insight into regulation of plant development.

  2. Comparative analysis of Phytophthora genes encoding secreted proteins reveals conserved synteny and lineage-specific gene duplications and deletions

    NARCIS (Netherlands)

    Jiang, R.H.Y.; Tyler, B.M.; Govers, F.

    2006-01-01

    Comparative analysis of two Phytophthora genomes revealed overall colinearity in four genomic regions consisting of a 1.5-Mb sequence of Phytophthora sojae and a 0.9-Mb sequence of R ramorum. In these regions with conserved synteny, the gene order is largely similar; however, genome rearrangements a

  3. CyanoLyase: a database of phycobilin lyase sequences, motifs and functions.

    Science.gov (United States)

    Bretaudeau, Anthony; Coste, François; Humily, Florian; Garczarek, Laurence; Le Corguillé, Gildas; Six, Christophe; Ratin, Morgane; Collin, Olivier; Schluchter, Wendy M; Partensky, Frédéric

    2013-01-01

    CyanoLyase (http://cyanolyase.genouest.org/) is a manually curated sequence and motif database of phycobilin lyases and related proteins. These enzymes catalyze the covalent ligation of chromophores (phycobilins) to specific binding sites of phycobiliproteins (PBPs). The latter constitute the building bricks of phycobilisomes, the major light-harvesting systems of cyanobacteria and red algae. Phycobilin lyases sequences are poorly annotated in public databases. Sequences included in CyanoLyase were retrieved from all available genomes of these organisms and a few others by similarity searches using biochemically characterized enzyme sequences and then classified into 3 clans and 32 families. Amino acid motifs were computed for each family using Protomata learner. CyanoLyase also includes BLAST and a novel pattern matching tool (Protomatch) that allow users to rapidly retrieve and annotate lyases from any new genome. In addition, it provides phylogenetic analyses of all phycobilin lyases families, describes their function, their presence/absence in all genomes of the database (phyletic profiles) and predicts the chromophorylation of PBPs in each strain. The site also includes a thorough bibliography about phycobilin lyases and genomes included in the database. This resource should be useful to scientists and companies interested in natural or artificial PBPs, which have a number of biotechnological applications, notably as fluorescent markers.

  4. Impact of different alginate lyases on combined cellulase–lyase saccharification of brown seaweed

    DEFF Research Database (Denmark)

    Manns, Dirk Martin; Nyffenegger, Christian; Saake, B.

    2016-01-01

    Two bacterial polysaccharide lyase (PL) family 7 alginate lyases (EC 4.2.2.-) from Sphingomonas sp. (SALy) and Flavobacterium sp. (FALy), respectively, were selected for heterologous, monocomponent expression in Escherichia coli. The thermal stability, pH, and temperature reaction optima and subs...... solubilization of sulfated fucoidan, whereas most of the nitrogen was recovered in the residual seaweed solids....

  5. Comparative analysis reveals conserved protein phosphorylation networks implicated in multiple diseases

    DEFF Research Database (Denmark)

    Tan, Chris Soon Heng; Bodenmiller, Bernd; Pasculescu, Adrian

    2009-01-01

    approximately 600 million years of evolution and hence are likely to be involved in fundamental cellular processes. This sequence-alignment analysis suggested that many phosphorylation sites evolve rapidly and therefore do not display strong evolutionary conservation in terms of sequence position in distantly...... related organisms. Thus, we devised a network-alignment approach to reconstruct conserved kinase-substrate networks, which identified 778 phosphorylation events in 698 human proteins. Both methods identified proteins tightly regulated by phosphorylation as well as signal integration hubs, and both types...... of phosphoproteins were enriched in proteins encoded by disease-associated genes. We analyzed the cellular functions and structural relationships for these conserved signaling events, noting the incomplete nature of current phosphoproteomes. Assessing phosphorylation conservation at both site and network levels...

  6. 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.

  7. Conserved intergenic sequences revealed by CTAG-profiling in Salmonella: thermodynamic modeling for function prediction

    Science.gov (United States)

    Tang, Le; Zhu, Songling; Mastriani, Emilio; Fang, Xin; Zhou, Yu-Jie; Li, Yong-Guo; Johnston, Randal N.; Guo, Zheng; Liu, Gui-Rong; Liu, Shu-Lin

    2017-01-01

    Highly conserved short sequences help identify functional genomic regions and facilitate genomic annotation. We used Salmonella as the model to search the genome for evolutionarily conserved regions and focused on the tetranucleotide sequence CTAG for its potentially important functions. In Salmonella, CTAG is highly conserved across the lineages and large numbers of CTAG-containing short sequences fall in intergenic regions, strongly indicating their biological importance. Computer modeling demonstrated stable stem-loop structures in some of the CTAG-containing intergenic regions, and substitution of a nucleotide of the CTAG sequence would radically rearrange the free energy and disrupt the structure. The postulated degeneration of CTAG takes distinct patterns among Salmonella lineages and provides novel information about genomic divergence and evolution of these bacterial pathogens. Comparison of the vertically and horizontally transmitted genomic segments showed different CTAG distribution landscapes, with the genome amelioration process to remove CTAG taking place inward from both terminals of the horizontally acquired segment. PMID:28262684

  8. Private development-based forest conservation in Patagonia: comparing mental models and revealing cultural truths

    Directory of Open Access Journals (Sweden)

    Christopher Serenari

    2015-09-01

    Full Text Available Private protected area (PPA conservation agents (CA engaging in development-based conservation in southern Chile have generated conflict with locals. Poor fit of dominant development-based conservation ideology in rural areas is commonly to blame. We developed and administered a cultural consensus survey near the Valdivian Coastal Reserve (RCV and Huilo Huilo Reserve (HH to examine fit of CA cultural truths with local residents. Cultural consensus analysis (CCA of 23 propositions reflecting CA cultural truths confirmed: (1 a single CA culture exists, and (2 RCV communities were more aligned with this culture than HH communities. Inadequate communication, inequitable decision making, divergent opinions about livelihood impacts and trajectories, and PPA purpose may explain differences between CAs and communities. Meanwhile, variability in response between and within communities may reflect differing environmental histories. Private protected area administrations might use CCA to confront cultural differences and thereby improve their community interactions.

  9. Conserved intergenic sequences revealed by CTAG-profiling in Salmonella: thermodynamic modeling for function prediction

    Science.gov (United States)

    Tang, Le; Zhu, Songling; Mastriani, Emilio; Fang, Xin; Zhou, Yu-Jie; Li, Yong-Guo; Johnston, Randal N.; Guo, Zheng; Liu, Gui-Rong; Liu, Shu-Lin

    2017-03-01

    Highly conserved short sequences help identify functional genomic regions and facilitate genomic annotation. We used Salmonella as the model to search the genome for evolutionarily conserved regions and focused on the tetranucleotide sequence CTAG for its potentially important functions. In Salmonella, CTAG is highly conserved across the lineages and large numbers of CTAG-containing short sequences fall in intergenic regions, strongly indicating their biological importance. Computer modeling demonstrated stable stem-loop structures in some of the CTAG-containing intergenic regions, and substitution of a nucleotide of the CTAG sequence would radically rearrange the free energy and disrupt the structure. The postulated degeneration of CTAG takes distinct patterns among Salmonella lineages and provides novel information about genomic divergence and evolution of these bacterial pathogens. Comparison of the vertically and horizontally transmitted genomic segments showed different CTAG distribution landscapes, with the genome amelioration process to remove CTAG taking place inward from both terminals of the horizontally acquired segment.

  10. Analysis of Schizosaccharomyces pombe mediator reveals a set of essential subunits conserved between yeast and metazoan cells

    DEFF Research Database (Denmark)

    Spåhr, H; Samuelsen, C O; Baraznenok, V;

    2001-01-01

    essential genes conserved between S. pombe and S. cerevisiae also have a metazoan homolog, indicating that an evolutionary conserved Mediator core is present in all eukaryotic cells. Our data suggest a closer functional relationship between yeast and metazoan Mediator than previously anticipated....... to any of the 10 S. cerevisiae components encoded by nonessential genes. S. pombe Mediator instead contains three unique components (Pmc2, -3, and -6), which lack homologs in other cell types. Presently, pmc2(+) and pmc3(+) have been shown to be nonessential genes. The data suggest that S. pombe and S....... cerevisiae share an essential protein module, which associates with nonessential speciesspecific subunits. In support of this view, sequence analysis of the conserved yeast Mediator components Med4 and Med8 reveals sequence homology to the metazoan Mediator components Trap36 and Arc32. Therefore, 8 of 10...

  11. Full-length RNA structure prediction of the HIV-1 genome reveals a conserved core domain

    DEFF Research Database (Denmark)

    Sukosd, Zsuzsanna; Andersen, Ebbe S.; Seemann, Stefan E.

    2015-01-01

    protein-coding regions the COS is supported by a particular high frequency of compensatory base changes, suggesting functional importance for this element. This new structural element potentially organizes the whole genome into three major domains protruding from a conserved core structure with potential...

  12. Full-length RNA structure prediction of the HIV-1 genome reveals a conserved core domain

    DEFF Research Database (Denmark)

    Sükösd, Zsuzsanna; Andersen, Ebbe Sloth; Seemann, Ernst Stefan;

    2015-01-01

    of the HIV-1 genome is highly variable in most regions, with a limited number of stable and conserved RNA secondary structures. Most interesting, a set of long distance interactions form a core organizing structure (COS) that organize the genome into three major structural domains. Despite overlapping...

  13. Genetic diversity and conservation implications of four Cupressus species in China as revealed by microsatellite markers.

    Science.gov (United States)

    Lu, Xu; Xu, Haiyan; Li, Zhonghu; Shang, Huiying; Adams, Robert P; Mao, Kangshan

    2014-04-01

    Understanding the extent and distribution of genetic diversity is crucial for the conservation and management of endangered species. Cupressus chengiana, C. duclouxiana, C. gigantea, and C. funebris are four ecologically and economically important species in China. We investigated their genetic diversity, population structure, and extant effective population size (35 populations, 484 individuals) employing six pairs of nuclear microsatellite markers (selected from 53). Their genetic diversity is moderate among conifers, and genetic differentiation among populations is much lower in C. gigantea than in the other three species; the estimated effective population size was largest for C. chengiana, at 1.70, 2.91, and 3.91 times the estimates for C. duclouxiana, C. funebris, and C. gigantea, respectively. According to Bayesian clustering analysis, the most plausible population subdivision scheme within species is two groups in C. chengiana, three groups in C. duclouxiana, and a single group for both C. funebris and C. gigantea. We propose a conservation strategy for these cypress species.

  14. The structure of a conserved piezo channel domain reveals a topologically distinct β sandwich fold.

    Science.gov (United States)

    Kamajaya, Aron; Kaiser, Jens T; Lee, Jonas; Reid, Michelle; Rees, Douglas C

    2014-10-07

    Piezo has recently been identified as a family of eukaryotic mechanosensitive channels composed of subunits containing over 2,000 amino acids, without recognizable sequence similarity to other channels. Here, we present the crystal structure of a large, conserved extramembrane domain located just before the last predicted transmembrane helix of C. elegans PIEZO, which adopts a topologically distinct β sandwich fold. The structure was also determined of a point mutation located on a conserved surface at the position equivalent to the human PIEZO1 mutation found in dehydrated hereditary stomatocytosis patients (M2225R). While the point mutation does not change the overall domain structure, it does alter the surface electrostatic potential that may perturb interactions with a yet-to-be-identified ligand or protein. The lack of structural similarity between this domain and any previously characterized fold, including those of eukaryotic and bacterial channels, highlights the distinctive nature of the Piezo family of eukaryotic mechanosensitive channels.

  15. Shadow response in the blind cavefish Astyanax reveals conservation of a functional pineal eye

    OpenAIRE

    2008-01-01

    The blind cavefish Astyanax mexicanus undergoes bilateral eye degeneration during embryonic development. Despite the absence of light in the cave environment, cavefish have retained a structurally intact pineal eye. We show here that contrary to visual degeneration in the bilateral eyes, the cavefish pineal eye has conserved the ability to detect light. Larvae of two different Astyanax cavefish populations and the con-specific sighted surface-dwelling form (surface fish) respond similarly to ...

  16. Isocitrate lyase localisation in Saccharomyces cerevisiae cells.

    Science.gov (United States)

    Chaves, R S; Herrero, P; Ordiz, I; Angeles del Brio, M; Moreno, F

    1997-10-01

    The isocitrate lyase from Saccharomyces cerevisiae was only located in the cell cytoplasm. This protein was found not to be associated with cell organelles, even under growth conditions that induce peroxisome proliferation. This conclusion is supported by experiments carried out by damaging the protoplast plasma membrane with DEAE-dextran, by differential centrifugation of osmotically lysed protoplast and by using the green fluorescent protein (GFP) of Aequorea victoria as a reporter fusion tag to localise the subcellular compartment to which isocitrate lyase is targeted.

  17. Characterization of Zebrafish von Willebrand Factor Reveals Conservation of Domain Structure, Multimerization, and Intracellular Storage

    Directory of Open Access Journals (Sweden)

    Arunima Ghosh

    2012-01-01

    Full Text Available von Willebrand disease (VWD is the most common inherited human bleeding disorder and is caused by quantitative or qualitative defects in von Willebrand factor (VWF. VWF is a secreted glycoprotein that circulates as large multimers. While reduced VWF is associated with bleeding, elevations in overall level or multimer size are implicated in thrombosis. The zebrafish is a powerful genetic model in which the hemostatic system is well conserved with mammals. The ability of this organism to generate thousands of offspring and its optical transparency make it unique and complementary to mammalian models of hemostasis. Previously, partial clones of zebrafish vwf have been identified, and some functional conservation has been demonstrated. In this paper we clone the complete zebrafish vwf cDNA and show that there is conservation of domain structure. Recombinant zebrafish Vwf forms large multimers and pseudo-Weibel-Palade bodies (WPBs in cell culture. Larval expression is in the pharyngeal arches, yolk sac, and intestinal epithelium. These results provide a foundation for continued study of zebrafish Vwf that may further our understanding of the mechanisms of VWD.

  18. The hand in motion of liberals and conservatives reveals the differential processing of positive and negative information.

    Science.gov (United States)

    Carraro, Luciana; Castelli, Luigi; Negri, Paolo

    2016-07-01

    Recent research revealed that political conservatives and liberals differ in the processing of valenced information. In particular, conservatives (vs. liberals) tend to weigh negative information more than positive information in their perception of the physical and social world. In the present work, we further investigated the ideology-based asymmetries in the processing of negative and positive information examining both the attention-grabbing power of negative information and the trajectories of the movements performed by respondents when required to categorize positive and negative stimuli. To this end we employed a modified version of the Mouse-Tracking procedure (Freeman & Ambady, 2010), recording hand movements during the execution of categorization tasks. Results showed that conservatives were indeed slower to start and execute response actions to negative stimuli, and, more specifically, the trajectories of their movements signaled avoidance tendencies aimed at increasing the distance from negative stimuli. In addition, this pattern of findings emerged both when participants were asked to categorize the stimuli according to their valence and when the same stimuli had to be categorized on the basis of irrelevant perceptual features. Overall, results demonstrate that conservatives and liberals process valenced information differently, perform different spontaneous movements when exposed to them, and that such asymmetries are largely independent from current processing goals.

  19. Identification of telomerase RNAs from filamentous fungi reveals conservation with vertebrates and yeasts.

    Directory of Open Access Journals (Sweden)

    Paulius V Kuprys

    Full Text Available Telomeres are the nucleoprotein complexes at eukaryotic chromosomal ends. Telomeric DNA is synthesized by the ribonucleoprotein telomerase, which comprises a telomerase reverse transcriptase (TERT and a telomerase RNA (TER. TER contains a template for telomeric DNA synthesis. Filamentous fungi possess extremely short and tightly regulated telomeres. Although TERT is well conserved between most organisms, TER is highly divergent and thus difficult to identify. In order to identify the TER sequence, we used the unusually long telomeric repeat sequence of Aspergillus oryzae together with reverse-transcription-PCR and identified a transcribed sequence that contains the potential template within a region predicted to be single stranded. We report the discovery of TERs from twelve other related filamentous fungi using comparative genomic analysis. These TERs exhibited strong conservation with the vertebrate template sequence, and two of these potentially use the identical template as humans. We demonstrate the existence of important processing elements required for the maturation of yeast TERs such as an Sm site, a 5' splice site and a branch point, within the newly identified TER sequences. RNA folding programs applied to the TER sequences show the presence of secondary structures necessary for telomerase activity, such as a yeast-like template boundary, pseudoknot, and a vertebrate-like three-way junction. These telomerase RNAs identified from filamentous fungi display conserved structural elements from both yeast and vertebrate TERs. These findings not only provide insights into the structure and evolution of a complex RNA but also provide molecular tools to further study telomere dynamics in filamentous fungi.

  20. Skinks (Reptilia: Scincidae) have highly conserved karyotypes as revealed by chromosome painting.

    Science.gov (United States)

    Giovannotti, M; Caputo, V; O'Brien, P C M; Lovell, F L; Trifonov, V; Cerioni, P Nisi; Olmo, E; Ferguson-Smith, M A; Rens, W

    2009-01-01

    Skinks represent the most diversified squamate reptiles with a great variation in body size and form, and are found worldwide in a variety of habitats. Their remarkable diversification has been accompanied by only a few chromosome rearrangements, resulting in highly-conservative chromosomal complements of these lizards. In this study cross-species chromosome painting using Scincus scincus (2n = 32) as the source genome, was used to detect the chromosomal rearrangements and homologies between the following skinks: Chalcides chalcides (2n = 28), C. ocellatus (2n = 28), Eumeces schneideri (2n = 32), Lepidothyris fernandi (2n = 30), Mabuya quinquetaeniata (2n = 32). The results of this study confirmed a high degree of chromosome conservation between these species. The main rearrangements in the studied skinks involve chromosomes 3, 5, 6 and 7 of S. scincus. These subtelocentric chromosomes are homologous to the p and q arms of metacentric pair 3 and 4 in C. chalcides, C. ocellatus, L. fernandi, and M. quinquetaeniata, while they are entirely conserved in E. schneideri. Other rearrangements involve S. scincus 11 in L. fernandi and M. quinquetaeniata, supporting the monophyly of Lygosominae, and one of the chromosomes S. scincus 12-16, in M. quinquetaeniata. In conclusion, our data support the monophyly of Scincidae and confirm that Scincus-Eumeces plus Chalcides do not form a monophyletic clade, suggesting that the Scincus-Eumeces clade is basal to other members of this family. This study represents the first time the whole genome of any reptile species has been used for cross-species chromosome painting to assess chromosomal evolution in this group of vertebrates.

  1. Comparative analysis of ABCB1 reveals novel structural and functional conservation between monocots and dicots

    Directory of Open Access Journals (Sweden)

    Amandeep Kaur Dhaliwal

    2014-11-01

    Full Text Available Phytohormone auxin plays a critical role in modulating plant architecture by creating a gradient regulated via its transporters such as ATP-binding cassette (ABC B1. Except for Arabidopsis and maize, where it was shown to interrupt auxin transport, ABCB1’s presence, structure and function in crop species is not known. Here we describe the structural and putative functional organization of ABCB1 among monocots relative to that of dicots. Identified from various plant species following specific and stringent criteria, ZmABCB1’s ‘true’ orthologs sequence identity ranged from 56-90% at the DNA and 75-91% at the predicted amino acid (aa level. Relative to ZmABCB1, the size of genomic copies ranged from -27 to +1.5% and aa from -7.7 to +0.6%. With the average gene size being similar (5.8 kb in monocots and 5.7 kb in dicots, dicots have about triple the number of introns with an average size of 194 bp (total 1743 bp compared to 556 bp (total 1667 bp in monocots. The intron-exon junctions across species were however conserved. N-termini of the predicted proteins were highly variable: in monocots due to mismatches and small deletions of 1-13 aa compared to large, species-specific deletions of up to 77 aa in dicots. The species- family-, and group- specific conserved motifs were identified in the N-terminus and linker regions of protein, possibly responsible for the specific functions. The near-identical conserved motifs of Nucleotide Binding Domains (NBDs in two halves of the protein showed subtle aa changes possibly favoring ATP binding to the N-terminus. Predicted 3-D protein structures showed remarkable similarity with each other and for the residues involved in auxin binding.

  2. Comparative proteomics reveals a significant bias toward alternative protein isoforms with conserved structure and function.

    Science.gov (United States)

    Ezkurdia, Iakes; del Pozo, Angela; Frankish, Adam; Rodriguez, Jose Manuel; Harrow, Jennifer; Ashman, Keith; Valencia, Alfonso; Tress, Michael L

    2012-09-01

    Advances in high-throughput mass spectrometry are making proteomics an increasingly important tool in genome annotation projects. Peptides detected in mass spectrometry experiments can be used to validate gene models and verify the translation of putative coding sequences (CDSs). Here, we have identified peptides that cover 35% of the genes annotated by the GENCODE consortium for the human genome as part of a comprehensive analysis of experimental spectra from two large publicly available mass spectrometry databases. We detected the translation to protein of "novel" and "putative" protein-coding transcripts as well as transcripts annotated as pseudogenes and nonsense-mediated decay targets. We provide a detailed overview of the population of alternatively spliced protein isoforms that are detectable by peptide identification methods. We found that 150 genes expressed multiple alternative protein isoforms. This constitutes the largest set of reliably confirmed alternatively spliced proteins yet discovered. Three groups of genes were highly overrepresented. We detected alternative isoforms for 10 of the 25 possible heterogeneous nuclear ribonucleoproteins, proteins with a key role in the splicing process. Alternative isoforms generated from interchangeable homologous exons and from short indels were also significantly enriched, both in human experiments and in parallel analyses of mouse and Drosophila proteomics experiments. Our results show that a surprisingly high proportion (almost 25%) of the detected alternative isoforms are only subtly different from their constitutive counterparts. Many of the alternative splicing events that give rise to these alternative isoforms are conserved in mouse. It was striking that very few of these conserved splicing events broke Pfam functional domains or would damage globular protein structures. This evidence of a strong bias toward subtle differences in CDS and likely conserved cellular function and structure is remarkable and

  3. Biogeographic comparison of Lophelia-associated bacterial communities in the Western Atlantic reveals conserved core microbiome

    Science.gov (United States)

    Kellogg, Christina A.; Goldsmith, Dawn; Gray, Michael A.

    2017-01-01

    Over the last decade, publications on deep-sea corals have tripled. Most attention has been paid to Lophelia pertusa, a globally distributed scleractinian coral that creates critical three-dimensional habitat in the deep ocean. The bacterial community associated with L. pertusa has been previously described by a number of studies at sites in the Mediterranean Sea, Norwegian fjords, off Great Britain, and in the Gulf of Mexico (GOM). However, use of different methodologies prevents direct comparisons in most cases. Our objectives were to address intra-regional variation and to identify any conserved bacterial core community. We collected samples from three distinct colonies of L. pertusa at each of four locations within the western Atlantic: three sites within the GOM and one off the east coast of the United States. Amplicon libraries of 16S rRNA genes were generated using primers targeting the V4–V5 hypervariable region and 454 pyrosequencing. The dominant phylum was Proteobacteria (75–96%). At the family level, 80–95% of each sample was comprised of five groups: Pirellulaceae, Pseudonocardiaceae, Rhodobacteraceae, Sphingomonadaceae, and unclassified Oceanospirillales. Principal coordinate analysis based on weighted UniFrac distances showed a clear distinction between the GOM and Atlantic samples. Interestingly, the replicate samples from each location did not always cluster together, indicating there is not a strong site-specific influence. The core bacterial community, conserved in 100% of the samples, was dominated by the operational taxonomic units of genera Novosphingobium and Pseudonocardia, both known degraders of aromatic hydrocarbons. The sequence of another core member, Propionibacterium, was also found in prior studies of L. pertusa from Norway and Great Britain, suggesting a role as a conserved symbiont. By examining more than 40,000 sequences per sample, we found that GOM samples were dominated by the identified conserved core sequences, whereas

  4. CDK1 structures reveal conserved and unique features of the essential cell cycle CDK

    Science.gov (United States)

    Brown, Nicholas R.; Korolchuk, Svitlana; Martin, Mathew P.; Stanley, Will A.; Moukhametzianov, Rouslan; Noble, Martin E. M.; Endicott, Jane A.

    2015-04-01

    CDK1 is the only essential cell cycle CDK in human cells and is required for successful completion of M-phase. It is the founding member of the CDK family and is conserved across all eukaryotes. Here we report the crystal structures of complexes of CDK1-Cks1 and CDK1-cyclin B-Cks2. These structures confirm the conserved nature of the inactive monomeric CDK fold and its ability to be remodelled by cyclin binding. Relative to CDK2-cyclin A, CDK1-cyclin B is less thermally stable, has a smaller interfacial surface, is more susceptible to activation segment dephosphorylation and shows differences in the substrate sequence features that determine activity. Both CDK1 and CDK2 are potential cancer targets for which selective compounds are required. We also describe the first structure of CDK1 bound to a potent ATP-competitive inhibitor and identify aspects of CDK1 structure and plasticity that might be exploited to develop CDK1-selective inhibitors.

  5. Comparative genomics reveals conservation of filaggrin and loss of caspase-14 in dolphins.

    Science.gov (United States)

    Strasser, Bettina; Mlitz, Veronika; Fischer, Heinz; Tschachler, Erwin; Eckhart, Leopold

    2015-05-01

    The expression of filaggrin and its stepwise proteolytic degradation are critical events in the terminal differentiation of epidermal keratinocytes and in the formation of the skin barrier to the environment. Here, we investigated whether the evolutionary transition from a terrestrial to a fully aquatic lifestyle of cetaceans, that is dolphins and whales, has been associated with changes in genes encoding filaggrin and proteins involved in the processing of filaggrin. We used comparative genomics, PCRs and re-sequencing of gene segments to screen for the presence and integrity of genes coding for filaggrin and proteases implicated in the maturation of (pro)filaggrin. Filaggrin has been conserved in dolphins (bottlenose dolphin, orca and baiji) but has been lost in whales (sperm whale and minke whale). All other S100 fused-type genes have been lost in cetaceans. Among filaggrin-processing proteases, aspartic peptidase retroviral-like 1 (ASPRV1), also known as saspase, has been conserved, whereas caspase-14 has been lost in all cetaceans investigated. In conclusion, our results suggest that filaggrin is dispensable for the acquisition of fully aquatic lifestyles of whales, whereas it appears to confer an evolutionary advantage to dolphins. The discordant evolution of filaggrin, saspase and caspase-14 in cetaceans indicates that the biological roles of these proteins are not strictly interdependent.

  6. Modular Architecture of Metabolic Pathways Revealed by Conserved Sequences of Reactions

    Science.gov (United States)

    2013-01-01

    The metabolic network is both a network of chemical reactions and a network of enzymes that catalyze reactions. Toward better understanding of this duality in the evolution of the metabolic network, we developed a method to extract conserved sequences of reactions called reaction modules from the analysis of chemical compound structure transformation patterns in all known metabolic pathways stored in the KEGG PATHWAY database. The extracted reaction modules are repeatedly used as if they are building blocks of the metabolic network and contain chemical logic of organic reactions. Furthermore, the reaction modules often correspond to traditional pathway modules defined as sets of enzymes in the KEGG MODULE database and sometimes to operon-like gene clusters in prokaryotic genomes. We identified well-conserved, possibly ancient, reaction modules involving 2-oxocarboxylic acids. The chain extension module that appears as the tricarboxylic acid (TCA) reaction sequence in the TCA cycle is now shown to be used in other pathways together with different types of modification modules. We also identified reaction modules and their connection patterns for aromatic ring cleavages in microbial biodegradation pathways, which are most characteristic in terms of both distinct reaction sequences and distinct gene clusters. The modular architecture of biodegradation modules will have a potential for predicting degradation pathways of xenobiotic compounds. The collection of these and many other reaction modules is made available as part of the KEGG database. PMID:23384306

  7. Genome-Scale Mapping of Escherichia coli σ54 Reveals Widespread, Conserved Intragenic Binding.

    Directory of Open Access Journals (Sweden)

    Richard P Bonocora

    2015-10-01

    Full Text Available Bacterial RNA polymerases must associate with a σ factor to bind promoter DNA and initiate transcription. There are two families of σ factor: the σ70 family and the σ54 family. Members of the σ54 family are distinct in their ability to bind promoter DNA sequences, in the context of RNA polymerase holoenzyme, in a transcriptionally inactive state. Here, we map the genome-wide association of Escherichia coli σ54, the archetypal member of the σ54 family. Thus, we vastly expand the list of known σ54 binding sites to 135. Moreover, we estimate that there are more than 250 σ54 sites in total. Strikingly, the majority of σ54 binding sites are located inside genes. The location and orientation of intragenic σ54 binding sites is non-random, and many intragenic σ54 binding sites are conserved. We conclude that many intragenic σ54 binding sites are likely to be functional. Consistent with this assertion, we identify three conserved, intragenic σ54 promoters that drive transcription of mRNAs with unusually long 5' UTRs.

  8. Shadow response in the blind cavefish Astyanax reveals conservation of a functional pineal eye.

    Science.gov (United States)

    Yoshizawa, Masato; Jeffery, William R

    2008-02-01

    The blind cavefish Astyanax mexicanus undergoes bilateral eye degeneration during embryonic development. Despite the absence of light in the cave environment, cavefish have retained a structurally intact pineal eye. We show here that contrary to visual degeneration in the bilateral eyes, the cavefish pineal eye has conserved the ability to detect light. Larvae of two different Astyanax cavefish populations and the con-specific sighted surface-dwelling form (surface fish) respond similarly to light dimming by shading the pineal eye. As a response to shading, cavefish larvae swim upward vertically. This behavior resembles that of amphibian tadpoles rather than other teleost larvae, which react to shadows by swimming downward. The shadow response is highest at 1.5-days post-fertilization (d.p.f.), gradually diminishes, and is virtually undetectable by 7.5 d.p.f. The shadow response was substantially reduced after surgical removal of the pineal gland from surface fish or cavefish larvae, indicating that it is based on pineal function. In contrast, removal of one or both bilateral eye primordia did not affect the shadow response. Consistent with its light detecting capacity, immunocytochemical studies indicate that surface fish and cavefish pineal eyes express a rhodopsin-like antigen, which is undetectable in the degenerating bilateral eyes of cavefish larvae. We conclude that light detection by the pineal eye has been conserved in cavefish despite a million or more years of evolution in complete darkness.

  9. Peptide Vocabulary Analysis Reveals Ultra-Conservation and Homonymity in Protein Sequences

    Directory of Open Access Journals (Sweden)

    Derek Gatherer

    2007-01-01

    Full Text Available A new algorithm is presented for vocabulary analysis (word detection in texts of human origin. It performs at 60%–70% overall accuracy and greater than 80% accuracy for longer words, and approximately 85% sensitivity on Alice in Wonderland, a considerable improvement on previous methods. When applied to protein sequences, it detects short sequences analogous to words in human texts, i.e. intolerant to changes in spelling (mutation, and relatively contextindependent in their meaning (function. Some of these are homonyms of up to 7 amino acids, which can assume different structures in different proteins. Others are ultra-conserved stretches of up to 18 amino acids within proteins of less than 40% overall identity, reflecting extreme constraint or convergent evolution. Different species are found to have qualitatively different major peptide vocabularies, e.g. some are dominated by large gene families, while others are rich in simple repeats or dominated by internally repetitive proteins. This suggests the possibility of a peptide vocabulary signature, analogous to genome signatures in DNA. Homonyms may be useful in detecting convergent evolution and positive selection in protein evolution. Ultra-conserved words may be useful in identifying structures intolerant to substitution over long periods of evolutionary time.

  10. Proteomic Analysis of Pathogenic Fungi Reveals Highly Expressed Conserved Cell Wall Proteins

    Directory of Open Access Journals (Sweden)

    Jackson Champer

    2016-01-01

    Full Text Available We are presenting a quantitative proteomics tally of the most commonly expressed conserved fungal proteins of the cytosol, the cell wall, and the secretome. It was our goal to identify fungi-typical proteins that do not share significant homology with human proteins. Such fungal proteins are of interest to the development of vaccines or drug targets. Protein samples were derived from 13 fungal species, cultured in rich or in minimal media; these included clinical isolates of Aspergillus, Candida, Mucor, Cryptococcus, and Coccidioides species. Proteomes were analyzed by quantitative MSE (Mass Spectrometry—Elevated Collision Energy. Several thousand proteins were identified and quantified in total across all fractions and culture conditions. The 42 most abundant proteins identified in fungal cell walls or supernatants shared no to very little homology with human proteins. In contrast, all but five of the 50 most abundant cytosolic proteins had human homologs with sequence identity averaging 59%. Proteomic comparisons of the secreted or surface localized fungal proteins highlighted conserved homologs of the Aspergillus fumigatus proteins 1,3-β-glucanosyltransferases (Bgt1, Gel1-4, Crf1, Ecm33, EglC, and others. The fact that Crf1 and Gel1 were previously shown to be promising vaccine candidates, underlines the value of the proteomics data presented here.

  11. Conservation of chromosomes syntenic with avian autosomes in squamate reptiles revealed by comparative chromosome painting.

    Science.gov (United States)

    Pokorná, Martina; Giovannotti, Massimo; Kratochvíl, Lukáš; Caputo, Vincenzo; Olmo, Ettore; Ferguson-Smith, Malcolm A; Rens, Willem

    2012-08-01

    In contrast to mammals, birds exhibit a slow rate of chromosomal evolution. It is not clear whether high chromosome conservation is an evolutionary novelty of birds or was inherited from an earlier avian ancestor. The evolutionary conservatism of macrochromosomes between birds and turtles supports the latter possibility; however, the rate of chromosomal evolution is largely unknown in other sauropsids. In squamates, we previously reported strong conservatism of the chromosomes syntenic with the avian Z, which could reflect a peculiarity of this part of the genome. The chromosome 1 of iguanians and snakes is largely syntenic with chromosomes 3, 5 and 7 of the avian ancestral karyotype. In this project, we used comparative chromosome painting to determine how widely this synteny is conserved across nine families covering most of the main lineages of Squamata. The results suggest that the association of the avian ancestral chromosomes 3, 5 and 7 can be dated back to at least the early Jurassic and could be an ancestral characteristic for Unidentata (Serpentes, Iguania, Anguimorpha, Laterata and Scinciformata). In Squamata chromosome conservatism therefore also holds for the parts of the genome which are homologous to bird autosomes, and following on from this, a slow rate of chromosomal evolution could be a common characteristic of all sauropsids. The large evolutionary stasis in chromosome organization in birds therefore seems to be inherited from their ancestors, and it is particularly striking in comparison with mammals, probably the only major tetrapod lineage with an increased rate of chromosomal rearrangements as a whole.

  12. The Large Mitochondrial Genome of Symbiodinium minutum Reveals Conserved Noncoding Sequences between Dinoflagellates and Apicomplexans.

    Science.gov (United States)

    Shoguchi, Eiichi; Shinzato, Chuya; Hisata, Kanako; Satoh, Nori; Mungpakdee, Sutada

    2015-07-20

    Even though mitochondrial genomes, which characterize eukaryotic cells, were first discovered more than 50 years ago, mitochondrial genomics remains an important topic in molecular biology and genome sciences. The Phylum Alveolata comprises three major groups (ciliates, apicomplexans, and dinoflagellates), the mitochondrial genomes of which have diverged widely. Even though the gene content of dinoflagellate mitochondrial genomes is reportedly comparable to that of apicomplexans, the highly fragmented and rearranged genome structures of dinoflagellates have frustrated whole genomic analysis. Consequently, noncoding sequences and gene arrangements of dinoflagellate mitochondrial genomes have not been well characterized. Here we report that the continuous assembled genome (∼326 kb) of the dinoflagellate, Symbiodinium minutum, is AT-rich (∼64.3%) and that it contains three protein-coding genes. Based upon in silico analysis, the remaining 99% of the genome comprises transcriptomic noncoding sequences. RNA edited sites and unique, possible start and stop codons clarify conserved regions among dinoflagellates. Our massive transcriptome analysis shows that almost all regions of the genome are transcribed, including 27 possible fragmented ribosomal RNA genes and 12 uncharacterized small RNAs that are similar to mitochondrial RNA genes of the malarial parasite, Plasmodium falciparum. Gene map comparisons show that gene order is only slightly conserved between S. minutum and P. falciparum. However, small RNAs and intergenic sequences share sequence similarities with P. falciparum, suggesting that the function of noncoding sequences has been preserved despite development of very different genome structures.

  13. Interspecies variation reveals a conserved repressor of alpha-specific genes in Saccharomyces yeasts.

    Science.gov (United States)

    Zill, Oliver A; Rine, Jasper

    2008-06-15

    The mating-type determination circuit in Saccharomyces yeast serves as a classic paradigm for the genetic control of cell type in all eukaryotes. Using comparative genetics, we discovered a central and conserved, yet previously undetected, component of this genetic circuit: active repression of alpha-specific genes in a cells. Upon inactivation of the SUM1 gene in Saccharomyces bayanus, a close relative of Saccharomyces cerevisiae, a cells acquired mating characteristics of alpha cells and displayed autocrine activation of their mating response pathway. Sum1 protein bound to the promoters of alpha-specific genes, repressing their transcription. In contrast to the standard model, alpha1 was important but not required for alpha-specific gene activation and mating of alpha cells in the absence of Sum1. Neither Sum1 protein expression, nor its association with target promoters was mating-type-regulated. Thus, the alpha1/Mcm1 coactivators did not overcome repression by occluding Sum1 binding to DNA. Surprisingly, the mating-type regulatory function of Sum1 was conserved in S. cerevisiae. We suggest that a comprehensive understanding of some genetic pathways may be best attained through the expanded phenotypic space provided by study of those pathways in multiple related organisms.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    –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......-octamer comprising an intertwined network of subunits with unexpected self-homologies. It contains two potential active sites that probably couple phosphonate compounds to ATP and subsequently hydrolyse the C–P bond. We map the binding site of PhnK on the complex using electron microscopy, and show that it binds...... to a conserved insertion domain of PhnJ. Our results provide a structural basis for understanding microbial phosphonate breakdown....

  15. 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.

  16. Significance of satellite DNA revealed by conservation of a widespread repeat DNA sequence among angiosperms.

    Science.gov (United States)

    Mehrotra, Shweta; Goel, Shailendra; Raina, Soom Nath; Rajpal, Vijay Rani

    2014-08-01

    The analysis of plant genome structure and evolution requires comprehensive characterization of repetitive sequences that make up the majority of plant nuclear DNA. In the present study, we analyzed the nature of pCtKpnI-I and pCtKpnI-II tandem repeated sequences, reported earlier in Carthamus tinctorius. Interestingly, homolog of pCtKpnI-I repeat sequence was also found to be present in widely divergent families of angiosperms. pCtKpnI-I showed high sequence similarity but low copy number among various taxa of different families of angiosperms analyzed. In comparison, pCtKpnI-II was specific to the genus Carthamus and was not present in any other taxa analyzed. The molecular structure of pCtKpnI-I was analyzed in various unrelated taxa of angiosperms to decipher the evolutionary conserved nature of the sequence and its possible functional role.

  17. Comparative Genomic and Phylogenomic Analyses Reveal a Conserved Core Genome Shared by Estuarine and Oceanic Cyanopodoviruses

    Science.gov (United States)

    Huang, Sijun; Zhang, Si; Jiao, Nianzhi; Chen, Feng

    2015-01-01

    Podoviruses are among the major viral groups that infect marine picocyanobacteria Prochlorococcus and Synechococcus. Here, we reported the genome sequences of five Synechococcus podoviruses isolated from the estuarine environment, and performed comparative genomic and phylogenomic analyses based on a total of 20 cyanopodovirus genomes. The genomes of all the known marine cyanopodoviruses are highly syntenic. A pan-genome of 349 clustered orthologous groups was determined, among which 15 were core genes. These core genes make up nearly half of each genome in length, reflecting the high level of genome conservation among this cyanophage type. The whole genome phylogenies based on concatenated core genes and gene content were highly consistent and confirmed the separation of two discrete marine cyanopodovirus clusters MPP-A and MPP-B. The genomes within cluster MPP-B grouped into subclusters mainly corresponding to Prochlorococcus or Synechococcus host types. Auxiliary metabolic genes tend to occur in a specific phylogenetic group of these cyanopodoviruses. All the MPP-B phages analyzed here encode the photosynthesis gene psbA, which are absent in all the MPP-A genomes thus far. Interestingly, all the MPP-B and two MPP-A Synechococcus podoviruses encode the thymidylate synthase gene thyX, while at the same genome locus all the MPP-B Prochlorococcus podoviruses encode the transaldolase gene talC. Both genes are hypothesized to have the potential to facilitate the biosynthesis of deoxynucleotide for phage replication. Inheritance of specific functional genes could be important to the evolution and ecological fitness of certain cyanophage genotypes. Our analyses demonstrate that cyanopodoviruses of estuarine and oceanic origins share a conserved core genome and suggest that accessory genes may be related to environmental adaptation. PMID:26569403

  18. Comparative Genomic and Phylogenomic Analyses Reveal a Conserved Core Genome Shared by Estuarine and Oceanic Cyanopodoviruses.

    Directory of Open Access Journals (Sweden)

    Sijun Huang

    Full Text Available Podoviruses are among the major viral groups that infect marine picocyanobacteria Prochlorococcus and Synechococcus. Here, we reported the genome sequences of five Synechococcus podoviruses isolated from the estuarine environment, and performed comparative genomic and phylogenomic analyses based on a total of 20 cyanopodovirus genomes. The genomes of all the known marine cyanopodoviruses are highly syntenic. A pan-genome of 349 clustered orthologous groups was determined, among which 15 were core genes. These core genes make up nearly half of each genome in length, reflecting the high level of genome conservation among this cyanophage type. The whole genome phylogenies based on concatenated core genes and gene content were highly consistent and confirmed the separation of two discrete marine cyanopodovirus clusters MPP-A and MPP-B. The genomes within cluster MPP-B grouped into subclusters mainly corresponding to Prochlorococcus or Synechococcus host types. Auxiliary metabolic genes tend to occur in a specific phylogenetic group of these cyanopodoviruses. All the MPP-B phages analyzed here encode the photosynthesis gene psbA, which are absent in all the MPP-A genomes thus far. Interestingly, all the MPP-B and two MPP-A Synechococcus podoviruses encode the thymidylate synthase gene thyX, while at the same genome locus all the MPP-B Prochlorococcus podoviruses encode the transaldolase gene talC. Both genes are hypothesized to have the potential to facilitate the biosynthesis of deoxynucleotide for phage replication. Inheritance of specific functional genes could be important to the evolution and ecological fitness of certain cyanophage genotypes. Our analyses demonstrate that cyanopodoviruses of estuarine and oceanic origins share a conserved core genome and suggest that accessory genes may be related to environmental adaptation.

  19. Annotation of Protein Domains Reveals Remarkable Conservation in the Functional Make up of Proteomes Across Superkingdoms

    Science.gov (United States)

    Nasir, Arshan; Naeem, Aisha; Khan, Muhammad Jawad; Lopez-Nicora, Horacio D.; Caetano-Anollés, Gustavo

    2011-01-01

    The functional repertoire of a cell is largely embodied in its proteome, the collection of proteins encoded in the genome of an organism. The molecular functions of proteins are the direct consequence of their structure and structure can be inferred from sequence using hidden Markov models of structural recognition. Here we analyze the functional annotation of protein domain structures in almost a thousand sequenced genomes, exploring the functional and structural diversity of proteomes. We find there is a remarkable conservation in the distribution of domains with respect to the molecular functions they perform in the three superkingdoms of life. In general, most of the protein repertoire is spent in functions related to metabolic processes but there are significant differences in the usage of domains for regulatory and extra-cellular processes both within and between superkingdoms. Our results support the hypotheses that the proteomes of superkingdom Eukarya evolved via genome expansion mechanisms that were directed towards innovating new domain architectures for regulatory and extra/intracellular process functions needed for example to maintain the integrity of multicellular structure or to interact with environmental biotic and abiotic factors (e.g., cell signaling and adhesion, immune responses, and toxin production). Proteomes of microbial superkingdoms Archaea and Bacteria retained fewer numbers of domains and maintained simple and smaller protein repertoires. Viruses appear to play an important role in the evolution of superkingdoms. We finally identify few genomic outliers that deviate significantly from the conserved functional design. These include Nanoarchaeum equitans, proteobacterial symbionts of insects with extremely reduced genomes, Tenericutes and Guillardia theta. These organisms spend most of their domains on information functions, including translation and transcription, rather than on metabolism and harbor a domain repertoire characteristic of

  20. Annotation of Protein Domains Reveals Remarkable Conservation in the Functional Make up of Proteomes Across Superkingdoms

    Directory of Open Access Journals (Sweden)

    Gustavo Caetano-Anollés

    2011-11-01

    Full Text Available The functional repertoire of a cell is largely embodied in its proteome, the collection of proteins encoded in the genome of an organism. The molecular functions of proteins are the direct consequence of their structure and structure can be inferred from sequence using hidden Markov models of structural recognition. Here we analyze the functional annotation of protein domain structures in almost a thousand sequenced genomes, exploring the functional and structural diversity of proteomes. We find there is a remarkable conservation in the distribution of domains with respect to the molecular functions they perform in the three superkingdoms of life. In general, most of the protein repertoire is spent in functions related to metabolic processes but there are significant differences in the usage of domains for regulatory and extra-cellular processes both within and between superkingdoms. Our results support the hypotheses that the proteomes of superkingdom Eukarya evolved via genome expansion mechanisms that were directed towards innovating new domain architectures for regulatory and extra/intracellular process functions needed for example to maintain the integrity of multicellular structure or to interact with environmental biotic and abiotic factors (e.g., cell signaling and adhesion, immune responses, and toxin production. Proteomes of microbial superkingdoms Archaea and Bacteria retained fewer numbers of domains and maintained simple and smaller protein repertoires. Viruses appear to play an important role in the evolution of superkingdoms. We finally identify few genomic outliers that deviate significantly from the conserved functional design. These include Nanoarchaeum equitans, proteobacterial symbionts of insects with extremely reduced genomes, Tenericutes and Guillardia theta. These organisms spend most of their domains on information functions, including translation and transcription, rather than on metabolism and harbor a domain

  1. The Trypanosome Exocyst: A Conserved Structure Revealing a New Role in Endocytosis

    Science.gov (United States)

    Boehm, Cordula M.; Obado, Samson; Gadelha, Catarina; Kaupisch, Alexandra; Manna, Paul T.; Gould, Gwyn W.; Rout, Michael P.; Field, Mark C.

    2017-01-01

    Membrane transport is an essential component of pathogenesis for most infectious organisms. In African trypanosomes, transport to and from the plasma membrane is closely coupled to immune evasion and antigenic variation. In mammals and fungi an octameric exocyst complex mediates late steps in exocytosis, but comparative genomics suggested that trypanosomes retain only six canonical subunits, implying mechanistic divergence. We directly determined the composition of the Trypanosoma brucei exocyst by affinity isolation and demonstrate that the parasite complex is nonameric, retaining all eight canonical subunits (albeit highly divergent at the sequence level) plus a novel essential subunit, Exo99. Exo99 and Sec15 knockdowns have remarkably similar phenotypes in terms of viability and impact on morphology and trafficking pathways. Significantly, both Sec15 and Exo99 have a clear function in endocytosis, and global proteomic analysis indicates an important role in maintaining the surface proteome. Taken together these data indicate additional exocyst functions in trypanosomes, which likely include endocytosis, recycling and control of surface composition. Knockdowns in HeLa cells suggest that the role in endocytosis is shared with metazoan cells. We conclude that, whilst the trypanosome exocyst has novel components, overall functionality appears conserved, and suggest that the unique subunit may provide therapeutic opportunities. PMID:28114397

  2. Comparative genomic analysis of innate immunity reveals novel and conserved components in crustacean food crop species.

    Science.gov (United States)

    Lai, Alvina G; Aboobaker, A Aziz

    2017-05-18

    Growing global demands for crustacean food crop species have driven large investments in aquaculture research worldwide. However, large-scale production is susceptible to pathogen-mediated destruction particularly in developing economies. Thus, a thorough understanding of the immune system components of food crop species is imperative for research to combat pathogens. Through a comparative genomics approach utilising extant data from 55 species, we describe the innate immune system of the class Malacostraca, which includes all food crop species. We identify 7407 malacostracan genes from 39 gene families implicated in different aspects of host defence and demonstrate dynamic evolution of innate immunity components within this group. Malacostracans have achieved flexibility in recognising infectious agents through divergent evolution and expansion of pathogen recognition receptors genes. Antiviral RNAi, Toll and JAK-STAT signal transduction pathways have remained conserved within Malacostraca, although the Imd pathway appears to lack several key components. Immune effectors such as the antimicrobial peptides (AMPs) have unique evolutionary profiles, with many malacostracan AMPs not found in other arthropods. Lastly, we describe four putative novel immune gene families, potentially representing important evolutionary novelties of the malacostracan immune system. Our analyses across the broader Malacostraca have allowed us to not only draw analogies with other arthropods but also to identify evolutionary novelties in immune modulation components and form strong hypotheses as to when key pathways have evolved or diverged. This will serve as a key resource for future immunology research in crustacean food crops.

  3. Hybridization capture reveals evolution and conservation across the entire Koala retrovirus genome.

    Directory of Open Access Journals (Sweden)

    Kyriakos Tsangaras

    Full Text Available The koala retrovirus (KoRV is the only retrovirus known to be in the midst of invading the germ line of its host species. Hybridization capture and next generation sequencing were used on modern and museum DNA samples of koala (Phascolarctos cinereus to examine ca. 130 years of evolution across the full KoRV genome. Overall, the entire proviral genome appeared to be conserved across time in sequence, protein structure and transcriptional binding sites. A total of 138 polymorphisms were detected, of which 72 were found in more than one individual. At every polymorphic site in the museum koalas, one of the character states matched that of modern KoRV. Among non-synonymous polymorphisms, radical substitutions involving large physiochemical differences between amino acids were elevated in env, potentially reflecting anti-viral immune pressure or avoidance of receptor interference. Polymorphisms were not detected within two functional regions believed to affect infectivity. Host sequences flanking proviral integration sites were also captured; with few proviral loci shared among koalas. Recently described variants of KoRV, designated KoRV-B and KoRV-J, were not detected in museum samples, suggesting that these variants may be of recent origin.

  4. High Throughput Sequencing of T Cell Antigen Receptors Reveals a Conserved TCR Repertoire

    Science.gov (United States)

    Hou, Xianliang; Lu, Chong; Chen, Sisi; Xie, Qian; Cui, Guangying; Chen, Jianing; Chen, Zhi; Wu, Zhongwen; Ding, Yulong; Ye, Ping; Dai, Yong; Diao, Hongyan

    2016-01-01

    Abstract The T-cell receptor (TCR) repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next-generation sequencing has become a powerful tool for deep TCR profiling. Herein, we used this technology to study the repertoire features of TCR beta chain in the blood of healthy individuals. Peripheral blood samples were collected from 10 healthy donors. T cells were isolated with anti-human CD3 magnetic beads according to the manufacturer's protocol. We then combined multiplex-PCR, Illumina sequencing, and IMGT/High V-QUEST to analyze the characteristics and polymorphisms of the TCR. Most of the individual T cell clones were present at very low frequencies, suggesting that they had not undergone clonal expansion. The usage frequencies of the TCR beta variable, beta joining, and beta diversity gene segments were similar among T cells from different individuals. Notably, the usage frequency of individual nucleotides and amino acids within complementarity-determining region (CDR3) intervals was remarkably consistent between individuals. Moreover, our data show that terminal deoxynucleotidyl transferase activity was biased toward the insertion of G (31.92%) and C (27.14%) over A (21.82%) and T (19.12%) nucleotides. Some conserved features could be observed in the composition of CDR3, which may inform future studies of human TCR gene recombination. PMID:26962778

  5. Comparative Assessment of Mediterranean Gorgonian-Associated Microbial Communities Reveals Conserved Core and Locally Variant Bacteria

    KAUST Repository

    van de Water, Jeroen A J M

    2016-10-10

    Gorgonians are key habitat-forming species of Mediterranean benthic communities, but their populations have suffered from mass mortality events linked to high summer seawater temperatures and microbial disease. However, our knowledge on the diversity, dynamics and function of gorgonian-associated microbial communities is limited. Here, we analysed the spatial variability of the microbiomes of five sympatric gorgonian species (Eunicella singularis, Eunicella cavolini, Eunicella verrucosa, Leptogorgia sarmentosa and Paramuricea clavata), collected from the Mediterranean Sea over a scale of ∼1100 km, using next-generation amplicon sequencing of the 16S rRNA gene. The microbiomes of all gorgonian species were generally dominated by members of the genus Endozoicomonas, which were at very low abundance in the surrounding seawater. Although the composition of the core microbiome (operational taxonomic units consistently present in a species) was found to be unique for each host species, significant overlap was observed. These spatially consistent associations between gorgonians and their core bacteria suggest intricate symbiotic relationships and regulation of the microbiome composition by the host. At the same time, local variations in microbiome composition were observed. Functional predictive profiling indicated that these differences could be attributed to seawater pollution. Taken together, our data indicate that gorgonian-associated microbiomes are composed of spatially conserved bacteria (core microbiome members) and locally variant members, and that local pollution may influence these local associations, potentially impacting gorgonian health.

  6. Macroscopic law of conservation revealed in the population dynamics of Toll-like receptor signaling

    Directory of Open Access Journals (Sweden)

    Selvarajoo Kumar

    2011-04-01

    Full Text Available Abstract Stimulating the receptors of a single cell generates stochastic intracellular signaling. The fluctuating response has been attributed to the low abundance of signaling molecules and the spatio-temporal effects of diffusion and crowding. At population level, however, cells are able to execute well-defined deterministic biological processes such as growth, division, differentiation and immune response. These data reflect biology as a system possessing microscopic and macroscopic dynamics. This commentary discusses the average population response of the Toll-like receptor (TLR 3 and 4 signaling. Without requiring detailed experimental data, linear response equations together with the fundamental law of information conservation have been used to decipher novel network features such as unknown intermediates, processes and cross-talk mechanisms. For single cell response, however, such simplicity seems far from reality. Thus, as observed in any other complex systems, biology can be considered to possess order and disorder, inheriting a mixture of predictable population level and unpredictable single cell outcomes.

  7. Comparative analyses of six solanaceous transcriptomes reveal a high degree of sequence conservation and species-specific transcripts

    Directory of Open Access Journals (Sweden)

    Ouyang Shu

    2005-09-01

    Full Text Available Abstract Background The Solanaceae is a family of closely related species with diverse phenotypes that have been exploited for agronomic purposes. Previous studies involving a small number of genes suggested sequence conservation across the Solanaceae. The availability of large collections of Expressed Sequence Tags (ESTs for the Solanaceae now provides the opportunity to assess sequence conservation and divergence on a genomic scale. Results All available ESTs and Expressed Transcripts (ETs, 449,224 sequences for six Solanaceae species (potato, tomato, pepper, petunia, tobacco and Nicotiana benthamiana, were clustered and assembled into gene indices. Examination of gene ontologies revealed that the transcripts within the gene indices encode a similar suite of biological processes. Although the ESTs and ETs were derived from a variety of tissues, 55–81% of the sequences had significant similarity at the nucleotide level with sequences among the six species. Putative orthologs could be identified for 28–58% of the sequences. This high degree of sequence conservation was supported by expression profiling using heterologous hybridizations to potato cDNA arrays that showed similar expression patterns in mature leaves for all six solanaceous species. 16–19% of the transcripts within the six Solanaceae gene indices did not have matches among Solanaceae, Arabidopsis, rice or 21 other plant gene indices. Conclusion Results from this genome scale analysis confirmed a high level of sequence conservation at the nucleotide level of the coding sequence among Solanaceae. Additionally, the results indicated that part of the Solanaceae transcriptome is likely to be unique for each species.

  8. Conservation through connectivity: can isotopic gradients in Africa reveal winter quarters of a migratory bird?

    Science.gov (United States)

    Reichlin, Thomas S; Hobson, Keith A; Van Wilgenburg, Steven L; Schaub, Michael; Wassenaar, Leonard I; Martín-Vivaldi, Manuel; Arlettaz, Raphaël; Jenni, Lukas

    2013-02-01

    Conservation of migratory wildlife requires knowledge of migratory connectivity between breeding and non-breeding locations. Stable isotopes in combination with geographical isotopic patterns (isoscapes) can provide inferences about migratory connectivity. This study examines whether such an approach can be used to infer wintering areas in sub-Saharan Africa, where we lack such knowledge for many species, but where this method has not been used widely. We measured δ (2)H, δ (13)C and δ (15)N in winter-grown feathers of a breeding Swiss and Spanish population of European hoopoe Upupa epops--a typical Palaearctic-Afrotropical migrant. δ (2)H values predicted that ~70 % of the hoopoes spent the non-breeding season in the western portion of their potential winter range. This was corroborated by a shallow east-west gradient in feather-δ (2)H values of museum specimens from known African origin across the potential winter range and by the recovery of Swiss hoopoes marked with geolocators. Hoopoes categorized as from eastern versus western regions of the wintering range were further delineated spatially using feather δ (13)C and δ (15)N. δ (15)N showed no trend, whereas adults were more enriched in (13)C in the western portion of the range, with eastern adults being in addition more depleted in (13)C than eastern juveniles. This suggests that eastern juveniles may have occupied more xeric habitats than sympatric adults. We demonstrated that stable isotopes, especially δ (2)H, could only very roughly delineate the winter distribution of a trans-Saharan Palaearctic migrant restricted primarily to the Sahelian and savanna belt south of the Sahara. Further refinements of precipitation isoscapes for Africa as well the development of isoscapes for δ (13)C and δ (15)N may improve assignment of this and other migrants.

  9. Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway.

    Science.gov (United States)

    Shi, Tujin; Niepel, Mario; McDermott, Jason E; Gao, Yuqian; Nicora, Carrie D; Chrisler, William B; Markillie, Lye M; Petyuk, Vladislav A; Smith, Richard D; Rodland, Karin D; Sorger, Peter K; Qian, Wei-Jun; Wiley, H Steven

    2016-07-12

    Various genetic mutations associated with cancer are known to alter cell signaling, but it is not clear whether they dysregulate signaling pathways by altering the abundance of pathway proteins. Using a combination of RNA sequencing and ultrasensitive targeted proteomics, we defined the primary components-16 core proteins and 10 feedback regulators-of the epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) pathway in normal human mammary epithelial cells and then quantified their absolute abundance across a panel of normal and breast cancer cell lines as well as fibroblasts. We found that core pathway proteins were present at very similar concentrations across all cell types, with a variance similar to that of proteins previously shown to display conserved abundances across species. In contrast, EGFR and transcriptionally controlled feedback regulators were present at highly variable concentrations. The absolute abundance of most core proteins was between 50,000 and 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower amounts (2000 to 5000 copies per cell). MAPK signaling showed saturation in all cells between 3000 and 10,000 occupied EGFRs, consistent with the idea that adaptors limit signaling. Our results suggest that the relative stoichiometry of core MAPK pathway proteins is very similar across different cell types, with cell-specific differences mostly restricted to variable amounts of feedback regulators and receptors. The low abundance of adaptors relative to EGFR could be responsible for previous observations that only a fraction of total cell surface EGFR is capable of rapid endocytosis, high-affinity binding, and mitogenic signaling.

  10. A suite of Lotus japonicus starch mutants reveals both conserved and novel features of starch metabolism.

    Science.gov (United States)

    Vriet, Cécile; Welham, Tracey; Brachmann, Andreas; Pike, Marilyn; Pike, Jodie; Perry, Jillian; Parniske, Martin; Sato, Shusei; Tabata, Satoshi; Smith, Alison M; Wang, Trevor L

    2010-10-01

    The metabolism of starch is of central importance for many aspects of plant growth and development. Information on leaf starch metabolism other than in Arabidopsis (Arabidopsis thaliana) is scarce. Furthermore, its importance in several agronomically important traits exemplified by legumes remains to be investigated. To address this issue, we have provided detailed information on the genes involved in starch metabolism in Lotus japonicus and have characterized a comprehensive collection of forward and TILLING (for Targeting Induced Local Lesions IN Genomes) reverse genetics mutants affecting five enzymes of starch synthesis and two enzymes of starch degradation. The mutants provide new insights into the structure-function relationships of ADP-glucose pyrophosphorylase and glucan, water dikinase1 in particular. Analyses of the mutant phenotypes indicate that the pathways of leaf starch metabolism in L. japonicus and Arabidopsis are largely conserved. However, the importance of these pathways for plant growth and development differs substantially between the two species. Whereas essentially starchless Arabidopsis plants lacking plastidial phosphoglucomutase grow slowly relative to wild-type plants, the equivalent mutant of L. japonicus grows normally even in a 12-h photoperiod. In contrast, the loss of GLUCAN, WATER DIKINASE1, required for starch degradation, has a far greater effect on plant growth and fertility in L. japonicus than in Arabidopsis. Moreover, we have also identified several mutants likely to be affected in new components or regulators of the pathways of starch metabolism. This suite of mutants provides a substantial new resource for further investigations of the partitioning of carbon and its importance for symbiotic nitrogen fixation, legume seed development, and perenniality and vegetative regrowth.

  11. The elephant shark methylome reveals conservation of epigenetic regulation across jawed vertebrates.

    Science.gov (United States)

    Peat, Julian R; Ortega-Recalde, Oscar; Kardailsky, Olga; Hore, Timothy A

    2017-01-01

    Methylation of CG dinucleotides constitutes a critical system of epigenetic memory in bony vertebrates, where it modulates gene expression and suppresses transposon activity. The genomes of studied vertebrates are pervasively hypermethylated, with the exception of regulatory elements such as transcription start sites (TSSs), where the presence of methylation is associated with gene silencing. This system is not found in the sparsely methylated genomes of invertebrates, and establishing how it arose during early vertebrate evolution is impeded by a paucity of epigenetic data from basal vertebrates.  We perform whole-genome bisulfite sequencing to generate the first genome-wide methylation profiles of a cartilaginous fish, the elephant shark Callorhinchus milii. Employing these to determine the elephant shark methylome structure and its relationship with expression, we compare this with higher vertebrates and an invertebrate chordate using published methylation and transcriptome data.  Results: Like higher vertebrates, the majority of elephant shark CG sites are highly methylated, and methylation is abundant across the genome rather than patterned in the mosaic configuration of invertebrates. This global hypermethylation includes transposable elements and the bodies of genes at all expression levels. Significantly, we document an inverse relationship between TSS methylation and expression in the elephant shark, supporting the presence of the repressive regulatory architecture shared by higher vertebrates.  Our demonstration that methylation patterns in a cartilaginous fish are characteristic of higher vertebrates imply the conservation of this epigenetic modification system across jawed vertebrates separated by 465 million years of evolution. In addition, these findings position the elephant shark as a valuable model to explore the evolutionary history and function of vertebrate methylation.

  12. Zebrafish eda and edar mutants reveal conserved and ancestral roles of ectodysplasin signaling in vertebrates.

    Directory of Open Access Journals (Sweden)

    Matthew P Harris

    2008-10-01

    Full Text Available The genetic basis of the development and variation of adult form of vertebrates is not well understood. To address this problem, we performed a mutant screen to identify genes essential for the formation of adult skeletal structures of the zebrafish. Here, we describe the phenotypic and molecular characterization of a set of mutants showing loss of adult structures of the dermal skeleton, such as the rays of the fins and the scales, as well as the pharyngeal teeth. The mutations represent adult-viable, loss of function alleles in the ectodysplasin (eda and ectodysplasin receptor (edar genes. These genes are frequently mutated in the human hereditary disease hypohidrotic ectodermal dysplasia (HED; OMIM 224900, 305100 that affects the development of integumentary appendages such as hair and teeth. We find mutations in zebrafish edar that affect similar residues as mutated in human cases of HED and show similar phenotypic consequences. eda and edar are not required for early zebrafish development, but are rather specific for the development of adult skeletal and dental structures. We find that the defects of the fins and scales are due to the role of Eda signaling in organizing epidermal cells into discrete signaling centers of the scale epidermal placode and fin fold. Our genetic analysis demonstrates dose-sensitive and organ-specific response to alteration in levels of Eda signaling. In addition, we show substantial buffering of the effect of loss of edar function in different genetic backgrounds, suggesting canalization of this developmental system. We uncover a previously unknown role of Eda signaling in teleosts and show conservation of the developmental mechanisms involved in the formation and variation of both integumentary appendages and limbs. Lastly, our findings point to the utility of adult genetic screens in the zebrafish in identifying essential developmental processes involved in human disease and in morphological evolution.

  13. Zebrafish eda and edar Mutants Reveal Conserved and Ancestral Roles of Ectodysplasin Signaling in Vertebrates

    Science.gov (United States)

    Harris, Matthew P.; Rohner, Nicolas; Schwarz, Heinz; Perathoner, Simon; Konstantinidis, Peter; Nüsslein-Volhard, Christiane

    2008-01-01

    The genetic basis of the development and variation of adult form of vertebrates is not well understood. To address this problem, we performed a mutant screen to identify genes essential for the formation of adult skeletal structures of the zebrafish. Here, we describe the phenotypic and molecular characterization of a set of mutants showing loss of adult structures of the dermal skeleton, such as the rays of the fins and the scales, as well as the pharyngeal teeth. The mutations represent adult-viable, loss of function alleles in the ectodysplasin (eda) and ectodysplasin receptor (edar) genes. These genes are frequently mutated in the human hereditary disease hypohidrotic ectodermal dysplasia (HED; OMIM 224900, 305100) that affects the development of integumentary appendages such as hair and teeth. We find mutations in zebrafish edar that affect similar residues as mutated in human cases of HED and show similar phenotypic consequences. eda and edar are not required for early zebrafish development, but are rather specific for the development of adult skeletal and dental structures. We find that the defects of the fins and scales are due to the role of Eda signaling in organizing epidermal cells into discrete signaling centers of the scale epidermal placode and fin fold. Our genetic analysis demonstrates dose-sensitive and organ-specific response to alteration in levels of Eda signaling. In addition, we show substantial buffering of the effect of loss of edar function in different genetic backgrounds, suggesting canalization of this developmental system. We uncover a previously unknown role of Eda signaling in teleosts and show conservation of the developmental mechanisms involved in the formation and variation of both integumentary appendages and limbs. Lastly, our findings point to the utility of adult genetic screens in the zebrafish in identifying essential developmental processes involved in human disease and in morphological evolution. PMID:18833299

  14. The strawberry (Fragariaxananassa) fruit-specific rhamnogalacturonate lyase 1 (FaRGLyase1) gene encodes an enzyme involved in the degradation of cell-wall middle lamellae.

    Science.gov (United States)

    Molina-Hidalgo, Francisco J; Franco, Antonio R; Villatoro, Carmen; Medina-Puche, Laura; Mercado, José A; Hidalgo, Miguel A; Monfort, Amparo; Caballero, José Luis; Muñoz-Blanco, Juan; Blanco-Portales, Rosario

    2013-04-01

    Pectins are essential components of primary plant cell walls and middle lamellae, and are related to the consistency of the fruit and its textural changes during ripening. In fact, strawberries become soft as the middle lamellae of cortical parenchyma cells are extensively degraded during ripening, leading to the observed short post-harvest shelf life. Using a custom-made oligonucleotide-based strawberry microarray platform, a putative rhamnogalacturonate lyase gene (FaRGlyase1) was identified. Bioinformatic analysis of the FaRGlyase1 sequence allowed the identification of a conserved rhamnogalacturonate lyase domain, which was also present in other putative RGlyase sequences deposited in the databases. Expression of FaRGlyase1 occurred mainly in the receptacle, concurrently with ripening, and it was positively regulated by abscisic acid and negatively by auxins. FaRGLyase1 gene expression was transiently silenced by injecting live Agrobacterium cells harbouring RNA interference constructs into fruit receptacles. Light and electron microscopy analyses of these transiently silenced fruits revealed that this gene is involved in the degradation of pectins present in the middle lamella region between parenchymatic cells. In addition, genetic linkage association analyses in a strawberry-segregating population showed that FaRGLyase1 is linked to a quantitative trait loci linkage group related to fruit hardness and firmness. The results showed that FaRGlyase1 could play an important role in the fruit ripening-related softening process that reduces strawberry firmness and post-harvest life.

  15. Evolutionarily Conserved Pattern of Interactions in a Protein Revealed by Local Thermal Expansion Properties.

    Science.gov (United States)

    Dellarole, Mariano; Caro, Jose A; Roche, Julien; Fossat, Martin; Barthe, Philippe; García-Moreno E, Bertrand; Royer, Catherine A; Roumestand, Christian

    2015-07-29

    The way in which the network of intramolecular interactions determines the cooperative folding and conformational dynamics of a protein remains poorly understood. High-pressure NMR spectroscopy is uniquely suited to examine this problem because it combines the site-specific resolution of the NMR experiments with the local character of pressure perturbations. Here we report on the temperature dependence of the site-specific volumetric properties of various forms of staphylococcal nuclease (SNase), including three variants with engineered internal cavities, as measured with high-pressure NMR spectroscopy. The strong temperature dependence of pressure-induced unfolding arises from poorly understood differences in thermal expansion between the folded and unfolded states. A significant inverse correlation was observed between the global thermal expansion of the folded proteins and the number of strong intramolecular hydrogen bonds, as determined by the temperature coefficient of the backbone amide chemical shifts. Comparison of the identity of these strong H-bonds with the co-evolution of pairs of residues in the SNase protein family suggests that the architecture of the interactions detected in the NMR experiments could be linked to a functional aspect of the protein. Moreover, the temperature dependence of the residue-specific volume changes of unfolding yielded residue-specific differences in expansivity and revealed how mutations impact intramolecular interaction patterns. These results show that intramolecular interactions in the folded states of proteins impose constraints against thermal expansion and that, hence, knowledge of site-specific thermal expansivity offers insight into the patterns of strong intramolecular interactions and other local determinants of protein stability, cooperativity, and potentially also of function.

  16. Characterization of C-S lyase from Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365 and its potential role in food flavour applications.

    Science.gov (United States)

    Allegrini, Alessandra; Astegno, Alessandra; La Verde, Valentina; Dominici, Paola

    2016-12-21

    Volatile thiols have substantial impact on the aroma of many beverages and foods. Thus, the control of their formation, which has been linked to C-S lyase enzymatic activities, is of great significance in industrial applications involving food flavours. Herein, we have carried out a spectroscopic and functional characterization of a putative pyridoxal 5'-phosphate (PLP)-dependent C-S lyase from the lactic acid bacterium Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365 (LDB C-S lyase). Recombinant LDB C-S lyase exists as a tetramer in solution and shows spectral properties of enzymes containing PLP as cofactor. The enzyme has a broad substrate specificity toward sulphur-containing amino acids with aminoethyl-L-cysteine and L-cystine being the most effective substrates over L-cysteine and L-cystathionine. Notably, the protein also reveals cysteine-S-conjugate β-lyase activity in vitro, and is able to cleave a cysteinylated substrate precursor into the corresponding flavour-contributing thiol, with a catalytic efficiency higher than L-cystathionine. Contrary to similar enzymes of other lactic acid bacteria however, LDB C-S lyase is not capable of α,γ-elimination activity towards L-methionine to produce methanethiol, which is a significant compound in flavour development. Based on our results, future developments can be expected regarding the flavour-forming potential of Lactobacillus C-S lyase and its use in enhancing food flavours.

  17. Structural and Biophysical Analysis of BST-2/Tetherin Ectodomains Reveals an Evolutionary Conserved Design to Inhibit Virus Release

    Energy Technology Data Exchange (ETDEWEB)

    Swiecki, M.; Allaire, M.; Scheaffer, S.; Fremont, D.H.; et.al.

    2011-01-28

    BST-2/tetherin is a host antiviral molecule that functions to potently inhibit the release of enveloped viruses from infected cells. In return, viruses have evolved antagonists to this activity. BST-2 traps budding virions by using two separate membrane-anchoring regions that simultaneously incorporate into the host and viral membranes. Here, we detailed the structural and biophysical properties of the full-length BST-2 ectodomain, which spans the two membrane anchors. The 1.6-{angstrom} crystal structure of the complete mouse BST-2 ectodomain reveals an {approx}145-{angstrom} parallel dimer in an extended {alpha}-helix conformation that predominantly forms a coiled coil bridged by three intermolecular disulfides that are required for stability. Sequence analysis in the context of the structure revealed an evolutionarily conserved design that destabilizes the coiled coil, resulting in a labile superstructure, as evidenced by solution x-ray scattering displaying bent conformations spanning 150 and 180 {angstrom} for the mouse and human BST-2 ectodomains, respectively. Additionally, crystal packing analysis revealed possible curvature-sensing tetrameric structures that may aid in proper placement of BST-2 during the genesis of viral progeny. Overall, this extended coiled-coil structure with inherent plasticity is undoubtedly necessary to accommodate the dynamics of viral budding while ensuring separation of the anchors.

  18. Structural and biophysical analysis of BST-2/tetherin ectodomains reveals an evolutionary conserved design to inhibit virus release.

    Science.gov (United States)

    Swiecki, Melissa; Scheaffer, Suzanne M; Allaire, Marc; Fremont, Daved H; Colonna, Marco; Brett, Tom J

    2011-01-28

    BST-2/tetherin is a host antiviral molecule that functions to potently inhibit the release of enveloped viruses from infected cells. In return, viruses have evolved antagonists to this activity. BST-2 traps budding virions by using two separate membrane-anchoring regions that simultaneously incorporate into the host and viral membranes. Here, we detailed the structural and biophysical properties of the full-length BST-2 ectodomain, which spans the two membrane anchors. The 1.6-Å crystal structure of the complete mouse BST-2 ectodomain reveals an ∼145-Å parallel dimer in an extended α-helix conformation that predominantly forms a coiled coil bridged by three intermolecular disulfides that are required for stability. Sequence analysis in the context of the structure revealed an evolutionarily conserved design that destabilizes the coiled coil, resulting in a labile superstructure, as evidenced by solution x-ray scattering displaying bent conformations spanning 150 and 180 Å for the mouse and human BST-2 ectodomains, respectively. Additionally, crystal packing analysis revealed possible curvature-sensing tetrameric structures that may aid in proper placement of BST-2 during the genesis of viral progeny. Overall, this extended coiled-coil structure with inherent plasticity is undoubtedly necessary to accommodate the dynamics of viral budding while ensuring separation of the anchors.

  19. High-Throughput Sequencing Reveals Diverse Sets of Conserved, Nonconserved, and Species-Specific miRNAs in Jute.

    Science.gov (United States)

    Islam, Md Tariqul; Ferdous, Ahlan Sabah; Najnin, Rifat Ara; Sarker, Suprovath Kumar; Khan, Haseena

    2015-01-01

    MicroRNAs play a pivotal role in regulating a broad range of biological processes, acting by cleaving mRNAs or by translational repression. A group of plant microRNAs are evolutionarily conserved; however, others are expressed in a species-specific manner. Jute is an agroeconomically important fibre crop; nonetheless, no practical information is available for microRNAs in jute to date. In this study, Illumina sequencing revealed a total of 227 known microRNAs and 17 potential novel microRNA candidates in jute, of which 164 belong to 23 conserved families and the remaining 63 belong to 58 nonconserved families. Among a total of 81 identified microRNA families, 116 potential target genes were predicted for 39 families and 11 targets were predicted for 4 among the 17 identified novel microRNAs. For understanding better the functions of microRNAs, target genes were analyzed by Gene Ontology and their pathways illustrated by KEGG pathway analyses. The presence of microRNAs identified in jute was validated by stem-loop RT-PCR followed by end point PCR and qPCR for randomly selected 20 known and novel microRNAs. This study exhaustively identifies microRNAs and their target genes in jute which will ultimately pave the way for understanding their role in this crop and other crops.

  20. High-Throughput Sequencing Reveals Diverse Sets of Conserved, Nonconserved, and Species-Specific miRNAs in Jute

    Directory of Open Access Journals (Sweden)

    Md. Tariqul Islam

    2015-01-01

    Full Text Available MicroRNAs play a pivotal role in regulating a broad range of biological processes, acting by cleaving mRNAs or by translational repression. A group of plant microRNAs are evolutionarily conserved; however, others are expressed in a species-specific manner. Jute is an agroeconomically important fibre crop; nonetheless, no practical information is available for microRNAs in jute to date. In this study, Illumina sequencing revealed a total of 227 known microRNAs and 17 potential novel microRNA candidates in jute, of which 164 belong to 23 conserved families and the remaining 63 belong to 58 nonconserved families. Among a total of 81 identified microRNA families, 116 potential target genes were predicted for 39 families and 11 targets were predicted for 4 among the 17 identified novel microRNAs. For understanding better the functions of microRNAs, target genes were analyzed by Gene Ontology and their pathways illustrated by KEGG pathway analyses. The presence of microRNAs identified in jute was validated by stem-loop RT-PCR followed by end point PCR and qPCR for randomly selected 20 known and novel microRNAs. This study exhaustively identifies microRNAs and their target genes in jute which will ultimately pave the way for understanding their role in this crop and other crops.

  1. Cloning and characterization of the first polysaccharide lyase family 6 oligoalginate lyase from marine Shewanella sp. Kz7.

    Science.gov (United States)

    Li, Shangyong; Wang, Linna; Han, Feng; Gong, Qianhong; Yu, Wengong

    2016-01-01

    Alginate, the most abundant carbohydrate in brown macroalgae, is widely used in the food and pharmaceutical industries. Recently, alginate has attracted increasing attention, as it may serve as an alternative biomass for the production of biofuel. The degradation of alginate into monomeric units is the prerequisite for bioethanol production. All known oligoalginate lyases belong to the polysaccharide lyase (PL) family 7, 14, 15 and 17, and most of them preferred to degrade the polyM blocks to yield 4-deoxy-l-erythro-5-hexoseulose uronic acid as the primary product. In this study, we cloned an oligoalginate lyase gene, oalS6, from Shewanella sp. Kz7 and expressed it in Escherichia coli. The PL family 6 oligoalginate lyase (OalS6) has no significant sequence similarity with other known oligoalginate lyases. OalS6 contains a chondroitinase-like domain and was assigned to the PL family 6. This lyase is an exo-type oligoalginate lyase and prefer to depolymerize polyG block into 2, 4, 5, 6-tetrahydroxytetrahydro-2H-pyran-2-carboxylic acid. All of these results indicate that OalS6 is a novel oligoalginate lyase that is structurally and functionally different from other known oligoalginate lyases. This finding provides new insights into the development of biofuel processing biotechnologies from seaweed.

  2. Conservative Management of a Congenital Seminal Vesicle Cyst Associated with Ipsilateral Renal Agenesis Revealed by Cystitis: One Case Report

    Directory of Open Access Journals (Sweden)

    Youness Ahallal

    2011-01-01

    Full Text Available Seminal vesicle cyst is an extremely rare disease. Its association with ipsilateral renal agenesis is even more exceptional. We present herein one case of a 16-year-old male who presented with a four-month history of lower urinary tract symptoms (LUTSs and micturition pain. The digital rectal examination revealed a small mass arising from the prostate. The urine culture showed that E. coli is sensitive to all antibiotics tested. Transrectal ultrasound (TRUS revealed a cystic mass in the outer prostate. Seminal vesicle cyst and left renal agenesis were confirmed by magnetic resonance imaging (MRI. Maximum flow (Qmax at uroflow was greater than 15 mL/sec. We therefore decided to manage this disease conservatively with alpha blockers and antibiotics. After 6-month' followup the patient did not report any complain and the uroflow test was similar to a normal urination. From one case report and literature review, the authors suggest a diagnostic and therapeutic strategy for the management of this rare condition.

  3. A novel fragile X syndrome mutation reveals a conserved role for the carboxy-terminus in FMRP localization and function.

    Science.gov (United States)

    Okray, Zeynep; de Esch, Celine E F; Van Esch, Hilde; Devriendt, Koen; Claeys, Annelies; Yan, Jiekun; Verbeeck, Jelle; Froyen, Guy; Willemsen, Rob; de Vrij, Femke M S; Hassan, Bassem A

    2015-02-17

    Loss of function of the FMR1 gene leads to fragile X syndrome (FXS), the most common form of intellectual disability. The loss of FMR1 function is usually caused by epigenetic silencing of the FMR1 promoter leading to expansion and subsequent methylation of a CGG repeat in the 5' untranslated region. Very few coding sequence variations have been experimentally characterized and shown to be causal to the disease. Here, we describe a novel FMR1 mutation and reveal an unexpected nuclear export function for the C-terminus of FMRP. We screened a cohort of patients with typical FXS symptoms who tested negative for CGG repeat expansion in the FMR1 locus. In one patient, we identified a guanine insertion in FMR1 exon 15. This mutation alters the open reading frame creating a short novel C-terminal sequence, followed by a stop codon. We find that this novel peptide encodes a functional nuclear localization signal (NLS) targeting the patient FMRP to the nucleolus in human cells. We also reveal an evolutionarily conserved nuclear export function associated with the endogenous C-terminus of FMRP. In vivo analyses in Drosophila demonstrate that a patient-mimetic mutation alters the localization and function of Dfmrp in neurons, leading to neomorphic neuronal phenotypes.

  4. Kinetic and thermodynamic properties of alginate lyase and cellulase co-produced by Exiguobacterium species Alg-S5.

    Science.gov (United States)

    Mohapatra, Bidyut R

    2017-05-01

    In an effort to screen out the alginolytic and cellulolytic bacteria from the putrefying invasive seaweed Sargassum species accumulated off Barbados' coast, a potent bacterial strain was isolated. This bacterium, which simultaneously produced alginate lyase and cellulase, was identified as Exiguobacterium sp. Alg-S5 via the phylogenetic approach targeting the 16S rRNA gene. The co-produced alginate lyase and cellulase exhibited maximal enzymatic activity at pH 7.5 and at 40°C and 45°C, respectively. The Km and Vmax values recorded as 0.91mg/mL and 21.8U/mg-protein, respectively, for alginate lyase, and 10.9mg/mL and 74.6U/mg-protein, respectively, for cellulase. First order kinetic analysis of the thermal denaturation of the co-produced alginate lyase and cellulase in the temperature range from 40°C to 55°C revealed that both the enzymes were thermodynamically efficient by displaying higher activation energy and enthalpy of denaturation. These enzymatic properties indicate the potential industrial importance of this bacterium in algal biomass conversion. This appears to be the first report on assessing the efficacy of a bacterium for the co-production of alginate lyase and cellulase.

  5. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site.

    Science.gov (United States)

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-04-20

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide.

  6. Combined Use of Systematic Conservation Planning, Species Distribution Modelling, and Connectivity Analysis Reveals Severe Conservation Gaps in a Megadiverse Country (Peru)

    Science.gov (United States)

    Fajardo, Javier; Lessmann, Janeth; Bonaccorso, Elisa; Devenish, Christian; Muñoz, Jesús

    2014-01-01

    Conservation planning is crucial for megadiverse countries where biodiversity is coupled with incomplete reserve systems and limited resources to invest in conservation. Using Peru as an example of a megadiverse country, we asked whether the national system of protected areas satisfies biodiversity conservation needs. Further, to complement the existing reserve system, we identified and prioritized potential conservation areas using a combination of species distribution modeling, conservation planning and connectivity analysis. Based on a set of 2,869 species, including mammals, birds, amphibians, reptiles, butterflies, and plants, we used species distribution models to represent species' geographic ranges to reduce the effect of biased sampling and partial knowledge about species' distributions. A site-selection algorithm then searched for efficient and complementary proposals, based on the above distributions, for a more representative system of protection. Finally, we incorporated connectivity among areas in an innovative post-hoc analysis to prioritize those areas maximizing connectivity within the system. Our results highlight severe conservation gaps in the Coastal and Andean regions, and we propose several areas, which are not currently covered by the existing network of protected areas. Our approach helps to find areas that contribute to creating a more representative, connected and efficient network. PMID:25479411

  7. Purification, stabilization and characterization of tomato fatty acid hydroperoxide lyase

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Suurmeijer, C.N.S.P.; Pérez-Gilabert, M.; Unen, D.-J. van; Hijden, H.T.W.M. van der; Veldink, G.A.

    2000-01-01

    Fatty acid hydroperoxide lyase (HPO-lyase) was purified 300-fold from tomatoes. The enzymatic activity appeared to be very unstable, but addition of Triton X100 and beta-mercaptoethanol to the buffer yielded an active enzyme that could be stored for several months at −80°C. The enzyme was inhibited

  8. Characterization of pectate lyase A from Aspergillus niger

    NARCIS (Netherlands)

    Benen, J.A.E.; Parenicova, L.; Kester, H.C.M.; Visser, J.

    2001-01-01

    The Aspergillus niger plyA gene encoding pectate lyase A (EC 4.2.99.3) was cloned from a chromosomal EMBL4 library using the Aspergillus nidulans pectate lyase encoding gene [Dean, R. A., and Timberlake, W. E. (1989) Plant Cell 1, 275-284] as a probe. The plyA gene was overexpressed using a promoter

  9. Cysteine S-conjugate β-lyases

    OpenAIRE

    Arthur J. L. Cooper; Krasnikov, Boris F.; Pinto, John T.; Bruschi, Sam A.

    2010-01-01

    Cysteine S-conjugate β-lyases are pyridoxal 5′-phosphate (PLP)-containing enzymes that catalyze the conversion of cysteine S-conjugates [RSCH2CH(NH3+)CO2−] and selenium Se-conjugates [RSeCH2CH(NH3+)CO2−] that contain a leaving group in the β position to pyruvate, ammonium and a sulfur-containing fragment (RSH) or selenium-containing fragment (RSeH), respectively. At least ten PLP enzymes catalyze β-elimination reactions with such cysteine S-conjugates. All are enzymes involved in amino acid m...

  10. Direct Spectrophotometric Assay for Benzaldehyde Lyase Activity

    Directory of Open Access Journals (Sweden)

    Dessy Natalia

    2011-01-01

    Full Text Available Benzaldehyde lyase from Pseudomonas fluorescens Biovar I. (BAL, EC 4.1.2.38 is a versatile catalyst for the organic synthesis of chiral α-hydroxy ketones. To allow fast assessment of enzyme activity, a direct spectrophotometric assay is desirable. Here, a new robust and easy-to-handle assay based on UV absorption is presented. The assay developed is based on the ligation of the α-hydroxy ketone (R-2,2′-furoin from 2-furaldehyde. A robust assay with direct monitoring of the product is facilitated with a convenient concentration working range minimising experimental associated with low concentrations.

  11. Transcriptomic analysis of tail regeneration in the lizard Anolis carolinensis reveals activation of conserved vertebrate developmental and repair mechanisms.

    Directory of Open Access Journals (Sweden)

    Elizabeth D Hutchins

    Full Text Available Lizards, which are amniote vertebrates like humans, are able to lose and regenerate a functional tail. Understanding the molecular basis of this process would advance regenerative approaches in amniotes, including humans. We have carried out the first transcriptomic analysis of tail regeneration in a lizard, the green anole Anolis carolinensis, which revealed 326 differentially expressed genes activating multiple developmental and repair mechanisms. Specifically, genes involved in wound response, hormonal regulation, musculoskeletal development, and the Wnt and MAPK/FGF pathways were differentially expressed along the regenerating tail axis. Furthermore, we identified 2 microRNA precursor families, 22 unclassified non-coding RNAs, and 3 novel protein-coding genes significantly enriched in the regenerating tail. However, high levels of progenitor/stem cell markers were not observed in any region of the regenerating tail. Furthermore, we observed multiple tissue-type specific clusters of proliferating cells along the regenerating tail, not localized to the tail tip. These findings predict a different mechanism of regeneration in the lizard than the blastema model described in the salamander and the zebrafish, which are anamniote vertebrates. Thus, lizard tail regrowth involves the activation of conserved developmental and wound response pathways, which are potential targets for regenerative medical therapies.

  12. Global Analysis of Gene Expression Profiles in Brassica napus Developing Seeds Reveals a Conserved Lipid Metabolism Regulation with Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Ya Niu; Guo-Zhang Wu; Rui Ye; Wen-Hui Lin; Qiu-Ming Shi; Liang-Jiao Xue; Xiao-Dong Xu; Yao Li; Yu-Guang; Hong-Wei Xue

    2009-01-01

    In order to study Brassica napus fatty acid (FA) metabolism and relevant regulatory networks, a systematic identification of fatty acid (FA) biosynthesis-related genes was conducted. Following gene identification, gene expression profiles during B. napus seed development and FA metabolism were performed by cDNA chip hybridization (>8000 EST clones from seed). The results showed that FA biosynthesis and regulation, and carbon flux, were conserved between B. napus and Arabidopsis. However, a more critical role of starch metabolism was detected for B. napus seed FA metabolism and storage-component accumulation when compared with Arabidopsis. In addition, a crucial stage for the transition of seed-to-sink tissue was 17-21 d after flowering (DAF), whereas FA biosynthesis-related genes were highly expressed pri-marily at 21 DAF. Hormone (auxin and jasmonate) signaling is found to be important for FA metabolism. This study helps to reveal the global regulatory network of FA metabolism in developing B. napus seeds.

  13. Transcriptomic analysis of tail regeneration in the lizard Anolis carolinensis reveals activation of conserved vertebrate developmental and repair mechanisms.

    Science.gov (United States)

    Hutchins, Elizabeth D; Markov, Glenn J; Eckalbar, Walter L; George, Rajani M; King, Jesse M; Tokuyama, Minami A; Geiger, Lauren A; Emmert, Nataliya; Ammar, Michael J; Allen, April N; Siniard, Ashley L; Corneveaux, Jason J; Fisher, Rebecca E; Wade, Juli; DeNardo, Dale F; Rawls, J Alan; Huentelman, Matthew J; Wilson-Rawls, Jeanne; Kusumi, Kenro

    2014-01-01

    Lizards, which are amniote vertebrates like humans, are able to lose and regenerate a functional tail. Understanding the molecular basis of this process would advance regenerative approaches in amniotes, including humans. We have carried out the first transcriptomic analysis of tail regeneration in a lizard, the green anole Anolis carolinensis, which revealed 326 differentially expressed genes activating multiple developmental and repair mechanisms. Specifically, genes involved in wound response, hormonal regulation, musculoskeletal development, and the Wnt and MAPK/FGF pathways were differentially expressed along the regenerating tail axis. Furthermore, we identified 2 microRNA precursor families, 22 unclassified non-coding RNAs, and 3 novel protein-coding genes significantly enriched in the regenerating tail. However, high levels of progenitor/stem cell markers were not observed in any region of the regenerating tail. Furthermore, we observed multiple tissue-type specific clusters of proliferating cells along the regenerating tail, not localized to the tail tip. These findings predict a different mechanism of regeneration in the lizard than the blastema model described in the salamander and the zebrafish, which are anamniote vertebrates. Thus, lizard tail regrowth involves the activation of conserved developmental and wound response pathways, which are potential targets for regenerative medical therapies.

  14. Characterisation of worldwide Helicobacter pylori strains reveals genetic conservation and essentiality of serine protease HtrA.

    Science.gov (United States)

    Tegtmeyer, Nicole; Moodley, Yoshan; Yamaoka, Yoshio; Pernitzsch, Sandy Ramona; Schmidt, Vanessa; Traverso, Francisco Rivas; Schmidt, Thomas P; Rad, Roland; Yeoh, Khay Guan; Bow, Ho; Torres, Javier; Gerhard, Markus; Schneider, Gisbert; Wessler, Silja; Backert, Steffen

    2016-03-01

    HtrA proteases and chaperones exhibit important roles in periplasmic protein quality control and stress responses. The genetic inactivation of htrA has been described for many bacterial pathogens. However, in some cases such as the gastric pathogen Helicobacter pylori, HtrA is secreted where it cleaves the tumour-suppressor E-cadherin interfering with gastric disease development, but the generation of htrA mutants is still lacking. Here, we show that the htrA gene locus is highly conserved in worldwide strains. HtrA presence was confirmed in 992 H. pylori isolates in gastric biopsy material from infected patients. Differential RNA-sequencing (dRNA-seq) indicated that htrA is encoded in an operon with two subsequent genes, HP1020 and HP1021. Genetic mutagenesis and complementation studies revealed that HP1020 and HP1021, but not htrA, can be mutated. In addition, we demonstrate that suppression of HtrA proteolytic activity with a newly developed inhibitor is sufficient to effectively kill H. pylori, but not other bacteria. We show that Helicobacter htrA is an essential bifunctional gene with crucial intracellular and extracellular functions. Thus, we describe here the first microbe in which htrA is an indispensable gene, a situation unique in the bacterial kingdom. HtrA can therefore be considered a promising new target for anti-bacterial therapy.

  15. 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

  16. Probing the Active Center of Benzaldehyde Lyase with Substitutions and the Pseudosubstrate Analogue Benzoylphosphonic Acid Methyl Ester

    Energy Technology Data Exchange (ETDEWEB)

    Brandt, Gabriel S.; Nemeria, Natalia; Chakraborty, Sumit; McLeish, Michael J.; Yep, Alejandra; Kenyon, George L.; Petsko, Gregory A.; Jordan, Frank; Ringe, Dagmar (Rutgers); (Michigan); (Brandeis)

    2008-07-28

    Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of (R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg{sup 2+} as cofactors. The enzyme is important for the chemoenzymatic synthesis of a wide range of compounds via its carboligation reaction mechanism. In addition to its principal functions, BAL can slowly decarboxylate aromatic amino acids such as benzoylformic acid. It is also intriguing mechanistically due to the paucity of acid-base residues at the active center that can participate in proton transfer steps thought to be necessary for these types of reactions. Here methyl benzoylphosphonate, an excellent electrostatic analogue of benzoylformic acid, is used to probe the mechanism of benzaldehyde lyase. The structure of benzaldehyde lyase in its covalent complex with methyl benzoylphosphonate was determined to 2.49 {angstrom} (Protein Data Bank entry 3D7K) and represents the first structure of this enzyme with a compound bound in the active site. No large structural reorganization was detected compared to the complex of the enzyme with thiamin diphosphate. The configuration of the predecarboxylation thiamin-bound intermediate was clarified by the structure. Both spectroscopic and X-ray structural studies are consistent with inhibition resulting from the binding of MBP to the thiamin diphosphate in the active centers. We also delineated the role of His29 (the sole potential acid-base catalyst in the active site other than the highly conserved Glu50) and Trp163 in cofactor activation and catalysis by benzaldehyde lyase.

  17. Probing the active center of benzaldehyde lyase with substitutions and the pseudosubstrate analogue benzoylphosphonic acid methyl ester.

    Science.gov (United States)

    Brandt, Gabriel S; Nemeria, Natalia; Chakraborty, Sumit; McLeish, Michael J; Yep, Alejandra; Kenyon, George L; Petsko, Gregory A; Jordan, Frank; Ringe, Dagmar

    2008-07-22

    Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of ( R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg (2+) as cofactors. The enzyme is important for the chemoenzymatic synthesis of a wide range of compounds via its carboligation reaction mechanism. In addition to its principal functions, BAL can slowly decarboxylate aromatic amino acids such as benzoylformic acid. It is also intriguing mechanistically due to the paucity of acid-base residues at the active center that can participate in proton transfer steps thought to be necessary for these types of reactions. Here methyl benzoylphosphonate, an excellent electrostatic analogue of benzoylformic acid, is used to probe the mechanism of benzaldehyde lyase. The structure of benzaldehyde lyase in its covalent complex with methyl benzoylphosphonate was determined to 2.49 A (Protein Data Bank entry 3D7K ) and represents the first structure of this enzyme with a compound bound in the active site. No large structural reorganization was detected compared to the complex of the enzyme with thiamin diphosphate. The configuration of the predecarboxylation thiamin-bound intermediate was clarified by the structure. Both spectroscopic and X-ray structural studies are consistent with inhibition resulting from the binding of MBP to the thiamin diphosphate in the active centers. We also delineated the role of His29 (the sole potential acid-base catalyst in the active site other than the highly conserved Glu50) and Trp163 in cofactor activation and catalysis by benzaldehyde lyase.

  18. Transcriptomes of Eight Arabidopsis thaliana Accessions Reveal Core Conserved, Genotype- and Organ-Specific Responses to Flooding Stress1[OPEN

    Science.gov (United States)

    van Veen, Hans; Vashisht, Divya; Akman, Melis; Girke, Thomas; Mustroph, Angelika; Reinen, Emilie; Kooiker, Maarten; van Tienderen, Peter; Voesenek, Laurentius A.C.J.

    2016-01-01

    Climate change has increased the frequency and severity of flooding events, with significant negative impact on agricultural productivity. These events often submerge plant aerial organs and roots, limiting growth and survival due to a severe reduction in light reactions and gas exchange necessary for photosynthesis and respiration, respectively. To distinguish molecular responses to the compound stress imposed by submergence, we investigated transcriptomic adjustments to darkness in air and under submerged conditions using eight Arabidopsis (Arabidopsis thaliana) accessions differing significantly in sensitivity to submergence. Evaluation of root and rosette transcriptomes revealed an early transcriptional and posttranscriptional response signature that was conserved primarily across genotypes, although flooding susceptibility-associated and genotype-specific responses also were uncovered. Posttranscriptional regulation encompassed darkness- and submergence-induced alternative splicing of transcripts from pathways involved in the alternative mobilization of energy reserves. The organ-specific transcriptome adjustments reflected the distinct physiological status of roots and shoots. Root-specific transcriptome changes included marked up-regulation of chloroplast-encoded photosynthesis and redox-related genes, whereas those of the rosette were related to the regulation of development and growth processes. We identified a novel set of tolerance genes, recognized mainly by quantitative differences. These included a transcriptome signature of more pronounced gluconeogenesis in tolerant accessions, a response that included stress-induced alternative splicing. This study provides organ-specific molecular resolution of genetic variation in submergence responses involving interactions between darkness and low-oxygen constraints of flooding stress and demonstrates that early transcriptome plasticity, including alternative splicing, is associated with the ability to cope

  19. Mapping the transcription start points of the Staphylococcus aureus eap, emp, and vwb promoters reveals a conserved octanucleotide sequence that is essential for expression of these genes.

    Science.gov (United States)

    Harraghy, Niamh; Homerova, Dagmar; Herrmann, Mathias; Kormanec, Jan

    2008-01-01

    Mapping the transcription start points of the eap, emp, and vwb promoters revealed a conserved octanucleotide sequence (COS). Deleting this sequence abolished the expression of eap, emp, and vwb. However, electrophoretic mobility shift assays gave no evidence that this sequence was a binding site for SarA or SaeR, known regulators of eap and emp.

  20. 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.

  1. The ppuI-rsaL-ppuR quorum-sensing system regulates cellular motility, pectate lyase activity, and virulence in potato opportunistic pathogen Pseudomonas sp. StFLB209.

    Science.gov (United States)

    Kato, Taro; Morohoshi, Tomohiro; Someya, Nobutaka; Ikeda, Tsukasa

    2015-01-01

    Pseudomonas sp. StFLB209 was isolated from potato leaf as an N-acylhomoserine lactone (AHL)-producing bacterium and showed a close phylogenetic relationship with P. cichorii, a known plant pathogen. Although there are no reports of potato disease caused by pseudomonads in Japan, StFLB209 was pathogenic to potato leaf. In this study, we reveal the complete genome sequence of StFLB209, and show that the strain possesses a ppuI-rsaL-ppuR quorum-sensing system, the sequence of which shares a high similarity with that of Pseudomonas putida. Disruption of ppuI results in a loss of AHL production as well as remarkable reduction in motility. StFLB209 possesses strong pectate lyase activity and causes maceration on potato tuber and leaf, which was slightly reduced in the ppuI mutant. These results suggest that the quorum-sensing system is well conserved between StFLB209 and P. putida and that the system is essential for motility, full pectate lyase activity, and virulence in StFLB209.

  2. cDNA cloning and bacterial expression of a PL-14 alginate lyase from a herbivorous marine snail Littorina brevicula.

    Science.gov (United States)

    Rahman, Mohammad Matiur; Wang, Ling; Inoue, Akira; Ojima, Takao

    2012-10-01

    Herbivorous marine snails like Littorina species are known to possess alginate lyases in their digestive tracts. The Littorina enzymes have been identified as endolytic polymannuronate (poly(M)) lyases (EC 4.2.2.3); however, it is still unclear which polysaccharide-lyase family (PL) the Littorina enzymes belong to, since no complete primary structure of Littorina enzymes has been determined. Thus, in the present study, we analyzed the primary structure of LbAly28, a 28kDa alginate lyase isozyme of Littorina brevicula, by the cDNA method. LbAly28 cDNAs were amplified by PCR followed by 5'- and 3'-RACE PCRs from the L. brevicula hepatopancreas cDNA. A cDNA covering entire coding region of LbAly28 consisted of 1129bp and encoded an amino-acid sequence of 291 residues. The deduced amino-acid sequence comprised an initiation methionine, a putative signal peptide of 14 residues, a propeptide-like region of 16 residues, and a mature LbAly28 domain of 260 residues. The mature LbAly28 domain showed 43-53% amino-acid identities with other molluscan PL-14 enzymes. The catalytically important residues in PL-14 enzymes, which were identified in the Chlorella virus glucuronate-specific lyase vAL-1 and Aplysia poly(M) lyase AkAly30, were also conserved in LbAly28. Site-directed mutagenesis regarding these residues, that is, replacements of Lys94, Lys97, Thr121, Arg 123, Tyr135, and Tyr137 to Ala, decreased the activity of recombinant LbAly28 to various degrees. From these results we concluded that LbAly28 is a member of PL-14 alginate lyases. Besides the effects of above mutations, we noticed that the replacement of T121 by Ala changed the substrate preference of LbAly28. Namely, the activities toward sodium alginate and poly(MG)-block substrate increased and became comparable with the activity toward poly(M)-block substrate. This suggests that the region including T121 of LbAly28 closely relates to the recognition of poly(MG) region of alginate.

  3. Potato signal molecules that activate pectate lyase synthesis in Pectobacterium atrosepticum SCRI1043.

    Science.gov (United States)

    Tarasova, Nadezhda; Gorshkov, Vladimir; Petrova, Olga; Gogolev, Yuri

    2013-07-01

    A new type of plant-derived signal molecules that activate extracellular pectate lyase activity in phytopathogenic bacterium Pectobacterium atrosepticum SCRI1043 was revealed. These compounds were characterized and partially purified by means of several approaches including RT-PCR analysis, luminescence bioassay and HPLC fractionation. They were smaller than 1 kDa, thermoresistant, nonproteinaceous, hydrophilic, and slightly negatively charged molecules. Using gene expression analysis and bacterial biosensor assay the mode of activity of revealed compounds was studied. The possibility of their action through quorum sensing- and KdgR-mediated pathways was analyzed.

  4. The essential tyrosine-containing loop conformation and the role of the C-terminal multi-helix region in eukaryotic phenylalanine ammonia-lyases.

    Science.gov (United States)

    Pilbák, Sarolta; Tomin, Anna; Rétey, János; Poppe, László

    2006-03-01

    Besides the post-translationally cyclizing catalytic Ala-Ser-Gly triad, Tyr110 and its equivalents are of the most conserved residues in the active site of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), histidine ammonia-lyase (HAL, EC 4.3.1.3) and other related enzymes. The Tyr110Phe mutation results in the most pronounced inactivation of PAL indicating the importance of this residue. The recently published X-ray structures of PAL revealed that the Tyr110-loop was either missing (for Rhodospridium toruloides) or far from the active site (for Petroselinum crispum). In bacterial HAL ( approximately 500 amino acids) and plant and fungal PALs ( approximately 710 amino acids), a core PAL/HAL domain ( approximately 480 amino acids) with >or= 30% sequence identity along the different species is common. In plant and fungal PAL a approximately 100-residue long C-terminal multi-helix domain is present. The ancestor bacterial HAL is thermostable and, in all of its known X-ray structures, a Tyr83-loop-in arrangement has been found. Based on the HAL structures, a Tyr110-loop-in conformation of the P. crispum PAL structure was constructed by partial homology modeling, and the static and dynamic behavior of the loop-in/loop-out structures were compared. To study the role of the C-terminal multi-helix domain, Tyr-loop-in/loop-out model structures of two bacterial PALs (Streptomyces maritimus, 523 amino acids and Photorhabdus luminescens, 532 amino acids) lacking this C-terminal domain were also built. Molecular dynamics studies indicated that the Tyr-loop-in conformation was more rigid without the C-terminal multi-helix domain. On this basis it is hypothesized that a role of this C-terminal extension is to decrease the lifetime of eukaryotic PAL by destabilization, which might be important for the rapid responses in the regulation of phenylpropanoid biosynthesis.

  5. Cloning and characterization of the first polysaccharide lyase family 6 oligoalginate lyase from marine Shewanella sp. Kz7

    OpenAIRE

    Li, Shangyong; Wang, LinNa; Han, Feng; Gong, Qianhong; Yu, Wengong

    2015-01-01

    Alginate, the most abundant carbohydrate in brown macroalgae, is widely used in the food and pharmaceutical industries. Recently, alginate has attracted increasing attention, as it may serve as an alternative biomass for the production of biofuel. The degradation of alginate into monomeric units is the prerequisite for bioethanol production. All known oligoalginate lyases belong to the polysaccharide lyase (PL) family 7, 14, 15 and 17, and most of them preferred to degrade the polyM blocks to...

  6. High-Throughput Sequencing and Characterization of the Small RNA Transcriptome Reveal Features of Novel and Conserved MicroRNAs in Panax ginseng

    Science.gov (United States)

    Ma, Yimian; Yuan, Lichai; Lu, Shanfa

    2012-01-01

    microRNAs (miRNAs) play vital regulatory roles in many organisms through direct cleavage of transcripts, translational repression, or chromatin modification. Identification of miRNAs has been carried out in various plant species. However, no information is available for miRNAs from Panax ginseng, an economically significant medicinal plant species. Using the next generation high-throughput sequencing technology, we obtained 13,326,328 small RNA reads from the roots, stems, leaves and flowers of P. ginseng. Analysis of these small RNAs revealed the existence of a large, diverse and highly complicated small RNA population in P. ginseng. We identified 73 conserved miRNAs, which could be grouped into 33 families, and 28 non-conserved ones belonging to 9 families. Characterization of P. ginseng miRNA precursors revealed many features, such as production of two miRNAs from distinct regions of a precursor, clusters of two precursors in a transcript, and generation of miRNAs from both sense and antisense transcripts. It suggests the complexity of miRNA production in P. gingseng. Using a computational approach, we predicted for the conserved and non-conserved miRNA families 99 and 31 target genes, respectively, of which eight were experimentally validated. Among all predicted targets, only about 20% are conserved among various plant species, whereas the others appear to be non-conserved, indicating the diversity of miRNA functions. Consistently, many miRNAs exhibited tissue-specific expression patterns. Moreover, we identified five dehydration- and ten heat-responsive miRNAs and found the existence of a crosstalk among some of the stress-responsive miRNAs. Our results provide the first clue to the elucidation of miRNA functions in P. ginseng. PMID:22962612

  7. Mutational Studies on Resurrected Ancestral Proteins Reveal Conservation of Site-Specific Amino Acid Preferences throughout Evolutionary History

    Science.gov (United States)

    Risso, Valeria A.; Manssour-Triedo, Fadia; Delgado-Delgado, Asunción; Arco, Rocio; Barroso-delJesus, Alicia; Ingles-Prieto, Alvaro; Godoy-Ruiz, Raquel; Gavira, Jose A.; Gaucher, Eric A.; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M.

    2015-01-01

    Local protein interactions (“molecular context” effects) dictate amino acid replacements and can be described in terms of site-specific, energetic preferences for any different amino acid. It has been recently debated whether these preferences remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and molecular evolution modeling. Here, we take advantage of the recent availability of phenotypically supported laboratory resurrections of Precambrian thioredoxins and β-lactamases to experimentally address the change of site-specific amino acid preferences over long geological timescales. Extensive mutational analyses support the notion that evolutionary adjustment to a new amino acid may occur, but to a large extent this is insufficient to erase the primitive preference for amino acid replacements. Generally, site-specific amino acid preferences appear to remain conserved throughout evolutionary history despite local sequence divergence. We show such preference conservation to be readily understandable in molecular terms and we provide crystallographic evidence for an intriguing structural-switch mechanism: Energetic preference for an ancestral amino acid in a modern protein can be linked to reorganization upon mutation to the ancestral local structure around the mutated site. Finally, we point out that site-specific preference conservation naturally leads to one plausible evolutionary explanation for the existence of intragenic global suppressor mutations. PMID:25392342

  8. Multi-species sequence comparison reveals conservation of ghrelin gene-derived splice variants encoding a truncated ghrelin peptide.

    Science.gov (United States)

    Seim, Inge; Jeffery, Penny L; Thomas, Patrick B; Walpole, Carina M; Maugham, Michelle; Fung, Jenny N T; Yap, Pei-Yi; O'Keeffe, Angela J; Lai, John; Whiteside, Eliza J; Herington, Adrian C; Chopin, Lisa K

    2016-06-01

    The peptide hormone ghrelin is a potent orexigen produced predominantly in the stomach. It has a number of other biological actions, including roles in appetite stimulation, energy balance, the stimulation of growth hormone release and the regulation of cell proliferation. Recently, several ghrelin gene splice variants have been described. Here, we attempted to identify conserved alternative splicing of the ghrelin gene by cross-species sequence comparisons. We identified a novel human exon 2-deleted variant and provide preliminary evidence that this splice variant and in1-ghrelin encode a C-terminally truncated form of the ghrelin peptide, termed minighrelin. These variants are expressed in humans and mice, demonstrating conservation of alternative splicing spanning 90 million years. Minighrelin appears to have similar actions to full-length ghrelin, as treatment with exogenous minighrelin peptide stimulates appetite and feeding in mice. Forced expression of the exon 2-deleted preproghrelin variant mirrors the effect of the canonical preproghrelin, stimulating cell proliferation and migration in the PC3 prostate cancer cell line. This is the first study to characterise an exon 2-deleted preproghrelin variant and to demonstrate sequence conservation of ghrelin gene-derived splice variants that encode a truncated ghrelin peptide. This adds further impetus for studies into the alternative splicing of the ghrelin gene and the function of novel ghrelin peptides in vertebrates.

  9. Functional characterization of GmBZL2 (AtBZR1 like gene) reveals the conserved BR signaling regulation in Glycine max

    Science.gov (United States)

    Zhang, Yu; Zhang, Yan-Jie; Yang, Bao-Jun; Yu, Xian-Xian; Wang, Dun; Zu, Song-Hao; Xue, Hong-Wei; Lin, Wen-Hui

    2016-01-01

    Brassinosteroids (BRs) play key roles in plant growth and development, and regulate various agricultural traits. Enhanced BR signaling leads to increased seed number and yield in Arabidopsis bzr1-1D (AtBZR1P234L, gain-of-function mutant of the important transcription factor in BR signaling/effects). BR signal transduction pathway is well elucidated in Arabidopsis but less known in other species. Soybean is an important dicot crop producing edible oil and protein. Phylogenetic analysis reveals AtBZR1-like genes are highly conserved in angiosperm and there are 4 orthologues in soybean (GmBZL1-4). We here report the functional characterization of GmBZL2 (relatively highly expresses in flowers). The P234 site in AtBZR1 is conserved in GmBZL2 (P216) and mutation of GmBZL2P216L leads to GmBZL2 accumulation. GmBZL2P216L (GmBZL2*) in Arabidopsis results in enhanced BR signaling; including increased seed number per silique. GmBZL2* partially rescued the defects of bri1-5, further demonstrating the conserved function of GmBZL2 with AtBZR1. BR treatment promotes the accumulation, nuclear localization and dephosphorylation/phosphorylation ratio of GmBZL2, revealing that GmBZL2 activity is regulated conservatively by BR signaling. Our studies not only indicate the conserved regulatory mechanism of GmBZL2 and BR signaling pathway in soybean, but also suggest the potential application of GmBZL2 in soybean seed yield. PMID:27498784

  10. Enhancing RGI lyase thermostability by targeted single point mutations

    DEFF Research Database (Denmark)

    Silva, Inês R.; Larsen, Dorte Møller; Jers, Carsten

    2013-01-01

    experimental verification of the thermal stability of the designed mutants versus the original wild-type RGI lyase, several promising single point mutations were obtained, particularly in position Glu434 on the surface of the enzyme protein. The best mutant, Glu434Leu, produced a half-life of 31 min at 60 °C......, corresponding to a 1.6-fold improvement of the thermal stability compared to the original RGI lyase. Gly55Val was the second best mutation with a thermostability half-life increase of 27 min at 60 °C, and the best mutations following were Glu434Trp, Glu434Phe, and Glu434Tyr, respectively. The data verify......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...

  11. A novel, inducible, citral lyase purified from spores of Penicillium digitatum

    NARCIS (Netherlands)

    Wolken, W.A.M.; Loo, W.J.V. van; Tramper, J.; Werf, M.J. van der

    2002-01-01

    A novel lyase, combining hydratase and aldolase activity, that converts citral into methylheptenone and acetaldehyde, was purified from spores of Penicillium digitatum. Remarkably, citral lyase activity was induced 118-fold by incubating nongerminating spores with the substrate, citral. This cofacto

  12. A novel, inducible, citral lyase purified from spores of Penicillium digitatum

    NARCIS (Netherlands)

    Wolken, W.A.M.; Loo, W.J.V. van; Tramper, J.; Werf, M.J. van der

    2002-01-01

    A novel lyase, combining hydratase and aldolase activity, that converts citral into methylheptenone and acetaldehyde, was purified from spores of Penicillium digitatum. Remarkably, citral lyase activity was induced 118-fold by incubating nongerminating spores with the substrate, citral. This

  13. Long non-coding RNA discovery across the genus anopheles reveals conserved secondary structures within and beyond the Gambiae complex.

    Science.gov (United States)

    Jenkins, Adam M; Waterhouse, Robert M; Muskavitch, Marc A T

    2015-04-23

    Long non-coding RNAs (lncRNAs) have been defined as mRNA-like transcripts longer than 200 nucleotides that lack significant protein-coding potential, and many of them constitute scaffolds for ribonucleoprotein complexes with critical roles in epigenetic regulation. Various lncRNAs have been implicated in the modulation of chromatin structure, transcriptional and post-transcriptional gene regulation, and regulation of genomic stability in mammals, Caenorhabditis elegans, and Drosophila melanogaster. The purpose of this study is to identify the lncRNA landscape in the malaria vector An. gambiae and assess the evolutionary conservation of lncRNAs and their secondary structures across the Anopheles genus. Using deep RNA sequencing of multiple Anopheles gambiae life stages, we have identified 2,949 lncRNAs and more than 300 previously unannotated putative protein-coding genes. The lncRNAs exhibit differential expression profiles across life stages and adult genders. We find that across the genus Anopheles, lncRNAs display much lower sequence conservation than protein-coding genes. Additionally, we find that lncRNA secondary structure is highly conserved within the Gambiae complex, but diverges rapidly across the rest of the genus Anopheles. This study offers one of the first lncRNA secondary structure analyses in vector insects. Our description of lncRNAs in An. gambiae offers the most comprehensive genome-wide insights to date into lncRNAs in this vector mosquito, and defines a set of potential targets for the development of vector-based interventions that may further curb the human malaria burden in disease-endemic countries.

  14. Vertebrate beta-thymosins: conserved synteny reveals the relationship between those of bony fish and of land vertebrates.

    Science.gov (United States)

    Edwards, John

    2010-03-05

    Using conservation of synteny I show how the four thymosins expressed by teleost fish are related to the three of tetrapods, which is not evident from their protein sequences. This clarification was aided by identification of a novel thymosin of reptilians that replaces the beta10 thymosin of mammals. Recent reconstruction of the ancestral vertebrate genome suggests that divergence of beta-thymosins began with duplication preceding the two rounds of whole genome duplication. Copyright (c) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2015-11-01

    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-mediated conversions would be possible. To identify enzymes involved in the degradation of β-valine, a PsSBV1 gene library was prepared and used to complement the β-valine growth deficiency of a closely related Pseudomonas strain. This resulted in the identification of a gene encoding β-valinyl-coenzyme A ligase (BvaA) and two genes encoding β-valinyl-CoA ammonia lyases (BvaB1 and BvaB2). The BvaA protein demonstrated high sequence identity to several known phenylacetate CoA ligases. Purified BvaA enzyme did not convert phenyl acetic acid but was able to activate β-valine in an adenosine triphosphate (ATP)- and CoA-dependent manner. The substrate range of the enzyme appears to be narrow, converting only β-valine and to a lesser extent, 3-aminobutyrate and β-alanine. Characterization of BvaB1 and BvaB2 revealed that both enzymes were able to deaminate β-valinyl-CoA to produce 3-methylcrotonyl-CoA, a common intermediate in the leucine degradation pathway. Interestingly, BvaB1 and BvaB2 demonstrated no significant sequence identity to known CoA-dependent ammonia lyases, suggesting they belong to a new family of enzymes. BLAST searches revealed that BvaB1 and BvaB2 show high sequence identity to each other and to several enoyl-CoA hydratases, a class of enzymes that catalyze a similar reaction with water instead of amine as the leaving group.

  16. All-atom molecular dynamics simulations reveal significant differences in interaction between antimycin and conserved amino acid residues in bovine and bacterial bc1 complexes.

    Science.gov (United States)

    Kokhan, Oleksandr; Shinkarev, Vladimir P

    2011-02-02

    Antimycin A is the most frequently used specific and powerful inhibitor of the mitochondrial respiratory chain. We used all-atom molecular dynamics (MD) simulations to study the dynamic aspects of the interaction of antimycin A with the Q(i) site of the bacterial and bovine bc(1) complexes embedded in a membrane. The MD simulations revealed considerable conformational flexibility of antimycin and significant mobility of antimycin, as a whole, inside the Q(i) pocket. We conclude that many of the differences in antimycin binding observed in high-resolution x-ray structures may have a dynamic origin and result from fluctuations of protein and antimycin between multiple conformational states of similar energy separated by low activation barriers, as well as from the mobility of antimycin within the Q(i) pocket. The MD simulations also revealed a significant difference in interaction between antimycin and conserved amino acid residues in bovine and bacterial bc(1) complexes. The strong hydrogen bond between antimycin and conserved Asp-228 (bovine numeration) was observed to be frequently broken in the bacterial bc(1) complex and only rarely in the bovine bc(1) complex. In addition, the distances between antimycin and conserved His-201 and Lys-227 were consistently larger in the bacterial bc(1) complex. The observed differences could be responsible for a weaker interaction of antimycin with the bacterial bc(1) complex.

  17. A spectacular new Philippine monitor lizard reveals a hidden biogeographic boundary and a novel flagship species for conservation

    Science.gov (United States)

    Welton, Luke J.; Siler, Cameron D.; Bennett, Daniel; Diesmos, Arvin; Duya, M. Roy; Dugay, Roldan; Rico, Edmund Leo B.; Van Weerd, Merlijn; Brown, Rafe M.

    2010-01-01

    As humans continue to explore the last uncharted regions of the planet, discoveries of previously unknown species of large vertebrates have become infrequent. Here, we report on the discovery of a spectacular new species of giant, secretive, frugivorous, forest monitor lizard (Genus: Varanus) from the forests of the northern Philippines. Using data from morphology and mitochondrial and nuclear DNA sequences, we demonstrate the taxonomic distinctiveness of this new 2 m long species and provide insight into its historical biogeography and systematic affinities. Our molecular phylogenetic analyses indicate that the new species is closely related to Varanus olivaceus (from southern Luzon and nearby islands), but it differs from this and other varanids with respect to characteristics of scalation, colour pattern, body size, anatomy of the reproductive organs and genetic divergence. The new species appears to be restricted to forests of the central and northern Sierra Madre mountain range; it is separated from the range of V. olivaceus by a more than 150 km stretch that includes at least three low-elevation river valley barriers to dispersal. This discovery identifies a seldom-perceived biogeographic boundary and emphasizes the need for continued biodiversity research in the megadiverse conservation hotspot of the Philippines. It is anticipated that the new species will serve as an important flagship species for conservation efforts aimed at preserving the remaining forests of northern Luzon. PMID:20375042

  18. Solution structure of the porcine sapovirus VPg core reveals a stable three-helical bundle with a conserved surface patch.

    Science.gov (United States)

    Hwang, Hyo-Jeong; Min, Hye Jung; Yun, Hyosuk; Pelton, Jeffery G; Wemmer, David E; Cho, Kyoung-Oh; Kim, Jeong-Sun; Lee, Chul Won

    2015-04-17

    Viral protein genome-linked (VPg) proteins play a critical role in the life cycle of vertebrate and plant positive-sense RNA viruses by acting as a protein primer for genome replication and as a protein cap for translation initiation. Here we report the solution structure of the porcine sapovirus VPg core (VPg(C)) determined by multi-dimensional NMR spectroscopy. The structure of VPg(C) is composed of three α-helices stabilized by several conserved hydrophobic residues that form a helical bundle core similar to that of feline calicivirus VPg. The putative nucleotide acceptor Tyr956 within the first helix of the core is completely exposed to solvent accessible surface to facilitate nucleotidylation by viral RNA polymerase. Comparison of VPg structures suggests that the surface for nucleotidylation site is highly conserved among the Caliciviridae family, whereas the backbone core structures are different. These structural features suggest that caliciviruses share common mechanisms of VPg-dependent viral replication and translation. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Genetic variation in the Solanaceae fruit bearing species lulo and tree tomato revealed by Conserved Ortholog (COSII markers

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    Felix Enciso-Rodríguez

    2010-01-01

    Full Text Available The Lulo or naranjilla (Solanum quitoense Lam. and the tree tomato or tamarillo (Solanum betaceum Cav. Sendt. are both Andean tropical fruit species with high nutritional value and the potential for becoming premium products in local and export markets. Herein, we present a report on the genetic characterization of 62 accessions of lulos (n = 32 and tree tomatoes (n = 30 through the use of PCR-based markers developed from single-copy conserved orthologous genes (COSII in other Solanaceae (Asterid species. We successfully PCR amplified a set of these markers for lulos (34 out of 46 initially tested and tree tomatoes (26 out of 41 for molecular studies. Six polymorphic COSII markers were found in lulo with a total of 47 alleles and five polymorphic markers in tree tomato with a total of 39 alleles in the two populations. Further genetic analyses indicated a high population structure (with F ST > 0.90, which may be a result of low migration between populations, adaptation to various niches and the number of markers evaluated. We propose COSII markers as sound tools for molecular studies, conservation and the breeding of these two fruit species.

  20. A DNA-Centric Protein Interaction Map of Ultraconserved Elements Reveals Contribution of Transcription Factor Binding Hubs to Conservation

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    Tar Viturawong

    2013-10-01

    Full Text Available Ultraconserved elements (UCEs have been the subject of great interest because of their extreme sequence identity and their seemingly cryptic and largely uncharacterized functions. Although in vivo studies of UCE sequences have demonstrated regulatory activity, protein interactors at UCEs have not been systematically identified. Here, we combined high-throughput affinity purification, high-resolution mass spectrometry, and SILAC quantification to map intrinsic protein interactions for 193 UCE sequences. The interactome contains over 400 proteins, including transcription factors with known developmental roles. We demonstrate based on our data that UCEs consist of strongly conserved overlapping binding sites. We also generated a fine-resolution interactome of a UCE, confirming the hub-like nature of the element. The intrinsic interactions mapped here are reflected in open chromatin, as indicated by comparison with existing ChIP data. Our study argues for a strong contribution of protein-DNA interactions to UCE conservation and provides a basis for further functional characterization of UCEs.

  1. Aberrant splicing in maize rough endosperm3 reveals a conserved role for U12 splicing in eukaryotic multicellular development

    Science.gov (United States)

    Barbazuk, W. Brad

    2017-01-01

    RNA splicing of U12-type introns functions in human cell differentiation, but it is not known whether this class of introns has a similar role in plants. The maize ROUGH ENDOSPERM3 (RGH3) protein is orthologous to the human splicing factor, ZRSR2. ZRSR2 mutations are associated with myelodysplastic syndrome (MDS) and cause U12 splicing defects. Maize rgh3 mutants have aberrant endosperm cell differentiation and proliferation. We found that most U12-type introns are retained or misspliced in rgh3. Genes affected in rgh3 and ZRSR2 mutants identify cell cycle and protein glycosylation as common pathways disrupted. Transcripts with retained U12-type introns can be found in polysomes, suggesting that splicing efficiency can alter protein isoforms. The rgh3 mutant protein disrupts colocalization with a known ZRSR2-interacting protein, U2AF2. These results indicate conserved function for RGH3/ZRSR2 in U12 splicing and a deeply conserved role for the minor spliceosome to promote cell differentiation from stem cells to terminal fates. PMID:28242684

  2. A conserved alternative splicing event in plants reveals an ancient exonization of 5S rRNA that regulates TFIIIA.

    Science.gov (United States)

    Barbazuk, W Brad

    2010-01-01

    Uncovering conserved alternative splicing (AS) events can identify AS events that perform important functions. This is especially useful for identifying premature stop codon containing (PTC) AS isoforms that may regulate protein expression by being targets for nonsense mediated decay. This report discusses the identification of a PTC containing splice isoform of the TFIIIA gene that is highly conserved in land plants. TFIIIA is essential for RNA Polymerase III-based transcription of 5S rRNA in eukaryotes. Two independent groups have determined that the PTC containing alternative exon is ultraconserved and is coupled with nonsense-mediated mRNA decay. The alternative exon appears to have been derived by the exonization of 5S ribosomal RNA (5S rRNA) within the gene of its own transcription regulator, TFIIIA. This provides the first evidence of ancient exaptation of 5S rRNA in plants, suggesting a novel gene regulation model mediated by the AS of an anciently exonized non-coding element.

  3. Genomic analysis of the Pacific oyster (Crassostrea gigas) reveals possible conservation of vertebrate sex determination in a mollusc.

    Science.gov (United States)

    Zhang, Na; Xu, Fei; Guo, Ximing

    2014-09-11

    Despite the prevalence of sex in animal kingdom, we have only limited understanding of how sex is determined and evolved in many taxa. The mollusc Pacific oyster Crassostrea gigas exhibits complex modes of sexual reproduction that consists of protandric dioecy, sex change, and occasional hermaphroditism. This complex system is controlled by both environmental and genetic factors through unknown molecular mechanisms. In this study, we investigated genes related to sex-determining pathways in C. gigas through transcriptome sequencing and analysis of female and male gonads. Our analysis identified or confirmed novel homologs in the oyster of key sex-determining genes (SoxH or Sry-like and FoxL2) that were thought to be vertebrate-specific. Their expression profile in C. gigas is consistent with conserved roles in sex determination, under a proposed model where a novel testis-determining CgSoxH may serve as a primary regulator, directly or indirectly interacting with a testis-promoting CgDsx and an ovary-promoting CgFoxL2. Our findings plus previous results suggest that key vertebrate sex-determining genes such as Sry and FoxL2 may not be inventions of vertebrates. The presence of such genes in a mollusc with expression profiles consistent with expected roles in sex determination suggest that sex determination may be deeply conserved in animals, despite rapid evolution of the regulatory pathways that in C. gigas may involve both genetic and environmental factors.

  4. A spectacular new Philippine monitor lizard reveals a hidden biogeographic boundary and a novel flagship species for conservation.

    Science.gov (United States)

    Welton, Luke J; Siler, Cameron D; Bennett, Daniel; Diesmos, Arvin; Duya, M Roy; Dugay, Roldan; Rico, Edmund Leo B; Van Weerd, Merlijn; Brown, Rafe M

    2010-10-23

    As humans continue to explore the last uncharted regions of the planet, discoveries of previously unknown species of large vertebrates have become infrequent. Here, we report on the discovery of a spectacular new species of giant, secretive, frugivorous, forest monitor lizard (Genus: Varanus) from the forests of the northern Philippines. Using data from morphology and mitochondrial and nuclear DNA sequences, we demonstrate the taxonomic distinctiveness of this new 2 m long species and provide insight into its historical biogeography and systematic affinities. Our molecular phylogenetic analyses indicate that the new species is closely related to Varanus olivaceus (from southern Luzon and nearby islands), but it differs from this and other varanids with respect to characteristics of scalation, colour pattern, body size, anatomy of the reproductive organs and genetic divergence. The new species appears to be restricted to forests of the central and northern Sierra Madre mountain range; it is separated from the range of V. olivaceus by a more than 150 km stretch that includes at least three low-elevation river valley barriers to dispersal. This discovery identifies a seldom-perceived biogeographic boundary and emphasizes the need for continued biodiversity research in the megadiverse conservation hotspot of the Philippines. It is anticipated that the new species will serve as an important flagship species for conservation efforts aimed at preserving the remaining forests of northern Luzon.

  5. TALEN Gene Knockouts Reveal No Requirement for the Conserved Human Shelterin Protein Rap1 in Telomere Protection and Length Regulation

    Directory of Open Access Journals (Sweden)

    Shaheen Kabir

    2014-11-01

    Full Text Available The conserved protein Rap1 functions at telomeres in fungi, protozoa, and vertebrates. Like yeast Rap1, human Rap1 has been implicated in telomere length regulation and repression of nonhomologous end-joining (NHEJ at telomeres. However, mouse telomeres lacking Rap1 do not succumb to NHEJ. To determine the functions of human Rap1, we generated several transcription activator-like effector nuclease (TALEN-mediated human cell lines lacking Rap1. Loss of Rap1 did not affect the other components of shelterin, the modification of telomeric histones, the subnuclear position of telomeres, or the 3′ telomeric overhang. Telomeres lacking Rap1 did not show a DNA damage response, NHEJ, or consistent changes in their length, indicating that Rap1 does not have an important function in protection or length regulation of human telomeres. As human Rap1, like its mouse and unicellular orthologs, affects gene expression, we propose that the conservation of Rap1 reflects its role in transcriptional regulation rather than a function at telomeres.

  6. Comparison of C. elegans and C. briggsae genome sequences reveals extensive conservation of chromosome organization and synteny.

    Directory of Open Access Journals (Sweden)

    LaDeana W Hillier

    2007-07-01

    Full Text Available To determine whether the distinctive features of Caenorhabditis elegans chromosomal organization are shared with the C. briggsae genome, we constructed a single nucleotide polymorphism-based genetic map to order and orient the whole genome shotgun assembly along the six C. briggsae chromosomes. Although these species are of the same genus, their most recent common ancestor existed 80-110 million years ago, and thus they are more evolutionarily distant than, for example, human and mouse. We found that, like C. elegans chromosomes, C. briggsae chromosomes exhibit high levels of recombination on the arms along with higher repeat density, a higher fraction of intronic sequence, and a lower fraction of exonic sequence compared with chromosome centers. Despite extensive intrachromosomal rearrangements, 1:1 orthologs tend to remain in the same region of the chromosome, and colinear blocks of orthologs tend to be longer in chromosome centers compared with arms. More strikingly, the two species show an almost complete conservation of synteny, with 1:1 orthologs present on a single chromosome in one species also found on a single chromosome in the other. The conservation of both chromosomal organization and synteny between these two distantly related species suggests roles for chromosome organization in the fitness of an organism that are only poorly understood presently.

  7. Cross-species transcriptional network analysis reveals conservation and variation in response to metal stress in cyanobacteria

    Science.gov (United States)

    2013-01-01

    Background As one of the most dominant bacterial groups on Earth, cyanobacteria play a pivotal role in the global carbon cycling and the Earth atmosphere composition. Understanding their molecular responses to environmental perturbations has important scientific and environmental values. Since important biological processes or networks are often evolutionarily conserved, the cross-species transcriptional network analysis offers a useful strategy to decipher conserved and species-specific transcriptional mechanisms that cells utilize to deal with various biotic and abiotic disturbances, and it will eventually lead to a better understanding of associated adaptation and regulatory networks. Results In this study, the Weighted Gene Co-expression Network Analysis (WGCNA) approach was used to establish transcriptional networks for four important cyanobacteria species under metal stress, including iron depletion and high copper conditions. Cross-species network comparison led to discovery of several core response modules and genes possibly essential to metal stress, as well as species-specific hub genes for metal stresses in different cyanobacteria species, shedding light on survival strategies of cyanobacteria responding to different environmental perturbations. Conclusions The WGCNA analysis demonstrated that the application of cross-species transcriptional network analysis will lead to novel insights to molecular response to environmental changes which will otherwise not be achieved by analyzing data from a single species. PMID:23421563

  8. Crystal Structure of PhnH: an Essential Component of Carbon-Phosphorus Lyase in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Adams,M.; Luo, Y.; Hove-Jensen, B.; He, S.; van Staalduinen, L.; Zechel, D.; Jia, Z.

    2008-01-01

    Organophosphonates are reduced forms of phosphorous that are characterized by the presence of a stable carbon-phosphorus (C-P) bond, which resists chemical hydrolysis, thermal decomposition, and photolysis. The chemically inert nature of the C-P bond has raised environmental concerns as toxic phosphonates accumulate in a number of ecosystems. Carbon-phosphorous lyase (CP lyase) is a multienzyme pathway encoded by the phn operon in gram-negative bacteria. In Escherichia coli 14 cistrons comprise the operon (phnCDEFGHIJKLMNOP) and collectively allow the internalization and degradation of phosphonates. Here we report the X-ray crystal structure of the PhnH component at 1.77 Angstroms resolution. The protein exhibits a novel fold, although local similarities with the pyridoxal 5'-phosphate-dependent transferase family of proteins are apparent. PhnH forms a dimer in solution and in the crystal structure, the interface of which is implicated in creating a potential ligand binding pocket. Our studies further suggest that PhnH may be capable of binding negatively charged cyclic compounds through interaction with strictly conserved residues. Finally, we show that PhnH is essential for C-P bond cleavage in the CP lyase pathway.

  9. Quantitation of heparosan with heparin lyase III and spectrophotometry.

    Science.gov (United States)

    Huang, Haichan; Zhao, Yingying; Lv, Shencong; Zhong, Weihong; Zhang, Fuming; Linhardt, Robert J

    2014-02-15

    Heparosan is Escherichia coli K5 capsule polysaccharide, which is the key precursor for preparing bioengineered heparin. A rapid and effective quantitative method for detecting heparosan is important in the large-scale production of heparosan. Heparin lyase III (Hep III) effectively catalyzes the heparosan depolymerization, forming unsaturated disaccharides that are measurable using a spectrophotometer at 232 nm. We report a new method for the quantitative detection of heparosan with heparin lyase III and spectrophotometry that is safer and more specific than the traditional carbazole assay. In an optimized detection system, heparosan at a minimum concentration of 0.60 g/L in fermentation broth can be detected.

  10. Analysis of the sulfur-regulated control of the cystathionine γ-lyase gene of Neurospora crassa

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    Reveal Brad S

    2012-07-01

    Full Text Available Abstract Background Cystathionine γ-lyase plays a key role in the transsulfuration pathway through its primary reaction of catalyzing the formation of cysteine from cystathionine. The Neurospora crassa cystathionine γ-lyase gene (cys-16+ is of particular interest in dissecting the regulation and dynamics of transsulfuration. The aim of this study was to determine the regulatory connection of cys-16+ to the Neurospora sulfur regulatory network. In addition, the cys-16+ promoter was characterized with the goal of developing a strongly expressed and regulatable gene expression tool. Findings The cystathionine γ-lyase cys-16+ gene was cloned and characterized. The gene, which contains no introns, encodes a protein of 417 amino acids with conserved pyridoxal 5’-phosphate binding site and substrate-cofactor binding pocket. Northern blot analysis using wild type cells showed that cys-16+ transcript levels increased under sulfur limiting (derepressing conditions and were present only at a low level under sulfur sufficient (repressing conditions. In contrast, cys-16+ transcript levels in a Δcys-3 regulatory mutant were present at a low level under either derepressing or repressing conditions. Gel mobility shift analysis demonstrated the presence of four CYS3 transcriptional activator binding sites on the cys-16+ promoter, which were close matches to the CYS3 consensus binding sequence. Conclusions In this work, we confirm the control of cystathionine γ-lyase gene expression by the CYS3 transcriptional activator through the loss of cys-16+ expression in a Δcys-3 mutant and through the in vitro binding of CYS3 to the cys-16+ promoter at four sites. The highly regulated cys-16+ promoter should be a useful tool for gene expression studies in Neurospora

  11. The First Myriapod Genome Sequence Reveals Conservative Arthropod Gene Content and Genome Organisation in the Centipede Strigamia maritima

    Science.gov (United States)

    Chipman, Ariel D.; Ferrier, David E. K.; Brena, Carlo; Qu, Jiaxin; Hughes, Daniel S. T.; Schröder, Reinhard; Torres-Oliva, Montserrat; Znassi, Nadia; Jiang, Huaiyang; Almeida, Francisca C.; Alonso, Claudio R.; Apostolou, Zivkos; Aqrawi, Peshtewani; Arthur, Wallace; Barna, Jennifer C. J.; Blankenburg, Kerstin P.; Brites, Daniela; Capella-Gutiérrez, Salvador; Coyle, Marcus; Dearden, Peter K.; Du Pasquier, Louis; Duncan, Elizabeth J.; Ebert, Dieter; Eibner, Cornelius; Erikson, Galina; Evans, Peter D.; Extavour, Cassandra G.; Francisco, Liezl; Gabaldón, Toni; Gillis, William J.; Goodwin-Horn, Elizabeth A.; Green, Jack E.; Griffiths-Jones, Sam; Grimmelikhuijzen, Cornelis J. P.; Gubbala, Sai; Guigó, Roderic; Han, Yi; Hauser, Frank; Havlak, Paul; Hayden, Luke; Helbing, Sophie; Holder, Michael; Hui, Jerome H. L.; Hunn, Julia P.; Hunnekuhl, Vera S.; Jackson, LaRonda; Javaid, Mehwish; Jhangiani, Shalini N.; Jiggins, Francis M.; Jones, Tamsin E.; Kaiser, Tobias S.; Kalra, Divya; Kenny, Nathan J.; Korchina, Viktoriya; Kovar, Christie L.; Kraus, F. Bernhard; Lapraz, François; Lee, Sandra L.; Lv, Jie; Mandapat, Christigale; Manning, Gerard; Mariotti, Marco; Mata, Robert; Mathew, Tittu; Neumann, Tobias; Newsham, Irene; Ngo, Dinh N.; Ninova, Maria; Okwuonu, Geoffrey; Ongeri, Fiona; Palmer, William J.; Patil, Shobha; Patraquim, Pedro; Pham, Christopher; Pu, Ling-Ling; Putman, Nicholas H.; Rabouille, Catherine; Ramos, Olivia Mendivil; Rhodes, Adelaide C.; Robertson, Helen E.; Robertson, Hugh M.; Ronshaugen, Matthew; Rozas, Julio; Saada, Nehad; Sánchez-Gracia, Alejandro; Scherer, Steven E.; Schurko, Andrew M.; Siggens, Kenneth W.; Simmons, DeNard; Stief, Anna; Stolle, Eckart; Telford, Maximilian J.; Tessmar-Raible, Kristin; Thornton, Rebecca; van der Zee, Maurijn; von Haeseler, Arndt; Williams, James M.; Willis, Judith H.; Wu, Yuanqing; Zou, Xiaoyan; Lawson, Daniel; Muzny, Donna M.; Worley, Kim C.; Gibbs, Richard A.; Akam, Michael; Richards, Stephen

    2014-01-01

    Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific

  12. Conserved synteny at the protein family level reveals genes underlying Shewanella species cold tolerance and predicts their novel phenotypes

    Energy Technology Data Exchange (ETDEWEB)

    Karpinets, Tatiana V.; Obraztsova, Anna; Wang, Yanbing; Schmoyer, Denise D.; Kora, Guruprasad; Park, Byung H.; Serres, Margrethe H.; Romine, Margaret F.; Land, Miriam L.; Kothe, Terence B.; Fredrickson, Jim K.; Nealson, Kenneth H.; Uberbacher, Edward

    2010-03-01

    Bacteria of the genus Shewanella can thrive in different environments and demonstrate significant variability in their metabolic and ecophysiological capabilities including cold and salt tolerance. Genomic characteristics underlying this variability across species are largely unknown. In this study we address the problem by a comparison of the physiological, metabolic and genomic characteristics of 19 sequenced Shewanella species. We have employed two novel approaches based on association of a phenotypic trait with the number of the trait-specific protein families (Pfam domains) and on the conservation of synteny (order in the genome) of the trait-related genes. Our first approach is top-down and involves experimental evaluation and quantification of the species’ cold tolerance followed by identification of the correlated Pfam domains and genes with a conserved synteny. The second, a bottom-up approach, predicts novel phenotypes of the species by calculating profiles of each Pfam domain among their genomes and following pair-wise correlation of the profiles and their network clustering. Using the first approach we find a link between cold and salt tolerance of the species and the presence in the genome of a Na+/H+ antiporter gene cluster. Other cold tolerance related genes includes peptidases, chemotaxis sensory transducer proteins, a cysteine exporter, and helicases. Using the bottom-up approach we found several novel phenotypes in the newly sequenced Shewanella species, including degradation of aromatic compounds by an aerobic hybrid pathway in S. woodyi, degradation of ethanolamine by S. benthica, and propanediol degradation by S. putrefaciens CN32 and S. sp. W3-18-1.

  13. The first myriapod genome sequence reveals conservative arthropod gene content and genome organisation in the centipede Strigamia maritima.

    Directory of Open Access Journals (Sweden)

    Ariel D Chipman

    2014-11-01

    Full Text Available Myriapods (e.g., centipedes and millipedes display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations

  14. Spatial dynamics and expanded vertical niche of blue sharks in oceanographic fronts reveal habitat targets for conservation.

    Science.gov (United States)

    Queiroz, Nuno; Humphries, Nicolas E; Noble, Leslie R; Santos, António M; Sims, David W

    2012-01-01

    Dramatic population declines among species of pelagic shark as a result of overfishing have been reported, with some species now at a fraction of their historical biomass. Advanced telemetry techniques enable tracking of spatial dynamics and behaviour, providing fundamental information on habitat preferences of threatened species to aid conservation. We tracked movements of the highest pelagic fisheries by-catch species, the blue shark Prionace glauca, in the North-east Atlantic using pop-off satellite-linked archival tags to determine the degree of space use linked to habitat and to examine vertical niche. Overall, blue sharks moved south-west of tagging sites (English Channel; southern Portugal), exhibiting pronounced site fidelity correlated with localized productive frontal areas, with estimated space-use patterns being significantly different from that of random walks. Tracked female sharks displayed behavioural variability in diel depth preferences, both within and between individuals. Diel depth use ranged from normal DVM (nDVM; dawn descent, dusk ascent), to reverse DVM (rDVM; dawn ascent, dusk descent), to behavioural patterns where no diel differences were apparent. Results showed that blue sharks occupy some of the most productive marine zones for extended periods and structure diel activity patterns across multiple spatio-temporal scales in response to particular habitat types. In so doing, sharks occupied an extraordinarily broad vertical depth range for their size (1.0-2.0 m fork length), from the surface into the bathypelagic realm (max. dive depth, 1160 m). The space-use patterns of blue sharks indicated they spend much of the time in areas where pelagic longlining activities are often highest, and in depth zones where these fisheries particularly target other species, which could account for the rapid declines recently reported for blue sharks in many parts of the world's oceans. Our results provide habitat targets for blue shark conservation that

  15. Spatial dynamics and expanded vertical niche of blue sharks in oceanographic fronts reveal habitat targets for conservation.

    Directory of Open Access Journals (Sweden)

    Nuno Queiroz

    Full Text Available Dramatic population declines among species of pelagic shark as a result of overfishing have been reported, with some species now at a fraction of their historical biomass. Advanced telemetry techniques enable tracking of spatial dynamics and behaviour, providing fundamental information on habitat preferences of threatened species to aid conservation. We tracked movements of the highest pelagic fisheries by-catch species, the blue shark Prionace glauca, in the North-east Atlantic using pop-off satellite-linked archival tags to determine the degree of space use linked to habitat and to examine vertical niche. Overall, blue sharks moved south-west of tagging sites (English Channel; southern Portugal, exhibiting pronounced site fidelity correlated with localized productive frontal areas, with estimated space-use patterns being significantly different from that of random walks. Tracked female sharks displayed behavioural variability in diel depth preferences, both within and between individuals. Diel depth use ranged from normal DVM (nDVM; dawn descent, dusk ascent, to reverse DVM (rDVM; dawn ascent, dusk descent, to behavioural patterns where no diel differences were apparent. Results showed that blue sharks occupy some of the most productive marine zones for extended periods and structure diel activity patterns across multiple spatio-temporal scales in response to particular habitat types. In so doing, sharks occupied an extraordinarily broad vertical depth range for their size (1.0-2.0 m fork length, from the surface into the bathypelagic realm (max. dive depth, 1160 m. The space-use patterns of blue sharks indicated they spend much of the time in areas where pelagic longlining activities are often highest, and in depth zones where these fisheries particularly target other species, which could account for the rapid declines recently reported for blue sharks in many parts of the world's oceans. Our results provide habitat targets for blue shark

  16. A zebrafish transgenic model of Ewing’s sarcoma reveals conserved mediators of EWS-FLI1 tumorigenesis

    Directory of Open Access Journals (Sweden)

    Stefanie W. Leacock

    2012-01-01

    Ewing’s sarcoma, a malignant bone tumor of children and young adults, is a member of the small-round-blue-cell tumor family. Ewing’s sarcoma family tumors (ESFTs, which include peripheral primitive neuroectodermal tumors (PNETs, are characterized by chromosomal translocations that generate fusions between the EWS gene and ETS-family transcription factors, most commonly FLI1. The EWS-FLI1 fusion oncoprotein represents an attractive therapeutic target for treatment of Ewing’s sarcoma. The cell of origin of ESFT and the molecular mechanisms by which EWS-FLI1 mediates tumorigenesis remain unknown, and few animal models of Ewing’s sarcoma exist. Here, we report the use of zebrafish as a vertebrate model of EWS-FLI1 function and tumorigenesis. Mosaic expression of the human EWS-FLI1 fusion protein in zebrafish caused the development of tumors with histology strongly resembling that of human Ewing’s sarcoma. The incidence of tumors increased in a p53 mutant background, suggesting that the p53 pathway suppresses EWS-FLI1-driven tumorigenesis. Gene expression profiling of the zebrafish tumors defined a set of genes that might be regulated by EWS-FLI1, including the zebrafish ortholog of a crucial EWS-FLI1 target gene in humans. Stable zebrafish transgenic lines expressing EWS-FLI1 under the control of the heat-shock promoter exhibit altered embryonic development and defective convergence and extension, suggesting that EWS-FLI1 interacts with conserved developmental pathways. These results indicate that functional targets of EWS-FLI1 that mediate tumorigenesis are conserved from zebrafish to human and provide a novel context in which to study the function of this fusion oncogene.

  17. Gene expression in chicken reveals correlation with structural genomic features and conserved patterns of transcription in the terrestrial vertebrates.

    Directory of Open Access Journals (Sweden)

    Haisheng Nie

    Full Text Available BACKGROUND: The chicken is an important agricultural and avian-model species. A survey of gene expression in a range of different tissues will provide a benchmark for understanding expression levels under normal physiological conditions in birds. With expression data for birds being very scant, this benchmark is of particular interest for comparative expression analysis among various terrestrial vertebrates. METHODOLOGY/PRINCIPAL FINDINGS: We carried out a gene expression survey in eight major chicken tissues using whole genome microarrays. A global picture of gene expression is presented for the eight tissues, and tissue specific as well as common gene expression were identified. A Gene Ontology (GO term enrichment analysis showed that tissue-specific genes are enriched with GO terms reflecting the physiological functions of the specific tissue, and housekeeping genes are enriched with GO terms related to essential biological functions. Comparisons of structural genomic features between tissue-specific genes and housekeeping genes show that housekeeping genes are more compact. Specifically, coding sequence and particularly introns are shorter than genes that display more variation in expression between tissues, and in addition intergenic space was also shorter. Meanwhile, housekeeping genes are more likely to co-localize with other abundantly or highly expressed genes on the same chromosomal regions. Furthermore, comparisons of gene expression in a panel of five common tissues between birds, mammals and amphibians showed that the expression patterns across tissues are highly similar for orthologous genes compared to random gene pairs within each pair-wise comparison, indicating a high degree of functional conservation in gene expression among terrestrial vertebrates. CONCLUSIONS: The housekeeping genes identified in this study have shorter gene length, shorter coding sequence length, shorter introns, and shorter intergenic regions, there seems

  18. Bioinformatic analysis of CaBP/calneuron proteins reveals a family of highly conserved vertebrate Ca2+-binding proteins

    Directory of Open Access Journals (Sweden)

    Burgoyne Robert D

    2010-04-01

    Full Text Available Abstract Background Ca2+-binding proteins are important for the transduction of Ca2+ signals into physiological outcomes. As in calmodulin many of the Ca2+-binding proteins bind Ca2+ through EF-hand motifs. Amongst the large number of EF-hand containing Ca2+-binding proteins are a subfamily expressed in neurons and retinal photoreceptors known as the CaBPs and the related calneuron proteins. These were suggested to be vertebrate specific but exactly which family members are expressed outside of mammalian species had not been examined. Findings We have carried out a bioinformatic analysis to determine when members of this family arose and the conserved aspects of the protein family. Sequences of human members of the family obtained from GenBank were used in Blast searches to identify corresponding proteins encoded in other species using searches of non-redundant proteins, genome sequences and mRNA sequences. Sequences were aligned and compared using ClustalW. Some families of Ca2+-binding proteins are known to show a progressive expansion in gene number as organisms increase in complexity. In contrast, the results for CaBPs and calneurons showed that a full complement of CaBPs and calneurons are present in the teleost fish Danio rerio and possibly in cartilaginous fish. These findings suggest that the entire family of genes may have arisen at the same time during vertebrate evolution. Certain members of the family (for example the short form of CaBP1 and calneuron 1 are highly conserved suggesting essential functional roles. Conclusions The findings support the designation of the calneurons as a distinct sub-family. While the gene number for CaBPs/calneurons does not increase, a distinctive evolutionary change in these proteins in vertebrates has been an increase in the number of splice variants present in mammals.

  19. The first myriapod genome sequence reveals conservative arthropod gene content and genome organisation in the centipede Strigamia maritima.

    Science.gov (United States)

    Chipman, Ariel D; Ferrier, David E K; Brena, Carlo; Qu, Jiaxin; Hughes, Daniel S T; Schröder, Reinhard; Torres-Oliva, Montserrat; Znassi, Nadia; Jiang, Huaiyang; Almeida, Francisca C; Alonso, Claudio R; Apostolou, Zivkos; Aqrawi, Peshtewani; Arthur, Wallace; Barna, Jennifer C J; Blankenburg, Kerstin P; Brites, Daniela; Capella-Gutiérrez, Salvador; Coyle, Marcus; Dearden, Peter K; Du Pasquier, Louis; Duncan, Elizabeth J; Ebert, Dieter; Eibner, Cornelius; Erikson, Galina; Evans, Peter D; Extavour, Cassandra G; Francisco, Liezl; Gabaldón, Toni; Gillis, William J; Goodwin-Horn, Elizabeth A; Green, Jack E; Griffiths-Jones, Sam; Grimmelikhuijzen, Cornelis J P; Gubbala, Sai; Guigó, Roderic; Han, Yi; Hauser, Frank; Havlak, Paul; Hayden, Luke; Helbing, Sophie; Holder, Michael; Hui, Jerome H L; Hunn, Julia P; Hunnekuhl, Vera S; Jackson, LaRonda; Javaid, Mehwish; Jhangiani, Shalini N; Jiggins, Francis M; Jones, Tamsin E; Kaiser, Tobias S; Kalra, Divya; Kenny, Nathan J; Korchina, Viktoriya; Kovar, Christie L; Kraus, F Bernhard; Lapraz, François; Lee, Sandra L; Lv, Jie; Mandapat, Christigale; Manning, Gerard; Mariotti, Marco; Mata, Robert; Mathew, Tittu; Neumann, Tobias; Newsham, Irene; Ngo, Dinh N; Ninova, Maria; Okwuonu, Geoffrey; Ongeri, Fiona; Palmer, William J; Patil, Shobha; Patraquim, Pedro; Pham, Christopher; Pu, Ling-Ling; Putman, Nicholas H; Rabouille, Catherine; Ramos, Olivia Mendivil; Rhodes, Adelaide C; Robertson, Helen E; Robertson, Hugh M; Ronshaugen, Matthew; Rozas, Julio; Saada, Nehad; Sánchez-Gracia, Alejandro; Scherer, Steven E; Schurko, Andrew M; Siggens, Kenneth W; Simmons, DeNard; Stief, Anna; Stolle, Eckart; Telford, Maximilian J; Tessmar-Raible, Kristin; Thornton, Rebecca; van der Zee, Maurijn; von Haeseler, Arndt; Williams, James M; Willis, Judith H; Wu, Yuanqing; Zou, Xiaoyan; Lawson, Daniel; Muzny, Donna M; Worley, Kim C; Gibbs, Richard A; Akam, Michael; Richards, Stephen

    2014-11-01

    Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific

  20. Comparative analysis of A-to-I editing in human and non-human primate brains reveals conserved patterns and context-dependent regulation of RNA editing.

    Science.gov (United States)

    O'Neil, Richard T; Wang, Xiaojing; Morabito, Michael V; Emeson, Ronald B

    2017-04-06

    A-to-I RNA editing is an important process for generating molecular diversity in the brain through modification of transcripts encoding several proteins important for neuronal signaling. We investigated the relationships between the extent of editing at multiple substrate transcripts (5HT2C, MGLUR4, CADPS, GLUR2, GLUR4, and GABRA3) in brain tissue obtained from adult humans and rhesus macaques. Several patterns emerged from these studies revealing conservation of editing across primate species. Additionally, variability in the human population allows us to make novel inferences about the co-regulation of editing at different editing sites and even across different brain regions.

  1. 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.

  2. Structural basis for streptogramin B resistance in Staphylococcus aureus by virginiamycin B lyase.

    Science.gov (United States)

    Korczynska, Magdalena; Mukhtar, Tariq A; Wright, Gerard D; Berghuis, Albert M

    2007-06-19

    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 Mg2+ at 1.65- and 2.8-A 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 Mg2+ -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.

  3. Functional characterization of a conserved archaeal viral operon revealing single-stranded DNA binding, annealing and nuclease activities

    DEFF Research Database (Denmark)

    Guo, Yang; Kragelund, Birthe Brandt; White, Malcolm F.

    2015-01-01

    The majority of archaeal viral genes are of unknown function hindering our understanding of the virus life cycle and viral interactions with their host. Here, we first describe functional characterization of ORF131b (gp17) and ORF436 (gp18) of Sulfolobus islandicus rod-shaped virus 2 (SIRV2), bot...... for rudiviruses and the close interaction among the ssDNA binding, annealing and nuclease proteins strongly point to a role of the gene operon in genome maturation and/or DNA recombination that may function in viral DNA replication/repair.......The majority of archaeal viral genes are of unknown function hindering our understanding of the virus life cycle and viral interactions with their host. Here, we first describe functional characterization of ORF131b (gp17) and ORF436 (gp18) of Sulfolobus islandicus rod-shaped virus 2 (SIRV2), both...... encoding proteins of unknown function and forming an operon with ORF207 (gp19). SIRV2 gp17 was found to be a single-stranded DNA (ssDNA) binding protein different in structure from all previously characterized ssDNA binding proteins. Mutagenesis of a few conserved basic residues suggested a U...

  4. Whole-organism cellular gene-expression atlas reveals conserved cell types in the ventral nerve cord of Platynereis dumerilii.

    Science.gov (United States)

    Vergara, Hernando Martínez; Bertucci, Paola Yanina; Hantz, Peter; Tosches, Maria Antonietta; Achim, Kaia; Vopalensky, Pavel; Arendt, Detlev

    2017-06-06

    The comparative study of cell types is a powerful approach toward deciphering animal evolution. To avoid selection biases, however, comparisons ideally involve all cell types present in a multicellular organism. Here, we use image registration and a newly developed "Profiling by Signal Probability Mapping" algorithm to generate a cellular resolution 3D expression atlas for an entire animal. We investigate three-segmented young worms of the marine annelid Platynereis dumerilii, with a rich diversity of differentiated cells present in relatively low number. Starting from whole-mount expression images for close to 100 neural specification and differentiation genes, our atlas identifies and molecularly characterizes 605 bilateral pairs of neurons at specific locations in the ventral nerve cord. Among these pairs, we identify sets of neurons expressing similar combinations of transcription factors, located at spatially coherent anterior-posterior, dorsal-ventral, and medial-lateral coordinates that we interpret as cell types. Comparison with motor and interneuron types in the vertebrate neural tube indicates conserved combinations, for example, of cell types cospecified by Gata1/2/3 and Tal transcription factors. These include V2b interneurons and the central spinal fluid-contacting Kolmer-Agduhr cells in the vertebrates, and several neuron types in the intermediate ventral ganglionic mass in the annelid. We propose that Kolmer-Agduhr cell-like mechanosensory neurons formed part of the mucociliary sole in protostome-deuterostome ancestors and diversified independently into several neuron types in annelid and vertebrate descendants.

  5. Characterization of the fibronectin-attachment protein of Mycobacterium avium reveals a fibronectin-binding motif conserved among mycobacteria.

    Science.gov (United States)

    Schorey, J S; Holsti, M A; Ratliff, T L; Allen, P M; Brown, E J

    1996-07-01

    Mycobacterium avium is an intracellular pathogen and a major opportunistic infectious agent observed in patients with acquired immune deficiency syndrome (AIDS). Evidence suggests that the initial portal of infection by M. avium is often the gastrointestinal tract. However, the mechanism by which the M. avium crosses the epithelial barrier is unclear. A possible mechanism is suggested by the ability of M. avium to bind fibronectin, an extracellular matrix protein that is a virulence factor for several extracellular pathogenic bacteria which bind to mucosal surfaces. To further characterize fibronectin binding by M. avium, we have cloned the M. avium fibronectin-attachment protein (FAP). The M. avium FAP (FAP-A) has an unusually large number of Pro and Ala residues (40% overall) and is 50% identical to FAP of both Mycobacterium leprae and Mycobacterium tuberculosis. Using recombinant FAP-A and FAP-A peptides, we show that two non-continuous regions in FAP-A bind fibronectin. Peptides from these regions and homologous sequences from M. leprae FAP inhibit fibronectin binding by both M. avium and Mycobacterium bovis Bacillus Calmette-Guerin (BCG). These regions have no homology to eukaryotic fibronectin-binding proteins and are only distantly related to fibronectin-binding peptides of Gram-positive bacteria. Nevertheless, these fibronectin-binding regions are highly conserved among the mycobacterial FAPs, suggesting an essential function for this interaction in mycobacteria infection of their metazoan hosts.

  6. A spring stopover of a migratory osprey (Pandion haliaetus in northern Spain as revealed by satellite tracking: implications for conservation

    Directory of Open Access Journals (Sweden)

    Galarza, A.

    2009-12-01

    Full Text Available Improvements in the accuracy of satellite telemetry locations now allow detailed studies on territorial behaviour or use of habitat that can be used to enhance bird conservation. In this paper we describe the behaviour of a satellite-tracked adult female osprey (Pandion haliaetus in the Urdaibai Biosphere Reserve (N Spain to evaluate the suitability of this protected area for the species. The data set consisted of 10 complete days with a total of 145 exact fixes received. Night roosts were mainly surrounded by high or intermediate level protected land, separated from roads or buildings by more than 200 m and located less than one km away from the feeding area. During daylight hours, most fixes (76.5% were located in wooded areas. We found that the bird selected holm oak woods and we suggest that this is related to low disturbance from human activity. We also suggest that northern Spanish estuaries are important as stopovers by migrating ospreys for feeding during migration.

  7. Comparative analysis of the ATRX promoter and 5' regulatory region reveals conserved regulatory elements which are linked to roles in neurodevelopment, alpha-globin regulation and testicular function

    Directory of Open Access Journals (Sweden)

    Argentaro Anthony

    2011-06-01

    Full Text Available Abstract Background ATRX is a tightly-regulated multifunctional protein with crucial roles in mammalian development. Mutations in the ATRX gene cause ATR-X syndrome, an X-linked recessive developmental disorder resulting in severe mental retardation and mild alpha-thalassemia with facial, skeletal and genital abnormalities. Although ubiquitously expressed the clinical features of the syndrome indicate that ATRX is not likely to be a global regulator of gene expression but involved in regulating specific target genes. The regulation of ATRX expression is not well understood and this is reflected by the current lack of identified upstream regulators. The availability of genomic data from a range of species and the very highly conserved 5' regulatory regions of the ATRX gene has allowed us to investigate putative transcription factor binding sites (TFBSs in evolutionarily conserved regions of the mammalian ATRX promoter. Results We identified 12 highly conserved TFBSs of key gene regulators involved in biologically relevant processes such as neural and testis development and alpha-globin regulation. Conclusions Our results reveal potentially important regulatory elements in the ATRX gene which may lead to the identification of upstream regulators of ATRX and aid in the understanding of the molecular mechanisms that underlie ATR-X syndrome.

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

    NARCIS (Netherlands)

    Heberling, Matthew M.; Wu, Bian; Bartsch, Sebastian; Janssen, Dick B.

    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

  9. Redesign of a Phenylalanine Aminomutase into a Phenylalanine Ammonia Lyase

    NARCIS (Netherlands)

    Bartsch, S.; Wybenga, G.G.; Jansen, M.; Heberling, M.M.; Wu, B.; Dijkstra, B.W.; Janssen, D.B.

    2013-01-01

    An aminomutase, naturally catalyzing the interconversion of (S)--phenylalanine and (R)--phenylalanine, was converted into an ammonia lyase catalyzing the nonoxidative deamination of phenylalanine to cinnamic acid by a rational single-point mutation. It could be shown by crystal structures and kineti

  10. Novel, in-natural-infection subdominant HIV-1 CD8+ T-cell epitopes revealed in human recipients of conserved-region T-cell vaccines.

    Science.gov (United States)

    Borthwick, Nicola; Lin, Zhansong; Akahoshi, Tomohiro; Llano, Anuska; Silva-Arrieta, Sandra; Ahmed, Tina; Dorrell, Lucy; Brander, Christian; Murakoshi, Hayato; Takiguchi, Masafumi; Hanke, Tomáš

    2017-01-01

    Fine definition of targeted CD8+ T-cell epitopes and their human leucocyte antigen (HLA) class I restriction informs iterative improvements of HIV-1 T-cell vaccine designs and may predict early vaccine success or failure. Here, lymphocytes from volunteers, who had received candidate HIVconsv vaccines expressing conserved sub-protein regions of HIV-1, were used to define the optimum-length target epitopes and their HLA restriction. In HIV-1-positive patients, CD8+ T-cell responses predominantly recognize immunodominant, but hypervariable and therefore less protective epitopes. The less variable, more protective epitopes in conserved regions are typically subdominant. Therefore, induction of strong responses to conserved regions by vaccination provides an opportunity to discover novel important epitopes. Cryopreserved lymphocytes from vaccine recipients were expanded by stimulation with 15-mer responder peptides for 10 days to establish short term-cell-line (STCL) effector cells. These were subjected to intracellular cytokine staining using serially truncated peptides and peptide-pulsed 721.221 cells expressing individual HLA class I alleles to define minimal epitope length and HLA restriction by stimulation of IFN-γ and TNF-α production and surface expression of CD107a. Using lymphocyte samples of 12 vaccine recipients, we defined 14 previously unreported optimal CD8+ T-cell HIV-1 epitopes and their four-digit HLA allele restriction (6 HLA-A, 7 HLA-B and 1 HLA-C alleles). Further 13 novel targets with incomplete information were revealed. The high rate of discovery of novel CD8+ T-cell effector epitopes warrants further epitope mining in recipients of the conserved-region vaccines in other populations and informs development of HIV-1/AIDS vaccines. ClinicalTrials.gov NCT01151319.

  11. Transcripts of pectin-degrading enzymes and isolation of complete cDNA sequence of a pectate lyase gene induced by coffee white stem borer (Xylotrechus quadripes) in the bark tissue of Coffea canephora (robusta coffee).

    Science.gov (United States)

    Bharathi, Kosaraju; Santosh, P; Sreenath, H L

    2017-05-01

    Of the two commercially cultivated coffee (Coffea) species, C. arabica (arabica) is highly susceptible and C. canephora (robusta) is highly resistant to the insect pest Xylotrechus quadripes (Coleoptera: Cerambycidae), commonly known as coffee white stem borer (CWSB). We constructed a forward-subtracted cDNA library by Suppression Subtractive Hybridization (SSH) from robusta bark tissue for profiling genes induced by CWSB infestation. Among the 265 unigenes of the SSH EST library, 7 unigenes (5 contigs and 2 singletons) matching different pectin-degrading enzymes were discovered. These ESTs matched one pectate lyase, three polygalacturonases, and one pectin acetylesterase gene. Quantitative real-time PCR (qRT-PCR) revealed that CWSB infestation strongly induces the pectate lyase gene at 72 h. Complete cDNA sequence of the pectate lyase gene was obtained through 3' and 5' RACE reactions. It was a 1595 bp long sequence that included full CDS and both UTRs. Against C. canephora genome sequences in Coffee Genome Hub database ( http://coffee-genome.org/ ), it had 22 matches to different pectate lyase genes mapped on 9 of the 11 pseudochromosomes, the top match being Cc07_g00190 Pectate lyase. In NCBI database, it matched pectate lyase sequences of several plants. Apart from C. canephora, the closest pectate lyase matches were from Sesamum indicum and Nicotiana tabacum. The pectinolytic enzymes discovered here are thought to play a role in the production of oligogalacturonides (OGs) which act as Damage-Associated Molecular Pattern (DAMP) signals eliciting innate immunity in plants. The pectate lyase gene, induced by CWSB infestation, along with other endogenous pectinolytic enzymes and CWSB-specific elicitors, may be involved in triggering basal defense responses to protect the CWSB-damaged tissue against pathogens, as well as to contain CWSB in robusta.

  12. A Comprehensive Phylogeny Reveals Functional Conservation of the UV-B Photoreceptor UVR8 from Green Algae to Higher Plants

    Science.gov (United States)

    Fernández, María B.; Tossi, Vanesa; Lamattina, Lorenzo; Cassia, Raúl

    2016-01-01

    Ultraviolet-B (UV-B) is present in sunlight (280–315 nm) and has diverse effects on living organisms. Low fluence rate of exposure induces a specific photomorphogenic response regulated by the UV-B response locus 8 (UVR8) receptor. UVR8 was first described in Arabidopsis thaliana. In the absence of stimuli it is located in the cytoplasm as a homodimer. However, upon UV-B irradiation, it switches to a monomer and interacts with the ubiquitin ligase E3 COP1 via the UVR8 β-propeller domain and the VP core. This induces the expression of the transcription factor HY5 leading to changes in the expression of genes associated with UV-B acclimation and stress tolerance. UVR8 senses UV-B through tryptophan residues being Trp233 and 285 the most important. Based on the comparison and analysis of UVR8 functionally important motifs, we report a comprehensive phylogeny of UVR8, trying to identify UVR8 homologs and the ancestral organism where this gene could be originated. Results obtained showed that Chlorophytes are the first organisms from the Viridiplantae group where UVR8 appears. UVR8 is present in green algae, bryophytes, lycophytes, and angiosperms. All the sequences identified contain tryptophans 233 and 285, arginines involved in homodimerization and the VP domain suggesting they are true UVR8 photoreceptors. We also determined that some species from bryophytes and angiosperms contain more than one UVR8 gene copy posing the question if UVR8 could constitute a gene family in these species. In conclusion, we described the functional conservation among UVR8 proteins from green algae to higher plants. PMID:27895654

  13. A Comprehensive Phylogeny Reveals Functional Conservation of the UV-B Photoreceptor UVR8 from Green Algae to Higher Plants.

    Science.gov (United States)

    Fernández, María B; Tossi, Vanesa; Lamattina, Lorenzo; Cassia, Raúl

    2016-01-01

    Ultraviolet-B (UV-B) is present in sunlight (280-315 nm) and has diverse effects on living organisms. Low fluence rate of exposure induces a specific photomorphogenic response regulated by the UV-B response locus 8 (UVR8) receptor. UVR8 was first described in Arabidopsis thaliana. In the absence of stimuli it is located in the cytoplasm as a homodimer. However, upon UV-B irradiation, it switches to a monomer and interacts with the ubiquitin ligase E3 COP1 via the UVR8 β-propeller domain and the VP core. This induces the expression of the transcription factor HY5 leading to changes in the expression of genes associated with UV-B acclimation and stress tolerance. UVR8 senses UV-B through tryptophan residues being Trp233 and 285 the most important. Based on the comparison and analysis of UVR8 functionally important motifs, we report a comprehensive phylogeny of UVR8, trying to identify UVR8 homologs and the ancestral organism where this gene could be originated. Results obtained showed that Chlorophytes are the first organisms from the Viridiplantae group where UVR8 appears. UVR8 is present in green algae, bryophytes, lycophytes, and angiosperms. All the sequences identified contain tryptophans 233 and 285, arginines involved in homodimerization and the VP domain suggesting they are true UVR8 photoreceptors. We also determined that some species from bryophytes and angiosperms contain more than one UVR8 gene copy posing the question if UVR8 could constitute a gene family in these species. In conclusion, we described the functional conservation among UVR8 proteins from green algae to higher plants.

  14. IDENTIFICATION OF NICOTINAMIDE MONONUCLEOTIDE DEAMIDASE OF THE BACTERIAL PYRIDINE NUCLEOTIDE CYCLE REVEALS A NOVEL BROADLY CONSERVED AMIDOHYDROLASE FAMILY

    Energy Technology Data Exchange (ETDEWEB)

    Galeazzi, Luca; Bocci, Paolo; Amici, Adolfo; Brunetti, Lucia; Ruggieri, Silverio; Romine, Margaret F.; Reed, Samantha B.; Osterman, Andrei; Rodionov, Dmitry A.; Sorci, Leonardo; Raffaelli, Nadia

    2011-09-27

    The pyridine nucleotide cycle (PNC) is a network of salvage and recycling routes maintaining homeostasis of NAD(P) cofactor pool in the cell. Nicotinamide mononucleotide (NMN) deamidase (EC 3.5.1.42), one of the key enzymes of the bacterial PNC was originally described in Enterobacteria, but the corresponding gene eluded identification for over 30 years. A genomics-based reconstruction of NAD metabolism across hundreds bacterial species suggested that NMN deamidase reaction is the only possible way of nicotinamide salvage in the marine bacterium Shewanella oneidensis. This prediction was verified via purification of native NMN deamidase from S. oneidensis followed by the identification of the respective gene, termed pncC. Enzymatic characterization of the PncC protein, as well as phenotype analysis of deletion mutants, confirmed its proposed biochemical and physiological function in S. oneidensis. Of the three PncC homologs present in E. coli, NMN deamidase activity was confirmed only for the recombinant purified product of the ygaD gene. A comparative analysis at the level of sequence and three dimensional structure, which is available for one of the PncC family member, shows no homology with any previously described amidohydrolases. Multiple alignment analysis of functional and non functional PncC homologs, together with NMN docking experiments, allowed us to tentatively identify the active site area and conserved residues therein. An observed broad phylogenomic distribution of predicted functional PncCs in bacterial kingdom is consistent with a possible role in detoxification of NMN, resulting from NAD utilization by DNA ligase.

  15. Identification of Nicotinamide Mononucleotide Deamidase of the Bacterial Pyridine Nucleotide Cycle Reveals a Novel Broadly Conserved Amidohydrolase Family*

    Science.gov (United States)

    Galeazzi, Luca; Bocci, Paola; Amici, Adolfo; Brunetti, Lucia; Ruggieri, Silverio; Romine, Margaret; Reed, Samantha; Osterman, Andrei L.; Rodionov, Dmitry A.; Sorci, Leonardo; Raffaelli, Nadia

    2011-01-01

    The pyridine nucleotide cycle is a network of salvage and recycling routes maintaining homeostasis of NAD(P) cofactor pool in the cell. Nicotinamide mononucleotide (NMN) deamidase (EC 3.5.1.42), one of the key enzymes of the bacterial pyridine nucleotide cycle, was originally described in Enterobacteria, but the corresponding gene eluded identification for over 30 years. A genomics-based reconstruction of NAD metabolism across hundreds of bacterial species suggested that NMN deamidase reaction is the only possible way of nicotinamide salvage in the marine bacterium Shewanella oneidensis. This prediction was verified via purification of native NMN deamidase from S. oneidensis followed by the identification of the respective gene, termed pncC. Enzymatic characterization of the PncC protein, as well as phenotype analysis of deletion mutants, confirmed its proposed biochemical and physiological function in S. oneidensis. Of the three PncC homologs present in Escherichia coli, NMN deamidase activity was confirmed only for the recombinant purified product of the ygaD gene. A comparative analysis at the level of sequence and three-dimensional structure, which is available for one of the PncC family member, shows no homology with any previously described amidohydrolases. Multiple alignment analysis of functional and nonfunctional PncC homologs, together with NMN docking experiments, allowed us to tentatively identify the active site area and conserved residues therein. An observed broad phylogenomic distribution of predicted functional PncCs in the bacterial kingdom is consistent with a possible role in detoxification of NMN, resulting from NAD utilization by DNA ligase. PMID:21953451

  16. Identification of nicotinamide mononucleotide deamidase of the bacterial pyridine nucleotide cycle reveals a novel broadly conserved amidohydrolase family.

    Science.gov (United States)

    Galeazzi, Luca; Bocci, Paola; Amici, Adolfo; Brunetti, Lucia; Ruggieri, Silverio; Romine, Margaret; Reed, Samantha; Osterman, Andrei L; Rodionov, Dmitry A; Sorci, Leonardo; Raffaelli, Nadia

    2011-11-18

    The pyridine nucleotide cycle is a network of salvage and recycling routes maintaining homeostasis of NAD(P) cofactor pool in the cell. Nicotinamide mononucleotide (NMN) deamidase (EC 3.5.1.42), one of the key enzymes of the bacterial pyridine nucleotide cycle, was originally described in Enterobacteria, but the corresponding gene eluded identification for over 30 years. A genomics-based reconstruction of NAD metabolism across hundreds of bacterial species suggested that NMN deamidase reaction is the only possible way of nicotinamide salvage in the marine bacterium Shewanella oneidensis. This prediction was verified via purification of native NMN deamidase from S. oneidensis followed by the identification of the respective gene, termed pncC. Enzymatic characterization of the PncC protein, as well as phenotype analysis of deletion mutants, confirmed its proposed biochemical and physiological function in S. oneidensis. Of the three PncC homologs present in Escherichia coli, NMN deamidase activity was confirmed only for the recombinant purified product of the ygaD gene. A comparative analysis at the level of sequence and three-dimensional structure, which is available for one of the PncC family member, shows no homology with any previously described amidohydrolases. Multiple alignment analysis of functional and nonfunctional PncC homologs, together with NMN docking experiments, allowed us to tentatively identify the active site area and conserved residues therein. An observed broad phylogenomic distribution of predicted functional PncCs in the bacterial kingdom is consistent with a possible role in detoxification of NMN, resulting from NAD utilization by DNA ligase.

  17. The elephant shark methylome reveals conservation of epigenetic regulation across jawed vertebrates [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Julian R. Peat

    2017-04-01

    Full Text Available Background: Methylation of CG dinucleotides constitutes a critical system of epigenetic memory in bony vertebrates, where it modulates gene expression and suppresses transposon activity. The genomes of studied vertebrates are pervasively hypermethylated, with the exception of regulatory elements such as transcription start sites (TSSs, where the presence of methylation is associated with gene silencing. This system is not found in the sparsely methylated genomes of invertebrates, and establishing how it arose during early vertebrate evolution is impeded by a paucity of epigenetic data from basal vertebrates. Methods: We perform whole-genome bisulfite sequencing to generate the first genome-wide methylation profiles of a cartilaginous fish, the elephant shark Callorhinchus milii. Employing these to determine the elephant shark methylome structure and its relationship with expression, we compare this with higher vertebrates and an invertebrate chordate using published methylation and transcriptome data.  Results: Like higher vertebrates, the majority of elephant shark CG sites are highly methylated, and methylation is abundant across the genome rather than patterned in the mosaic configuration of invertebrates. This global hypermethylation includes transposable elements and the bodies of genes at all expression levels. Significantly, we document an inverse relationship between TSS methylation and expression in the elephant shark, supporting the presence of the repressive regulatory architecture shared by higher vertebrates. Conclusions: Our demonstration that methylation patterns in a cartilaginous fish are characteristic of higher vertebrates imply the conservation of this epigenetic modification system across jawed vertebrates separated by 465 million years of evolution. In addition, these findings position the elephant shark as a valuable model to explore the evolutionary history and function of vertebrate methylation.

  18. Functional characterization of a conserved archaeal viral operon revealing single-stranded DNA binding, annealing and nuclease activities.

    Science.gov (United States)

    Guo, Yang; Kragelund, Birthe B; White, Malcolm F; Peng, Xu

    2015-06-19

    The majority of archaeal viral genes are of unknown function hindering our understanding of the virus life cycle and viral interactions with their host. Here, we first describe functional characterization of ORF131b (gp17) and ORF436 (gp18) of Sulfolobus islandicus rod-shaped virus 2 (SIRV2), both encoding proteins of unknown function and forming an operon with ORF207 (gp19). SIRV2 gp17 was found to be a single-stranded DNA (ssDNA) binding protein different in structure from all previously characterized ssDNA binding proteins. Mutagenesis of a few conserved basic residues suggested a U-shaped binding path for ssDNA. The recombinant gp18 showed an ssDNA annealing activity often associated with helicases and recombinases. To gain insight into the biological role of the entire operon, we characterized SIRV2 gp19 and showed it to possess a 5' → 3' ssDNA exonuclease activity, in addition to the previously demonstrated ssDNA endonuclease activity. Further, in vitro pull-down assay demonstrated interactions between gp17 and gp18 and between gp18 and gp19 with the former being mediated by the intrinsically disordered C-terminus of gp17. The strand-displacement replication mode proposed previously for rudiviruses and the close interaction among the ssDNA binding, annealing and nuclease proteins strongly point to a role of the gene operon in genome maturation and/or DNA recombination that may function in viral DNA replication/repair.

  19. A comprehensive phylogeny reveals functional conservation of the UV-B photoreceptor UVR8 from green algae to higher plants

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    María Belén Fernández

    2016-11-01

    Full Text Available UV-B is present in sunlight (280- 315 nm and has diverse effects on living organisms. Low fluence rate of exposure induces a specific photomorphogenic response regulated by the UV-B response locus 8 UVR8 receptor. UVR8 was first described in Arabidopsis thaliana. In the absence of stimuli is located in the cytoplasm as a homodimer, however, upon UV-B irradiation, it switches to a monomer and interacts with the ubiquitin ligase E3 COP1 via the UVR8 β- propeller domain and the VP core. This induces the expression of the transcription factor HY5 leading to changes in the expression of genes associated with UV-B acclimation and stress tolerance. UVR8 senses UV-B through tryptophan residues being Trp233 and 285 the most important. Here we report a comprehensive phylogeny of UVR8, trying to identify UVR8 homologs and the ancestral organism where this gene could be originated based on the comparison and analysis of UVR8 functionally important motifs. Results obtained showed that Chlorophytes are the first organisms from the Viridiplantae group where UVR8 appear. UVR8 is present in green algae, bryophytes, lycophytes and angiosperms. All the sequences identified contain tryptophans 233 and 285, arginines involved in homodimerization and the VP domain suggesting they are true UVR8 photoreceptors. We also determined that some species from bryophytes and angiosperms contain more than one UVR8 gene copy opening the question if UVR8 could constitute a gene family in these species. In conclusion, we described the functional conservation among UVR8 proteins from green algae to higher plants.

  20. Proteomic analyses of human cytomegalovirus strain AD169 derivatives reveal highly conserved patterns of viral and cellular proteins in infected fibroblasts.

    Science.gov (United States)

    Reyda, Sabine; Büscher, Nicole; Tenzer, Stefan; Plachter, Bodo

    2014-01-07

    Human cytomegalovirus (HCMV) particle morphogenesis in infected cells is an orchestrated process that eventually results in the release of enveloped virions. Proteomic analysis has been employed to reveal the complexity in the protein composition of these extracellular particles. Only limited information is however available regarding the proteome of infected cells preceding the release of HCMV virions. We used quantitative mass spectrometry to address the pattern of viral and cellular proteins in cells, infected with derivatives of the AD169 laboratory strain. Our analyses revealed a remarkable conservation in the patterns of viral and of abundant cellular proteins in cells, infected for 2 hours, 2 days, or 4 days. Most viral proteins increased in abundance as the infection progressed over time. Of the proteins that were reliably detectable by mass spectrometry, only IE1 (pUL123), pTRS1, and pIRS1 were downregulated at 4 days after infection. In addition, little variation of viral proteins in the virions of the different viruses was detectable, independent of the expression of the major tegument protein pp65. Taken together these data suggest that there is little variation in the expression program of viral and cellular proteins in cells infected with related HCMVs, resulting in a conserved pattern of viral proteins ultimately associated with extracellular virions.

  1. Structure and characterization of a cDNA clone for phenylalanine ammonia-lyase from cut-injured roots of sweet potato

    Energy Technology Data Exchange (ETDEWEB)

    Tanaka, Yoshiyuki; Matsuoka, Makoto; Yamanoto, Naoki; Ohashi, Yuko; Kano-Murakami, Yuriko; Ozeki, Yoshihiro (National Institute of Agro-Environmental Sciences, Ibaraki (Japan) Univ. of Tokyo (Japan))

    1989-08-01

    A cDNA clone for phenylalanine ammonia-lyase (PAL) induced in wounded sweet potato (Ipomoea batatas Lam.) root was obtained by immunoscreening a cDNA library. The protein produced in Escherichia coli cells containing the plasmid pPAL02 was indistinguishable from sweet potato PAL as judged by Ouchterlony double diffusion assays. The M{sub r} of its subunit was 77,000. The cells converted ({sup 14}C)-L-phenylalanine into ({sup 14}C)-t-cinnamic acid and PAL activity was detected in the homogenate of the cells. The activity was dependent on the presence of the pPAL02 plasmid DNA. The nucleotide sequence of the cDNA contained a 2,121-base pair (bp) open-reading frame capable of coding for a polypeptide with 707 amino acids (M{sub r} 77,137), a 22-bp 5{prime}-noncoding region and a 207-bp 3{prime}-noncoding region. The results suggest that the insert DNA fully encoded the amino acid sequence for sweet potato PAL that is induced by wounding. Comparison of the deduced amino acid sequence with that of a PAL cDNA fragment from Phaseolus vulgaris revealed 78.9% homology. The sequence from amino acid residues 258 to 494 was highly conserved, showing 90.7% homology.

  2. A novel thermostable, alkaline pectate lyase from Bacillus tequilensis SV11 with potential in textile industry.

    Science.gov (United States)

    Chiliveri, Swarupa Rani; Linga, Venkateswar Rao

    2014-10-13

    An extracellular pectate lyase was purified and characterized from a UV mutant of Bacillus tequilensis SV11. Purification resulted in a 16.2-fold improvement in the enzyme specific activity, with approximately 40.2% yield. SDS-PAGE showed that the enzyme had two subunits with molecular masses of 135 ± 2 and 43 ± 2 kDa. Further, MALDI-TOF MS experiments revealed that the mass spectrum of the second peptide significantly (91% score) matched with the unsaturated rhamnogalacturonyl hydrolase YteR OS-Bacillus subtilis (strain 168) by 27% sequence coverage, nominal mass 43,231 Da, and PI 5.91. The enzyme was optimally active at 60 °C, pH 9. Km and Vmax of the purified pectate lyase was found to be 1.220 mg/mL and 1773 U/mL, respectively. The enzyme was studied for its applicability in bioscouring and found to be efficient in the removal of 97.91% pectin of cotton fabric when compared with alkali-treated fabric.

  3. Genome-wide characterization of the Pectate Lyase-like (PLL) genes in Brassica rapa.

    Science.gov (United States)

    Jiang, Jingjing; Yao, Lina; Miao, Ying; Cao, Jiashu

    2013-11-01

    Pectate lyases (PL) depolymerize demethylated pectin (pectate, EC 4.2.2.2) by catalyzing the eliminative cleavage of α-1,4-glycosidic linked galacturonan. Pectate Lyase-like (PLL) genes are one of the largest and most complex families in plants. However, studies on the phylogeny, gene structure, and expression of PLL genes are limited. To understand the potential functions of PLL genes in plants, we characterized their intron-exon structure, phylogenetic relationships, and protein structures, and measured their expression patterns in various tissues, specifically the reproductive tissues in Brassica rapa. Sequence alignments revealed two characteristic motifs in PLL genes. The chromosome location analysis indicated that 18 of the 46 PLL genes were located in the least fractionated sub-genome (LF) of B. rapa, while 16 were located in the medium fractionated sub-genome (MF1) and 12 in the more fractionated sub-genome (MF2). Quantitative RT-PCR analysis showed that BrPLL genes were expressed in various tissues, with most of them being expressed in flowers. Detailed qRT-PCR analysis identified 11 pollen specific PLL genes and several other genes with unique spatial expression patterns. In addition, some duplicated genes showed similar expression patterns. The phylogenetic analysis identified three PLL gene subfamilies in plants, among which subfamily II might have evolved from gene neofunctionalization or subfunctionalization. Therefore, this study opens the possibility for exploring the roles of PLL genes during plant development.

  4. Comparative Study of Regulatory Circuits in Two Sea Urchin Species Reveals Tight Control of Timing and High Conservation of Expression Dynamics.

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    Tsvia Gildor

    2015-07-01

    Full Text Available Accurate temporal control of gene expression is essential for normal development and must be robust to natural genetic and environmental variation. Studying gene expression variation within and between related species can delineate the level of expression variability that development can tolerate. Here we exploit the comprehensive model of sea urchin gene regulatory networks and generate high-density expression profiles of key regulatory genes of the Mediterranean sea urchin, Paracentrotus lividus (Pl. The high resolution of our studies reveals highly reproducible gene initiation times that have lower variation than those of maximal mRNA levels between different individuals of the same species. This observation supports a threshold behavior of gene activation that is less sensitive to input concentrations. We then compare Mediterranean sea urchin gene expression profiles to those of its Pacific Ocean relative, Strongylocentrotus purpuratus (Sp. These species shared a common ancestor about 40 million years ago and show highly similar embryonic morphologies. Our comparative analyses of five regulatory circuits operating in different embryonic territories reveal a high conservation of the temporal order of gene activation but also some cases of divergence. A linear ratio of 1.3-fold between gene initiation times in Pl and Sp is partially explained by scaling of the developmental rates with temperature. Scaling the developmental rates according to the estimated Sp-Pl ratio and normalizing the expression levels reveals a striking conservation of relative dynamics of gene expression between the species. Overall, our findings demonstrate the ability of biological developmental systems to tightly control the timing of gene activation and relative dynamics and overcome expression noise induced by genetic variation and growth conditions.

  5. Indigenous knowledge and science unite to reveal spatial and temporal dimensions of distributional shift in wildlife of conservation concern.

    Science.gov (United States)

    Service, Christina N; Adams, Megan S; Artelle, Kyle A; Paquet, Paul; Grant, Laura V; Darimont, Chris T

    2014-01-01

    Range shifts among wildlife can occur rapidly and impose cascading ecological, economic, and cultural consequences. However, occurrence data used to define distributional limits derived from scientific approaches are often outdated for wide ranging and elusive species, especially in remote environments. Accordingly, our aim was to amalgamate indigenous and western scientific evidence of grizzly bear (Ursus arctos horribilis) records and detail a potential range shift on the central coast of British Columbia, Canada. In addition, we test the hypothesis that data from each method yield similar results, as well as illustrate the complementary nature of this coupled approach. Combining information from traditional and local ecological knowledge (TEK/LEK) interviews with remote camera, genetic, and hunting data revealed that grizzly bears are now present on 10 islands outside their current management boundary. LEK interview data suggested this expansion has accelerated over the last 10 years. Both approaches provided complementary details and primarily affirmed one another: all islands with scientific evidence for occupation had consistent TEK/LEK evidence. Moreover, our complementary methods approach enabled a more spatially and temporally detailed account than either method would have afforded alone. In many cases, knowledge already held by local indigenous people could provide timely and inexpensive data about changing ecological processes. However, verifying the accuracy of scientific and experiential knowledge by pairing sources at the same spatial scale allows for increased confidence and detail. A similarly coupled approach may be useful across taxa in many regions.

  6. Heart of endosymbioses: transcriptomics reveals a conserved genetic program among arbuscular mycorrhizal, actinorhizal and legume-rhizobial symbioses.

    Directory of Open Access Journals (Sweden)

    Alexandre Tromas

    Full Text Available To improve their nutrition, most plants associate with soil microorganisms, particularly fungi, to form mycorrhizae. A few lineages, including actinorhizal plants and legumes are also able to interact with nitrogen-fixing bacteria hosted intracellularly inside root nodules. Fossil and molecular data suggest that the molecular mechanisms involved in these root nodule symbioses (RNS have been partially recycled from more ancient and widespread arbuscular mycorrhizal (AM symbiosis. We used a comparative transcriptomics approach to identify genes involved in establishing these 3 endosymbioses and their functioning. We analysed global changes in gene expression in AM in the actinorhizal tree C. glauca. A comparison with genes induced in AM in Medicago truncatula and Oryza sativa revealed a common set of genes induced in AM. A comparison with genes induced in nitrogen-fixing nodules of C. glauca and M. truncatula also made it possible to define a common set of genes induced in these three endosymbioses. The existence of this core set of genes is in accordance with the proposed recycling of ancient AM genes for new functions related to nodulation in legumes and actinorhizal plants.

  7. Indigenous knowledge and science unite to reveal spatial and temporal dimensions of distributional shift in wildlife of conservation concern.

    Directory of Open Access Journals (Sweden)

    Christina N Service

    Full Text Available Range shifts among wildlife can occur rapidly and impose cascading ecological, economic, and cultural consequences. However, occurrence data used to define distributional limits derived from scientific approaches are often outdated for wide ranging and elusive species, especially in remote environments. Accordingly, our aim was to amalgamate indigenous and western scientific evidence of grizzly bear (Ursus arctos horribilis records and detail a potential range shift on the central coast of British Columbia, Canada. In addition, we test the hypothesis that data from each method yield similar results, as well as illustrate the complementary nature of this coupled approach. Combining information from traditional and local ecological knowledge (TEK/LEK interviews with remote camera, genetic, and hunting data revealed that grizzly bears are now present on 10 islands outside their current management boundary. LEK interview data suggested this expansion has accelerated over the last 10 years. Both approaches provided complementary details and primarily affirmed one another: all islands with scientific evidence for occupation had consistent TEK/LEK evidence. Moreover, our complementary methods approach enabled a more spatially and temporally detailed account than either method would have afforded alone. In many cases, knowledge already held by local indigenous people could provide timely and inexpensive data about changing ecological processes. However, verifying the accuracy of scientific and experiential knowledge by pairing sources at the same spatial scale allows for increased confidence and detail. A similarly coupled approach may be useful across taxa in many regions.

  8. The genome sequence of black cottonwood (Populus trichocarpa) reveals 18 conserved cellulose synthase (CesA) genes.

    Science.gov (United States)

    Djerbi, Soraya; Lindskog, Mats; Arvestad, Lars; Sterky, Fredrik; Teeri, Tuula T

    2005-07-01

    The genome sequence of Populus trichocarpa was screened for genes encoding cellulose synthases by using full-length cDNA sequences and ESTs previously identified in the tissue specific cDNA libraries of other poplars. The data obtained revealed 18 distinct CesA gene sequences in P. trichocarpa. The identified genes were grouped in seven gene pairs, one group of three sequences and one single gene. Evidence from gene expression studies of hybrid aspen suggests that both copies of at least one pair, CesA3-1 and CesA3-2, are actively transcribed. No sequences corresponding to the gene pair, CesA6-1 and CesA6-2, were found in Arabidopsis or hybrid aspen, while one homologous gene has been identified in the rice genome and an active transcript in Populus tremuloides. A phylogenetic analysis suggests that the CesA genes previously associated with secondary cell wall synthesis originate from a single ancestor gene and group in three distinct subgroups. The newly identified copies of CesA genes in P. trichocarpa give rise to a number of new questions concerning the mechanism of cellulose synthesis in trees.

  9. Molecular and functional analyses of the metC gene of Lactococcus lactis, encoding cystathionine beta-lyase.

    Science.gov (United States)

    Fernández, M; van Doesburg, W; Rutten, G A; Marugg, J D; Alting, A C; van Kranenburg, R; Kuipers, O 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 beta-lyase (CBL) can convert cystathionine to homocysteine but is also able to catalyze an alpha, gamma 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 strain B78, isolated from a cheese starter culture and known to have a high capacity to produce volatile compounds. The metC gene was found to be cotranscribed with a downstream cysK gene, which encodes a putative cysteine synthase. The MetC proteins of both strains were overproduced in strain MG1363 with the NICE (nisin-controlled expression) system, resulting in a >25-fold increase in cystathionine lyase activity. A disruption of the metC gene was achieved in strain MG1363. Determination of enzymatic activities in the overproducing and knockout strains revealed that MetC is essential for the degradation of cystathionine but that at least one lyase other than CBL contributes to methionine degradation via alpha, gamma elimination to form volatile aroma compounds.

  10. Structural motif screening reveals a novel, conserved carbohydrate-binding surface in the pathogenesis-related protein PR-5d

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    Moffatt Barbara A

    2010-08-01

    Full Text Available Abstract Background Aromatic amino acids play a critical role in protein-glycan interactions. Clusters of surface aromatic residues and their features may therefore be useful in distinguishing glycan-binding sites as well as predicting novel glycan-binding proteins. In this work, a structural bioinformatics approach was used to screen the Protein Data Bank (PDB for coplanar aromatic motifs similar to those found in known glycan-binding proteins. Results The proteins identified in the screen were significantly associated with carbohydrate-related functions according to gene ontology (GO enrichment analysis, and predicted motifs were found frequently within novel folds and glycan-binding sites not included in the training set. In addition to numerous binding sites predicted in structural genomics proteins of unknown function, one novel prediction was a surface motif (W34/W36/W192 in the tobacco pathogenesis-related protein, PR-5d. Phylogenetic analysis revealed that the surface motif is exclusive to a subfamily of PR-5 proteins from the Solanaceae family of plants, and is absent completely in more distant homologs. To confirm PR-5d's insoluble-polysaccharide binding activity, a cellulose-pulldown assay of tobacco proteins was performed and PR-5d was identified in the cellulose-binding fraction by mass spectrometry. Conclusions Based on the combined results, we propose that the putative binding site in PR-5d may be an evolutionary adaptation of Solanaceae plants including potato, tomato, and tobacco, towards defense against cellulose-containing pathogens such as species of the deadly oomycete genus, Phytophthora. More generally, the results demonstrate that coplanar aromatic clusters on protein surfaces are a structural signature of glycan-binding proteins, and can be used to computationally predict novel glycan-binding proteins from 3 D structure.

  11. Crystal structure of Zebrafish interferons I and II reveals conservation of type I interferon structure in vertebrates.

    Science.gov (United States)

    Hamming, Ole Jensen; Lutfalla, Georges; Levraud, Jean-Pierre; Hartmann, Rune

    2011-08-01

    Interferons (IFNs) play a major role in orchestrating the innate immune response toward viruses in vertebrates, and their defining characteristic is their ability to induce an antiviral state in responsive cells. Interferons have been reported in a multitude of species, from bony fish to mammals. However, our current knowledge about the molecular function of fish IFNs as well as their evolutionary relationship to tetrapod IFNs is limited. Here we establish the three-dimensional (3D) structure of zebrafish IFNϕ1 and IFNϕ2 by crystallography. These high-resolution structures offer the first structural insight into fish cytokines. Tetrapods possess two types of IFNs that play an immediate antiviral role: type I IFNs (e.g., alpha interferon [IFN-α] and beta interferon [IFN-β]) and type III IFNs (lambda interferon [IFN-λ]), and each type is characterized by its specific receptor usage. Similarly, two groups of antiviral IFNs with distinct receptors exist in fish, including zebrafish. IFNϕ1 and IFNϕ2 represent group I and group II IFNs, respectively. Nevertheless, both structures reported here reveal a characteristic type I IFN architecture with a straight F helix, as opposed to the remaining class II cytokines, including IFN-λ, where helix F contains a characteristic bend. Phylogenetic trees derived from structure-guided multiple alignments confirmed that both groups of fish IFNs are evolutionarily closer to type I than to type III tetrapod IFNs. Thus, these fish IFNs belong to the type I IFN family. Our results also imply that a dual antiviral IFN system has arisen twice during vertebrate evolution.

  12. High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states.

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    Kevin A Wilkinson

    2008-04-01

    Full Text Available Replication and pathogenesis of the human immunodeficiency virus (HIV is tightly linked to the structure of its RNA genome, but genome structure in infectious virions is poorly understood. We invent high-throughput SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension technology, which uses many of the same tools as DNA sequencing, to quantify RNA backbone flexibility at single-nucleotide resolution and from which robust structural information can be immediately derived. We analyze the structure of HIV-1 genomic RNA in four biologically instructive states, including the authentic viral genome inside native particles. Remarkably, given the large number of plausible local structures, the first 10% of the HIV-1 genome exists in a single, predominant conformation in all four states. We also discover that noncoding regions functioning in a regulatory role have significantly lower (p-value < 0.0001 SHAPE reactivities, and hence more structure, than do viral coding regions that function as the template for protein synthesis. By directly monitoring protein binding inside virions, we identify the RNA recognition motif for the viral nucleocapsid protein. Seven structurally homologous binding sites occur in a well-defined domain in the genome, consistent with a role in directing specific packaging of genomic RNA into nascent virions. In addition, we identify two distinct motifs that are targets for the duplex destabilizing activity of this same protein. The nucleocapsid protein destabilizes local HIV-1 RNA structure in ways likely to facilitate initial movement both of the retroviral reverse transcriptase from its tRNA primer and of the ribosome in coding regions. Each of the three nucleocapsid interaction motifs falls in a specific genome domain, indicating that local protein interactions can be organized by the long-range architecture of an RNA. High-throughput SHAPE reveals a comprehensive view of HIV-1 RNA genome structure, and further

  13. The genome and linkage map of the northern pike (Esox lucius): conserved synteny revealed between the salmonid sister group and the Neoteleostei.

    Science.gov (United States)

    Rondeau, Eric B; Minkley, David R; Leong, Jong S; Messmer, Amber M; Jantzen, Johanna R; von Schalburg, Kristian R; Lemon, Craig; Bird, Nathan H; Koop, Ben F

    2014-01-01

    The northern pike is the most frequently studied member of the Esociformes, the closest order to the diverse and economically important Salmoniformes. The ancestor of all salmonids purportedly experienced a whole-genome duplication (WGD) event, making salmonid species ideal for studying the early impacts of genome duplication while complicating their use in wider analyses of teleost evolution. Studies suggest that the Esociformes diverged from the salmonid lineage prior to the WGD, supporting the use of northern pike as a pre-duplication outgroup. Here we present the first genome assembly, reference transcriptome and linkage map for northern pike, and evaluate the suitability of this species to provide a representative pre-duplication genome for future studies of salmonid and teleost evolution. The northern pike genome sequence is composed of 94,267 contigs (N50 = 16,909 bp) contained in 5,688 scaffolds (N50 = 700,535 bp); the total scaffolded genome size is 878 million bases. Multiple lines of evidence suggest that over 96% of the protein-coding genome is present in the genome assembly. The reference transcriptome was constructed from 13 tissues and contains 38,696 transcripts, which are accompanied by normalized expression data in all tissues. Gene-prediction analysis produced a total of 19,601 northern pike-specific gene models. The first-generation linkage map identifies 25 linkage groups, in agreement with northern pike's diploid karyotype of 2N = 50, and facilitates the placement of 46% of assembled bases onto linkage groups. Analyses reveal a high degree of conserved synteny between northern pike and other model teleost genomes. While conservation of gene order is limited to smaller syntenic blocks, the wider conservation of genome organization implies the northern pike exhibits a suitable approximation of a non-duplicated Protacanthopterygiian genome. This dataset will facilitate future studies of esocid biology and empower ongoing examinations of the

  14. Chromosome-wide mapping of DNA methylation patterns in normal and malignant prostate cells reveals pervasive methylation of gene-associated and conserved intergenic sequences

    Science.gov (United States)

    2011-01-01

    Background DNA methylation has been linked to genome regulation and dysregulation in health and disease respectively, and methods for characterizing genomic DNA methylation patterns are rapidly emerging. We have developed/refined methods for enrichment of methylated genomic fragments using the methyl-binding domain of the human MBD2 protein (MBD2-MBD) followed by analysis with high-density tiling microarrays. This MBD-chip approach was used to characterize DNA methylation patterns across all non-repetitive sequences of human chromosomes 21 and 22 at high-resolution in normal and malignant prostate cells. Results Examining this data using computational methods that were designed specifically for DNA methylation tiling array data revealed widespread methylation of both gene promoter and non-promoter regions in cancer and normal cells. In addition to identifying several novel cancer hypermethylated 5' gene upstream regions that mediated epigenetic gene silencing, we also found several hypermethylated 3' gene downstream, intragenic and intergenic regions. The hypermethylated intragenic regions were highly enriched for overlap with intron-exon boundaries, suggesting a possible role in regulation of alternative transcriptional start sites, exon usage and/or splicing. The hypermethylated intergenic regions showed significant enrichment for conservation across vertebrate species. A sampling of these newly identified promoter (ADAMTS1 and SCARF2 genes) and non-promoter (downstream or within DSCR9, C21orf57 and HLCS genes) hypermethylated regions were effective in distinguishing malignant from normal prostate tissues and/or cell lines. Conclusions Comparison of chromosome-wide DNA methylation patterns in normal and malignant prostate cells revealed significant methylation of gene-proximal and conserved intergenic sequences. Such analyses can be easily extended for genome-wide methylation analysis in health and disease. PMID:21669002

  15. Conservation in the mechanism of glucuronoxylan hydrolysis revealed by the structure of glucuronoxylan xylanohydrolase (CtXyn30A) from Clostridium thermocellum.

    Science.gov (United States)

    Freire, Filipe; Verma, Anil; Bule, Pedro; Alves, Victor D; Fontes, Carlos M G A; Goyal, Arun; Najmudin, Shabir

    2016-11-01

    Glucuronoxylan endo-β-1,4-xylanases cleave the xylan chain specifically at sites containing 4-O-methylglucuronic acid substitutions. These enzymes have recently received considerable attention owing to their importance in the cooperative hydrolysis of heteropolysaccharides. However, little is known about the hydrolysis of glucuronoxylans in extreme environments. Here, the structure of a thermostable family 30 glucuronoxylan endo-β-1,4-xylanase (CtXyn30A) from Clostridium thermocellum is reported. CtXyn30A is part of the cellulosome, a highly elaborate multi-enzyme complex secreted by the bacterium to efficiently deconstruct plant cell-wall carbohydrates. CtXyn30A preferably hydrolyses glucuronoxylans and displays maximum activity at pH 6.0 and 70°C. The structure of CtXyn30A displays a (β/α)8 TIM-barrel core with a side-associated β-sheet domain. Structural analysis of the CtXyn30A mutant E225A, solved in the presence of xylotetraose, revealed xylotetraose-cleavage oligosaccharides partially occupying subsites -3 to +2. The sugar ring at the +1 subsite is held in place by hydrophobic stacking interactions between Tyr139 and Tyr200 and hydrogen bonds to the OH group of Tyr227. Although family 30 glycoside hydrolases are retaining enzymes, the xylopyranosyl ring at the -1 subsite of CtXyn30A-E225A appears in the α-anomeric configuration. A set of residues were found to be strictly conserved in glucuronoxylan endo-β-1,4-xylanases and constitute the molecular determinants of the restricted specificity displayed by these enzymes. CtXyn30A is the first thermostable glucuronoxylan endo-β-1,4-xylanase described to date. This work reveals that substrate recognition by both thermophilic and mesophilic glucuronoxylan endo-β-1,4-xylanases is modulated by a conserved set of residues.

  16. 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

  17. 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.

  18. Refeeding syndrome in a young woman with argininosuccinate lyase deficiency

    Directory of Open Access Journals (Sweden)

    M. Stuy

    2015-09-01

    Full Text Available A severely chronically protein and calorie restricted young woman with argininosuccinate lyase deficiency developed transient refeeding syndrome (RFS and hyperammonemia after modest diet liberalization following initiation of glycerol phenylbutyrate (GPB. The patient required IV supportive care and supplementation with potassium, magnesium and calcium. She is now doing well on GPB and an appropriate maintenance diet. Susceptibility to RFS should be considered in chronically nutritionally restricted patients with metabolic disorders after liberalization of diet.

  19. Refeeding syndrome in a young woman with argininosuccinate lyase deficiency.

    Science.gov (United States)

    Stuy, M; Chen, G-F; Masonek, J M; Scharschmidt, B F

    2015-09-01

    A severely chronically protein and calorie restricted young woman with argininosuccinate lyase deficiency developed transient refeeding syndrome (RFS) and hyperammonemia after modest diet liberalization following initiation of glycerol phenylbutyrate (GPB). The patient required IV supportive care and supplementation with potassium, magnesium and calcium. She is now doing well on GPB and an appropriate maintenance diet. Susceptibility to RFS should be considered in chronically nutritionally restricted patients with metabolic disorders after liberalization of diet.

  20. Refeeding syndrome in a young woman with argininosuccinate lyase deficiency☆

    Science.gov (United States)

    Stuy, M.; Chen, G.-F.; Masonek, J.M.; Scharschmidt, B.F.

    2015-01-01

    A severely chronically protein and calorie restricted young woman with argininosuccinate lyase deficiency developed transient refeeding syndrome (RFS) and hyperammonemia after modest diet liberalization following initiation of glycerol phenylbutyrate (GPB). The patient required IV supportive care and supplementation with potassium, magnesium and calcium. She is now doing well on GPB and an appropriate maintenance diet. Susceptibility to RFS should be considered in chronically nutritionally restricted patients with metabolic disorders after liberalization of diet. PMID:26937403

  1. Bioscouring Knitted Cotton Fabric with an Experimental Pectate Lyase

    Institute of Scientific and Technical Information of China (English)

    D K Appiah; MAO Zhi-ping; L(U) Jia-hua

    2007-01-01

    An experimental pectate lyase enzyme was used toscour knitted cotton fabric and the emphasis was on pectinremoval. Using an enzyme dosage of 0.2 g/L at temperature55℃ and pH 6.35 for 30 rain, good scouring properties wereobtained. When appropriate concentrations of 1 - HydroxyEthylidene- 1, 1 - Diphosphonic Acid(HEDP) and CaCl2were added, the percentage pectin removal improvedsignificantly.

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

  3. The Crystal Structure of the Extracellular 11-heme Cytochrome UndA Reveals a Conserved 10-heme Motif and Defined Binding Site for Soluble Iron Chelates.

    Energy Technology Data Exchange (ETDEWEB)

    Edwards, Marcus; Hall, Andrea; Shi, Liang; Fredrickson, Jim K.; Zachara, John M.; Butt, Julea N.; Richardson, David; Clarke, Thomas A.

    2012-07-03

    Members of the genus Shewanella translocate deca- or undeca-heme cytochromes to the external cell surface thus enabling respiration using extracellular minerals and polynuclear Fe(III) chelates. The high resolution structure of the first undeca-heme outer membrane cytochrome, UndA, reveals a crossed heme chain with four potential electron ingress/egress sites arranged within four domains. Sequence and structural alignment of UndA and the deca-heme MtrF reveals the extra heme of UndA is inserted between MtrF hemes 6 and 7. The remaining UndA hemes can be superposed over the heme chain of the decaheme MtrF, suggesting that a ten heme core is conserved between outer membrane cytochromes. The UndA structure is the first outer membrane cytochrome to be crystallographically resolved in complex with substrates, an Fe(III)-nitrilotriacetate dimer or an Fe(III)-citrate trimer. The structural resolution of these UndA-Fe(III)-chelate complexes provides a rationale for previous kinetic measurements on UndA and other outer membrane cytochromes.

  4. Comparative sequence analysis of Solanum and Arabidopsis in a hot spot for pathogen resistance on potato chromosome V reveals a patchwork of conserved and rapidly evolving genome segments

    Directory of Open Access Journals (Sweden)

    Bruggmann Rémy

    2007-05-01

    Full Text Available Abstract Background Quantitative phenotypic variation of agronomic characters in crop plants is controlled by environmental and genetic factors (quantitative trait loci = QTL. To understand the molecular basis of such QTL, the identification of the underlying genes is of primary interest and DNA sequence analysis of the genomic regions harboring QTL is a prerequisite for that. QTL mapping in potato (Solanum tuberosum has identified a region on chromosome V tagged by DNA markers GP21 and GP179, which contains a number of important QTL, among others QTL for resistance to late blight caused by the oomycete Phytophthora infestans and to root cyst nematodes. Results To obtain genomic sequence for the targeted region on chromosome V, two local BAC (bacterial artificial chromosome contigs were constructed and sequenced, which corresponded to parts of the homologous chromosomes of the diploid, heterozygous genotype P6/210. Two contiguous sequences of 417,445 and 202,781 base pairs were assembled and annotated. Gene-by-gene co-linearity was disrupted by non-allelic insertions of retrotransposon elements, stretches of diverged intergenic sequences, differences in gene content and gene order. The latter was caused by inversion of a 70 kbp genomic fragment. These features were also found in comparison to orthologous sequence contigs from three homeologous chromosomes of Solanum demissum, a wild tuber bearing species. Functional annotation of the sequence identified 48 putative open reading frames (ORF in one contig and 22 in the other, with an average of one ORF every 9 kbp. Ten ORFs were classified as resistance-gene-like, 11 as F-box-containing genes, 13 as transposable elements and three as transcription factors. Comparing potato to Arabidopsis thaliana annotated proteins revealed five micro-syntenic blocks of three to seven ORFs with A. thaliana chromosomes 1, 3 and 5. Conclusion Comparative sequence analysis revealed highly conserved collinear regions

  5. Inactivating effects of the lactoperoxidase system on bacterial lyases involved in oral malodour production.

    Science.gov (United States)

    Nakano, Manabu; Shin, Kouichirou; Wakabayashi, Hiroyuki; Yamauchi, Koji; Abe, Fumiaki; Hironaka, Shouji

    2015-10-01

    The main components of oral malodour have been identified as volatile sulfur compounds (VSCs), including hydrogen sulfide (H(2)S) and methyl mercaptan (CH(3)SH). The lactoperoxidase (LPO) system (consisting of LPO, glucose oxidase, glucose and thiocyanate) was previously shown to exhibit antimicrobial activities against some oral bacteria in vitro and suppressive effects on VSCs in mouth air in a clinical trial. Here, we examined the in vitro effects of the LPO system on the activities of the bacterial lyases involved in the production of VSCs by oral anaerobes. The exposure of crude bacterial extracts of Fusobacterium nucleatum and Porphyromonas gingivalis or purified methionine γ-lyase to the LPO system resulted in the inactivation of their lyase activities through l-cysteine and l-methionine, which was linked to the production of H(2)S and CH(3)SH, respectively. The exposure of living F. nucleatum and P. gingivalis cells to the LPO system resulted in the suppression of cell numbers and lyase activities. The inactivation of the crude bacterial extracts of F. nucleatum and purified methionine γ-lyase by the LPO system was partly recovered by the addition of DTT. Therefore, the LPO system may inactivate bacterial lyases including methionine γ-lyase by reacting with the free cysteine residues of lyases. These results suggested that the LPO system suppresses the production of VSCs not only through its antimicrobial effects, but also by its inactivating effects on the bacterial lyases of F. nucleatum and P. gingivalis.

  6. From Amazonia to the Atlantic forest: molecular phylogeny of Phyzelaphryninae frogs reveals unexpected diversity and a striking biogeographic pattern emphasizing conservation challenges.

    Science.gov (United States)

    Fouquet, Antoine; Loebmann, Daniel; Castroviejo-Fisher, Santiago; Padial, José M; Orrico, Victor G D; Lyra, Mariana L; Roberto, Igor Joventino; Kok, Philippe J R; Haddad, Célio F B; Rodrigues, Miguel T

    2012-11-01

    Documenting the Neotropical amphibian diversity has become a major challenge facing the threat of global climate change and the pace of environmental alteration. Recent molecular phylogenetic studies have revealed that the actual number of species in South American tropical forests is largely underestimated, but also that many lineages are millions of years old. The genera Phyzelaphryne (1 sp.) and Adelophryne (6 spp.), which compose the subfamily Phyzelaphryninae, include poorly documented, secretive, and minute frogs with an unusual distribution pattern that encompasses the biotic disjunction between Amazonia and the Atlantic forest. We generated >5.8 kb sequence data from six markers for all seven nominal species of the subfamily as well as for newly discovered populations in order to (1) test the monophyly of Phyzelaphryninae, Adelophryne and Phyzelaphryne, (2) estimate species diversity within the subfamily, and (3) investigate their historical biogeography and diversification. Phylogenetic reconstruction confirmed the monophyly of each group and revealed deep subdivisions within Adelophryne and Phyzelaphryne, with three major clades in Adelophryne located in northern Amazonia, northern Atlantic forest and southern Atlantic forest. Our results suggest that the actual number of species in Phyzelaphryninae is, at least, twice the currently recognized species diversity, with almost every geographically isolated population representing an anciently divergent candidate species. Such results highlight the challenges for conservation, especially in the northern Atlantic forest where it is still degraded at a fast pace. Molecular dating revealed that Phyzelaphryninae originated in Amazonia and dispersed during early Miocene to the Atlantic forest. The two Atlantic forest clades of Adelophryne started to diversify some 7 Ma minimum, while the northern Amazonian Adelophryne diversified much earlier, some 13 Ma minimum. This striking biogeographic pattern coincides with

  7. A comparative transcriptome analysis reveals expression profiles conserved across three Eimeria spp. of domestic fowl and associated with multiple developmental stages.

    Science.gov (United States)

    Novaes, Jeniffer; Rangel, Luiz Thibério L D; Ferro, Milene; Abe, Ricardo Y; Manha, Alessandra P S; de Mello, Joana C M; Varuzza, Leonardo; Durham, Alan M; Madeira, Alda Maria B N; Gruber, Arthur

    2012-01-01

    Coccidiosis of the domestic fowl is a worldwide disease caused by seven species of protozoan parasites of the genus Eimeria. The genome of the model species, Eimeria tenella, presents a complexity of 55-60MB distributed in 14 chromosomes. Relatively few studies have been undertaken to unravel the complexity of the transcriptome of Eimeria parasites. We report here the generation of more than 45,000 open reading frame expressed sequence tag (ORESTES) cDNA reads of E. tenella, Eimeria maxima and Eimeria acervulina, covering several developmental stages: unsporulated oocysts, sporoblastic oocysts, sporulated oocysts, sporozoites and second generation merozoites. All reads were assembled to constitute gene indices and submitted to a comprehensive functional annotation pipeline. In the case of E. tenella, we also incorporated publicly available ESTs to generate an integrated body of information. Orthology analyses have identified genes conserved across different apicomplexan parasites, as well as genes restricted to the genus Eimeria. Digital expression profiles obtained from ORESTES/EST countings, submitted to clustering analyses, revealed a high conservation pattern across the three Eimeria spp. Distance trees showed that unsporulated and sporoblastic oocysts constitute a distinct clade in all species, with sporulated oocysts forming a more external branch. This latter stage also shows a close relationship with sporozoites, whereas first and second generation merozoites are more closely related to each other than to sporozoites. The profiles were unambiguously associated with the distinct developmental stages and strongly correlated with the order of the stages in the parasite life cycle. Finally, we present The Eimeria Transcript Database (http://www.coccidia.icb.usp.br/eimeriatdb), a website that provides open access to all sequencing data, annotation and comparative analysis. We expect this repository to represent a useful resource to the Eimeria scientific

  8. Discovery of the Selective CYP17A1 Lyase Inhibitor BMS-351 for the Treatment of Prostate Cancer.

    Science.gov (United States)

    Huang, Audris; Jayaraman, Lata; Fura, Aberra; Vite, Gregory D; Trainor, George L; Gottardis, Marco M; Spires, Thomas E; Spires, Vanessa M; Rizzo, Cheryl A; Obermeier, Mary T; Elzinga, Paul A; Todderud, Gordon; Fan, Yi; Newitt, John A; Beyer, Sophie M; Zhu, Yongxin; Warrack, Bethanne M; Goodenough, Angela K; Tebben, Andrew J; Doweyko, Arthur M; Gold, David L; Balog, Aaron

    2016-01-14

    Efforts to identify a potent, reversible, nonsteroidal CYP17A1 lyase inhibitor with good selectivity over CYP17A1 hydroxylase and CYPs 11B1 and 21A2 for the treatment of castration-resistant prostate cancer (CRPC) culminated in the discovery of BMS-351 (compound 18), a pyridyl biaryl benzimidazole with an excellent in vivo profile. Biological evaluation of BMS-351 at a dose of 1.5 mg in castrated cynomolgus monkeys revealed a remarkable reduction in testosterone levels with minimal glucocorticoid and mineralcorticoid perturbation. Based on a favorable profile, BMS-351 was selected as a candidate for further preclinical evaluation.

  9. Iodothyronine deiodinase gene analysis of the Pacific oyster Crassostrea gigas reveals possible conservation of thyroid hormone feedback regulation mechanism in mollusks

    Science.gov (United States)

    Huang, Wen; Xu, Fei; Qu, Tao; Li, Li; Que, Huayong; Zhang, Guofan

    2015-07-01

    Iodothyronine deiodinase catalyzes the initiation and termination of thyroid hormones (THs) effects, and plays a central role in the regulation of thyroid hormone level in vertebrates. In non-chordate invertebrates, only one deiodinase has been identified in the scallop Chlamys farreri. Here, two deiodinases were cloned in the Pacific oyster Crassostrea gigas ( CgDx and CgDy). The characteristic in-frame TGA codons and selenocysteine insertion sequence elements in the oyster deiodinase cDNAs supported the activity of them. Furthermore, seven orthologs of deiodinases were found by a tblastn search in the mollusk Lottia gigantea and the annelid Capitella teleta. A phylogenetic analysis revealed that the deiodinase gene originated from an common ancestor and a clade-specific gene duplication occurred independently during the differentiation of the mollusk, annelid, and vertebrate lineages. The distinct spatiotemporal expression patterns implied functional divergence of the two deiodinases. The expression of CgDx and CgDy was influenced by L-thyroxine T4, and putative thyroid hormone responsive elements were found in their promoters, which suggested that the oyster deiodinases were feedback regulated by TH. Epinephrine stimulated the expression level of CgDx and CgDy, suggesting an interaction effect between different hormones. This study provides the first evidence for the existence of a conserved TH feedback regulation mechanism in mollusks, providing insights into TH evolution.

  10. Targeted mutation of the talpid3 gene in zebrafish reveals its conserved requirement for ciliogenesis and Hedgehog signalling across the vertebrates.

    Science.gov (United States)

    Ben, Jin; Elworthy, Stone; Ng, Ashley Shu Mei; van Eeden, Freek; Ingham, Philip W

    2011-11-01

    Using zinc-finger nuclease-mediated mutagenesis, we have generated mutant alleles of the zebrafish orthologue of the chicken talpid3 (ta3) gene, which encodes a centrosomal protein that is essential for ciliogenesis. Animals homozygous for these mutant alleles complete embryogenesis normally, but manifest a cystic kidney phenotype during the early larval stages and die within a month of hatching. Elimination of maternally derived Ta3 activity by germline replacement resulted in embryonic lethality of ta3 homozygotes. The phenotype of such maternal and zygotic (MZta3) mutant zebrafish showed strong similarities to that of chick ta3 mutants: absence of primary and motile cilia as well as aberrant Hedgehog (Hh) signalling, the latter manifest by the expanded domains of engrailed and ptc1 expression in the somites, reduction of nkx2.2 expression in the neural tube, symmetric pectoral fins, cyclopic eyes and an ectopic lens. GFP-tagged Gli2a localised to the basal bodies in the absence of the primary cilia and western blot analysis showed that Gli2a protein is aberrantly processed in MZta3 embryos. Zygotic expression of ta3 largely rescued the effects of maternal depletion, but the motile cilia of Kupffer's vesicle remained aberrant, resulting in laterality defects. Our findings underline the importance of the primary cilium for Hh signaling in zebrafish and reveal the conservation of Ta3 function during vertebrate evolution.

  11. 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).

  12. Lactic acid bacteria involved in cocoa beans fermentation from Ivory Coast: Species diversity and citrate lyase production.

    Science.gov (United States)

    Ouattara, Hadja D; Ouattara, Honoré G; Droux, Michel; Reverchon, Sylvie; Nasser, William; Niamke, Sébastien L

    2017-09-01

    Microbial fermentation is an indispensable process for high quality chocolate from cocoa bean raw material. lactic acid bacteria (LAB) are among the major microorganisms responsible for cocoa fermentation but their exact role remains to be elucidated. In this study, we analyzed the diversity of LAB in six cocoa producing regions of Ivory Coast. Ribosomal 16S gene sequence analysis showed that Lactobacillus plantarum and Leuconostoc mesenteroides are the dominant LAB species in these six regions. In addition, other species were identified as the minor microbial population, namely Lactobacillus curieae, Enterococcus faecium, Fructobacillus pseudoficulneus, Lactobacillus casei, Weissella paramesenteroides and Weissella cibaria. However, in each region, the LAB microbial population was composed of a restricted number of species (maximum 5 species), which varied between the different regions. LAB implication in the breakdown of citric acid was investigated as a fundamental property for a successful cocoa fermentation process. High citrate lyase producer strains were characterized by rapid citric acid consumption, as revealed by a 4-fold decrease in citric acid concentration in the growth medium within 12h, concomitant with an increase in acetic acid and lactic acid concentration. The production of citrate lyase was strongly dependent on environmental conditions, with optimum production at acidic pH (pH<5), and moderate temperature (30-40°C), which corresponds to conditions prevailing in the early stage of natural cocoa fermentation. This study reveals that one of the major roles of LAB in the cocoa fermentation process involves the breakdown of citric acid during the early stage of cocoa fermentation through the activity of citrate lyase. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. New Mechanistic Insight from Substrate- and Product-Bound Structures of the Metal-Dependent Dimethylsulfoniopropionate Lyase DddQ.

    Science.gov (United States)

    Brummett, Adam E; Dey, Mishtu

    2016-11-08

    The marine microbial catabolism of dimethylsulfoniopropionate (DMSP) by the lyase pathway liberates ∼300 million tons of dimethyl sulfide (DMS) per year, which plays a major role in the biogeochemical cycling of sulfur. Recent biochemical and structural studies of some DMSP lyases, including DddQ, reveal the importance of divalent transition metal ions in assisting DMSP cleavage. While DddQ is believed to be zinc-dependent primarily on the basis of structural studies, excess zinc inhibits the enzyme. We examine the importance of iron in regulating the DMSP β-elimination reaction catalyzed by DddQ as our as-isolated purple-colored enzyme possesses ∼0.5 Fe/subunit. The UV-visible spectrum exhibited a feature at 550 nm, consistent with a tyrosinate-Fe(III) ligand-to-metal charge transfer transition. Incubation of as-isolated DddQ with added iron increases the intensity of the 550 nm peak, whereas addition of dithionite causes a bleaching as Fe(III) is reduced. Both the Fe(III) oxidized and Fe(II) reduced species are active, with similar kcat values and 2-fold differences in their Km values for DMSP. The slow turnover of Fe(III)-bound DddQ allowed us to capture a substrate-bound form of the enzyme. Our DMSP-Fe(III)-DddQ structure reveals conformational changes associated with substrate binding and shows that DMSP is positioned optimally to bind iron and is in the proximity of Tyr 120 that acts as a Lewis base to initiate catalysis. The structures of Tris-, DMSP-, and acrylate-bound forms of Fe(III)-DddQ reported here illustrate various states of the enzyme along the reaction pathway. These results provide new insights into DMSP lyase catalysis and have broader significance for understanding the mechanism of oceanic DMS production.

  14. 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

  15. Role of phosphoenolpyruvate in the NADP-isocitrate dehydrogenase and isocitrate lyase reaction in Escherichia coli.

    Science.gov (United States)

    Ogawa, Tadashi; Murakami, Keiko; Mori, Hirotada; Ishii, Nobuyoshi; Tomita, Masaru; Yoshin, Masataka

    2007-02-01

    Phosphoenolpyruvate inhibited Escherichia coli NADP-isocitrate dehydrogenase allosterically (Ki of 0.31 mM) and isocitrate lyase uncompetitively (Ki' of 0.893 mM). Phosphoenolpyruvate enhances the uncompetitive inhibition of isocitrate lyase by increasing isocitrate, which protects isocitrate dehydrogenase from the inhibition, and contributes to the control through the tricarboxylic acid cycle and glyoxylate shunt.

  16. Role of Phosphoenolpyruvate in the NADP-Isocitrate Dehydrogenase and Isocitrate Lyase Reaction in Escherichia coli▿

    OpenAIRE

    2006-01-01

    Phosphoenolpyruvate inhibited Escherichia coli NADP-isocitrate dehydrogenase allosterically (Ki of 0.31 mM) and isocitrate lyase uncompetitively (Ki′ of 0.893 mM). Phosphoenolpyruvate enhances the uncompetitive inhibition of isocitrate lyase by increasing isocitrate, which protects isocitrate dehydrogenase from the inhibition, and contributes to the control through the tricarboxylic acid cycle and glyoxylate shunt.

  17. Possible role of cysteine-S-conjugate β-lyase in species differences in cisplatin nephrotoxicity.

    Science.gov (United States)

    Katayama, Rieko; Nagata, Saori; Iida, Hiroko; Yamagishi, Norio; Yamashita, Tetsuro; Furuhama, Kazuhisa

    2011-09-01

    To better understand species differences in cisplatin nephrotoxicity, we focused on renal cysteine-S-conjugate β-lyase (C-S lyase), which may play a crucial role in the metabolism of platinum (Pt)-cysteine conjugates. Aminooxyacetic acid hemihydrochloride (AOAA), an inhibitor of C-S lyase, reduced renal injuries due to cisplatin in rats, suggesting involvement of C-S lyase. On day 5 following a bolus cisplatin injection, three species showed in vivo nephrotoxic potentials in the order of rats>mice=rabbits (the highest to lowest), based on body surface. The levels of renal Pt residue at the nephrotoxic dose were in order of rabbits>rats>mice. Meanwhile, the activity of endogenous (basal) mitochondrial aspartate aminotransferase (AST), one of the C-S lyases, in the renal cortex of naive animals was rats>mice=rabbits. In a qualitative Western blot analysis, expression of mitochondrial C-S lyase in the kidney was observed at approximately 37kDa in all five species used. In in vitro studies, the cytotoxicity of cisplatin was dependent on the expression level of C-S lyase mRNA in the respective renal cells. These results demonstrate that species differences in cisplatin nephrotoxicity are attributable to an interaction of renal Pt transition with C-S lyase activity.

  18. Spectroscopic studies on the active site of hydroperoxide lyase : the influence of detergents on its conformation

    NARCIS (Netherlands)

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

    2001-01-01

    Expression of high quantities of alfalfa hydroperoxide lyase in Escherichia coli made it possible to study its active site and structure in more detail. Circular dichroism (CD) spectra showed that hydroperoxide lyase consists for about 75% of alpha-helices. Electron paramagnetic resonance (EPR) spec

  19. 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

  20. Structure and Mechanism of PhnP, a Phosphodiesterase of the Carbon-Phosphorus Lyase Pathway

    DEFF Research Database (Denmark)

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

    2011-01-01

    PhnP is a phosphodiesterase that plays an important role within the bacterial carbon-phosphorus lyase (CP-lyase) pathway by recycling a "dead-end" intermediate, 5-phospho-α-d-ribosyl 1,2-cyclic phosphate, that is formed during organophosphonate catabolism. As a member of the metallo-β-lactamase s...

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

    NARCIS (Netherlands)

    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 g

  2. 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

  3. Hydroxynitrile Lyases: Biological Sources and Application as Biocatalysts

    Directory of Open Access Journals (Sweden)

    Herfried Griengl

    2004-01-01

    Full Text Available We review the state of the art regarding the application of hydroxynitrile lyases to obtain, enantioselectively, (R- and (S-cyanohydrins of aldehydes and ketones. Special emphasis is given to recent preparative applications and to research for extending the number of plants serving as sources for the enzyme. Depending on the plant family, the mechanism of the enzyme-catalysed reaction can be different. A novel area of research is the consideration of evolutionary aspects on the basis of structure comparisons.

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

    DEFF Research Database (Denmark)

    Brodersen, Ditlev Egeskov

    structural features. The complex contains at least two different active sites and suggest a revision of current models of carbon-phosphorus bond cleavage. Using electron microscopy, we map the binding site of an additional protein subunit, which may use ATP for driving conformational changes during...... the proteins encoded in the phn operon act in concert to catabolise phosphonate remain unknown. We have determined the crystal structure of a 240 kDa Escherichia coli carbon-phosphorus lyase core complex at 1.7 Å and show that it comprises a highly intertwined network of subunits with several unexpected...

  5. An active site homology model of phenylalanine ammonia-lyase from Petroselinum crispum.

    Science.gov (United States)

    Röther, Dagmar; Poppe, László; Morlock, Gaby; Viergutz, Sandra; Rétey, János

    2002-06-01

    The plant enzyme phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) shows homology to histidine ammonia-lyase (HAL) whose structure has been solved by X-ray crystallography. Based on amino-acid sequence alignment of the two enzymes, mutagenesis was performed on amino-acid residues that were identical or similar to the active site residues in HAL to gain insight into the importance of this residues in PAL for substrate binding or catalysis. We mutated the following amino-acid residues: S203, R354, Y110, Y351, N260, Q348, F400, Q488 and L138. Determination of the kinetic constants of the overexpressed and purified enzymes revealed that mutagenesis led in each case to diminished activity. Mutants S203A, R354A and Y351F showed a decrease in kcat by factors of 435, 130 and 235, respectively. Mutants F400A, Q488A and L138H showed a 345-, 615- and 14-fold lower kcat, respectively. The greatest loss of activity occurred in the PAL mutants N260A, Q348A and Y110F, which were 2700, 2370 and 75 000 times less active than wild-type PAL. To elucidate the possible function of the mutated amino-acid residues in PAL we built a homology model of PAL based on structural data of HAL and mutagenesis experiments with PAL. The homology model of PAL showed that the active site of PAL resembles the active site of HAL. This allowed us to propose possible roles for the corresponding residues in PAL catalysis.

  6. Alginate lyase: Review of major sources and classification, properties, structure-function analysis and applications

    Science.gov (United States)

    Zhu, Benwei; Yin, Heng

    2015-01-01

    Alginate lyases catalyze the degradation of alginate, a complex copolymer of α-L-guluronate and its C5 epimer β-D-mannuronate. The enzymes have been isolated from various kinds of organisms with different substrate specificities, including algae, marine mollusks, marine and terrestrial bacteria, and some viruses and fungi. With the progress of structural biology, many kinds of alginate lyases of different polysaccharide lyases families have been characterized by obtaining crystal structures, and the catalytic mechanism has also been elucidated. Combined with various studies, we summarized the source, classification and properties of the alginate lyases from different polysaccharide lyases families. The relationship between substrate specificity and protein sequence was also investigated. PMID:25831216

  7. Screening of Alginate Lyase-Producing Bacteria and Optimization of Media Compositions for Extracellular Alginate Lyase Production.

    Science.gov (United States)

    Tavafi, Hadis; Abdi-Ali, Ahya; Ghadam, Parinaz; Gharavi, Sara

    2017-01-01

    Alginate is a linear polysaccharide consisting of guluronate (polyG) and mannuronate (polyM) subunits. In the initial screening of alginate-degrading bacteria from soil, 10 isolates were able to grow on minimal medium containing alginate. The optimization of cell growth and alginate lyase (algL) production was carried out by the addition of 0.8% alginate and 0.2-0.3 M NaCl to the culture medium. Of 10 isolates, one was selected based on its fast growth rate on minimal 9 medium containing 0.4% sodium alginate. The selected bacterium, identified based on morphological and biochemical characteristics as well as 16S rDNA sequence data, was confirmed to be an isolate belonging to the genus Bacillus and designated as Bacillus sp. TAG8. Resuls: The results showed the ability of Bacillus sp. TAG8 to utilize alginate as a sole carbon source. Bacillus sp. TAG8 growth and algL production were augmented with an increase in sodium alginate concentration and also by the addition of 0.2-0.3 M NaCl. Molecular analysis of TAG8 algL gene showed 99% sequence identity with algL of Pseudomonas aeruginosa PAO1. algL produced by Bacillus sp. TAG8 cleaved both polyM and polyG blocks in alginate molecule as well as acetylated alginate residues, confirming the bifunctionality of the isolated lyase. The identification of novel algL genes from microbial communities constitutes a new approach for exploring lyases with specific activity against bacterial alginates and may thus contribute to the eradication of persistent biofilms from clinical samples.

  8. Screening of Alginate Lyase-Producing Bacteria and Optimization of Media Compositions for Extracellular Alginate Lyase Production

    Science.gov (United States)

    Tavafi, Hadis; Abdi- Ali, Ahya A; Ghadam, Parinaz; Gharavi, Sara

    2017-01-01

    Background: Alginate is a linear polysaccharide consisting of guluronate (polyG) and mannuronate (polyM) subunits. Methods: In the initial screening of alginate-degrading bacteria from soil, 10 isolates were able to grow on minimal medium containing alginate. The optimization of cell growth and alginate lyase (algL) production was carried out by the addition of 0.8% alginate and 0.2-0.3 M NaCl to the culture medium. Of 10 isolates, one was selected based on its fast growth rate on minimal 9 medium containing 0.4% sodium alginate. The selected bacterium, identified based on morphological and biochemical characteristics, as well as 16S rDNA sequence data, was confirmed to be an isolate belonging to the genus Bacillus and designated as Bacillus sp. TAG8. Results: The results showed the ability of Bacillus sp. TAG8 in utilizing alginate as a sole carbon source. Bacillus sp. TAG8 growth and algL production were augmented with an increase in sodium alginate concentration and also by the addition of 0.2-0.3 M NaCl. Molecular analysis of TAG8 algL gene showed 99% sequence identity with algL of Pseudomonas aeruginosa PAO1. The algL produced by Bacillus sp. TAG8 cleaved both polyM and polyG blocks in alginate molecule, as well as acetylated alginate residues, confirming the bifunctionality of the isolated lyase. Conclusion: The identification of novel algL genes from microbial communities constitutes a new approach for exploring lyases with specific activity against bacterial alginates and may thus contribute to the eradication of persistent biofilms from clinical samples. PMID:27432784

  9. The phenylalanine ammonia-lyase gene family in Isatis indigotica Fort.: molecular cloning, characterization, and expression analysis.

    Science.gov (United States)

    Ma, Rui-Fang; Liu, Qian-Zi; Xiao, Ying; Zhang, Lei; Li, Qing; Yin, Jun; Chen, Wan-Sheng

    2016-11-01

    Phenolic compounds, metabolites of the phenylpropanoid pathway, play an important role in the growth and environmental adaptation of many plants. Phenylalanine ammonia-lyase (PAL) is the first key enzyme of the phenylpropanoid pathway. The present study was designed to investigate whether there is a multi-gene family in I. Indigotic and, if so, to characterize their properties. We conducted a comprehensive survey on the transcription profiling database by using tBLASTn analysis. Several bioinformatics methods were employed to perform the prediction of composition and physicochemical characters. The expression levels of IiPAL genes in various tissues of I. indigotica with stress treatment were examined by quantitative real-time PCR. Protoplast transient transformation was used to observe the locations of IiPALs. IiPALs were functionally characterized by expression with pET-32a vector in Escherichia colis strain BL21 (DE3). Integration of transcripts and metabolite accumulations was used to reveal the relation between IiPALs and target compounds. An new gene (IiPAL2) was identified and both IiPALs had the conserved enzymatic active site Ala-Ser-Gly and were classified as members of dicotyledon. IiPAL1 and IiPAL2 were expressed in roots, stems, leaves, and flowers, with the highest expression levels of IiPAL1 and IiPAL2 being observed in stems and roots, respectively. The two genes responded to the exogenous elicitor in different manners. Subcellular localization experiment showed that both IiPALs were localized in the cytosol. The recombinant proteins were shown to catalyze the conversion of L-Phe to trans-cinnamic acid. Correlation analysis indicated that IiPAL1 was more close to the biosynthesis of secondary metabolites than IiPAL2. In conclusion, the present study provides a basis for the elucidation of the role of IiPALs genes in the biosynthesis of phenolic compounds, which will help further metabolic engineering to improve the accumulation of bioactive

  10. Functional Insights into Human HMG-CoA Lyase from Structures of Acyl-CoA-containing Ternary Complexes

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Zhuji; Runquist, Jennifer A.; Montgomery, Christa; Miziorko, Henry M.; Kim, Jung-Ja P. (MCW); (UMKC)

    2010-08-16

    HMG-CoA lyase (HMGCL) is crucial to ketogenesis, and inherited human mutations are potentially lethal. Detailed understanding of the HMGCL reaction mechanism and the molecular basis for correlating human mutations with enzyme deficiency have been limited by the lack of structural information for enzyme liganded to an acyl-CoA substrate or inhibitor. Crystal structures of ternary complexes of WT HMGCL with the competitive inhibitor 3-hydroxyglutaryl-CoA and of the catalytically deficient HMGCL R41M mutant with substrate HMG-CoA have been determined to 2.4 and 2.2 {angstrom}, respectively. Comparison of these {beta}/{alpha}-barrel structures with those of unliganded HMGCL and R41M reveals substantial differences for Mg{sup 2+} coordination and positioning of the flexible loop containing the conserved HMGCL 'signature' sequence. In the R41M-Mg{sup 2+}-substrate ternary complex, loop residue Cys{sup 266} (implicated in active-site function by mechanistic and mutagenesis observations) is more closely juxtaposed to the catalytic site than in the case of unliganded enzyme or the WT enzyme-Mg{sup 2+}-3-hydroxyglutaryl-CoA inhibitor complex. In both ternary complexes, the S-stereoisomer of substrate or inhibitor is specifically bound, in accord with the observed Mg{sup 2+} liganding of both C3 hydroxyl and C5 carboxyl oxygens. In addition to His{sup 233} and His{sup 235} imidazoles, other Mg{sup 2+} ligands are the Asp{sup 42} carboxyl oxygen and an ordered water molecule. This water, positioned between Asp{sup 42} and the C3 hydroxyl of bound substrate/inhibitor, may function as a proton shuttle. The observed interaction of Arg{sup 41} with the acyl-CoA C1 carbonyl oxygen explains the effects of Arg{sup 41} mutation on reaction product enolization and explains why human Arg{sup 41} mutations cause drastic enzyme deficiency.

  11. Genetic Structure and Diversity Analysis Revealed by AFLP Markers on Different Glycyrrhiza glabra L. an Endangered Medicinal Species from South of Iran and Implications for Conservation.

    Science.gov (United States)

    Hakimi, Atieh; Zolfaghari, Maryam; Sorkheh, Karim

    2016-09-28

    Glycyrrhiza glabra is an endangered and national-protected medicinal plant species distributed in semi-arid and arid areas of South of Iran. This study addresses the genetic diversity and relationship between populations in different habitats by amplified fragment length polymorphism (AFLP). The plant materials consisted of 90 individuals from nine different populating areas of Dezful, Ramhormoz, Ahvaz, Abadan, Khorramshahr, Behbahan, Haft-tapeh, Andimeshk, and Shushtar. Twenty-three AFLP primer combinations generated a total of 1019 bands with 94.80 % polymorphism. Unweighted pair group method based on arithmetic average (UPGMA) analysis was performed on Jaccard's similarity coefficient matrix. According to results, the genetic resources and diversity in wild populations of G. glabra were rich. The number of polymorphic fragments per primer combination detected ranged from 18 to 65 bands with an average of 41.95 bands. Average polymorphic information content (PIC) was 0.81 in overall primer combinations. M-GTC+P-AGC primer combination showed the highest PIC (0.94) which can be a good candidate primer combination to verify genetic diversity in G. glabra. The UPGMA and principal coordinate analysis showed a clear distinction among the genotypes and the genotypes divided into three clusters in the dendrogram results. A model-based structure analysis revealed the presence of three groups. The study showed that genetic variation and population structure are determined among the accessions of G. glabra collected from different locations. High level of genetic variation in both intra- and inter-species was detected. Conservational efforts have to be strengthened for all populations of the plant species in different habitats.

  12. Establishment of transgenic lines to monitor and manipulate Yap/Taz-Tead activity in zebrafish reveals both evolutionarily conserved and divergent functions of the Hippo pathway.

    Science.gov (United States)

    Miesfeld, Joel B; Link, Brian A

    2014-08-01

    To investigate the role of Hippo pathway signaling during vertebrate development transgenic zebrafish lines were generated and validated to dynamically monitor and manipulate Yap/Taz-Tead activity. Spatial and temporal analysis of Yap/Taz-Tead activity suggested the importance of Hippo signaling during cardiac precursor migration and other developmental processes. When the transcriptional co-activators, Yap and Taz were restricted from interacting with DNA-binding Tead transcription factors through expression of a dominant negative transgene, cardiac precursors failed to migrate completely to the midline resulting in strong cardia bifida. Yap/Taz-Tead activity reporters also allowed us to investigate upstream and downstream factors known to regulate Hippo signaling output in Drosophila. While Crumbs mutations in Drosophila eye disc epithelia increase nuclear translocation and activity of Yorkie (the fly homolog of Yap/Taz), zebrafish crb2a mutants lacked nuclear Yap positive cells and down-regulated Yap/Taz-Tead activity reporters in the eye epithelia, despite the loss of apical-basal cell polarity in those cells. However, as an example of evolutionary conservation, the Tondu-domain containing protein Vestigial-like 4b (Vgll4b) was found to down-regulate endogenous Yap/Taz-Tead activity in the retinal pigment epithelium, similar to Drosophila Tgi in imaginal discs. In conclusion, the Yap/Taz-Tead activity reporters revealed the dynamics of Yap/Taz-Tead signaling and novel insights into Hippo pathway regulation for vertebrates. These studies highlight the utility of this transgenic tool-suite for ongoing analysis into the mechanisms of Hippo pathway regulation and the consequences of signaling output.

  13. Sequence-based Screening for Rare Enzymes: New Insights into the World of AMDases Reveal a Conserved Motif and 58 Novel Enzymes Clustering in Eight Distinct Families.

    Directory of Open Access Journals (Sweden)

    Janine Maimanakos

    2016-08-01

    Full Text Available Arylmalonate-Decarboxylases (AMDases, EC 4.1.1.76 are very rare and mostly underexplored enzymes. Currently only four known and biochemically characterized representatives exist. However, their ability to decarboxylate α-disubstituted malonic acid derivatives to optically pure products without cofactors makes them attractive and promising candidates for the use as biocatalysts in industrial processes. Until now, AMDases could not be separated from other members of the aspartate/glutamate racemase superfamily based on their gene sequences. Within this work, a search algorithm was developed that enables a reliable prediction of AMDase activity for potential candidates. Based on specific sequence patterns and screening methods 58 novel AMDase candidate genes could be identified in this work. Thereby, AMDases with the conserved sequence pattern of Bordetella bronchiseptica’s prototype appeared to be limited to the classes of Alpha-, Beta- and Gammaproteobacteria. Amino acid homologies and comparison of gene surrounding sequences enabled the classification of eight enzyme clusters. Particularly striking is the accumulation of genes coding for different transporters of the TTT family, TRAP transporters and ABC transporters as well as genes coding for mandelate racemases/muconate lactonizing enzymes that might be involved in substrate uptake or degradation of AMDase products. Further, three novel AMDases were characterized which showed a high enantiomeric excess (>99% of the (R-enantiomer of flurbiprofen. These are the recombinant AmdA and AmdV from Variovorax sp. strains HH01 and HH02, originated from soil, and AmdP from Polymorphum gilvum found by a data base search. Altogether our findings give new insights into the class of AMDases and reveal many previously unknown enzyme candidates with high potential for bioindustrial processes.

  14. Sequence-Based Screening for Rare Enzymes: New Insights into the World of AMDases Reveal a Conserved Motif and 58 Novel Enzymes Clustering in Eight Distinct Families

    Science.gov (United States)

    Maimanakos, Janine; Chow, Jennifer; Gaßmeyer, Sarah K.; Güllert, Simon; Busch, Florian; Kourist, Robert; Streit, Wolfgang R.

    2016-01-01

    Arylmalonate Decarboxylases (AMDases, EC 4.1.1.76) are very rare and mostly underexplored enzymes. Currently only four known and biochemically characterized representatives exist. However, their ability to decarboxylate α-disubstituted malonic acid derivatives to optically pure products without cofactors makes them attractive and promising candidates for the use as biocatalysts in industrial processes. Until now, AMDases could not be separated from other members of the aspartate/glutamate racemase superfamily based on their gene sequences. Within this work, a search algorithm was developed that enables a reliable prediction of AMDase activity for potential candidates. Based on specific sequence patterns and screening methods 58 novel AMDase candidate genes could be identified in this work. Thereby, AMDases with the conserved sequence pattern of Bordetella bronchiseptica’s prototype appeared to be limited to the classes of Alpha-, Beta-, and Gamma-proteobacteria. Amino acid homologies and comparison of gene surrounding sequences enabled the classification of eight enzyme clusters. Particularly striking is the accumulation of genes coding for different transporters of the tripartite tricarboxylate transporters family, TRAP transporters and ABC transporters as well as genes coding for mandelate racemases/muconate lactonizing enzymes that might be involved in substrate uptake or degradation of AMDase products. Further, three novel AMDases were characterized which showed a high enantiomeric excess (>99%) of the (R)-enantiomer of flurbiprofen. These are the recombinant AmdA and AmdV from Variovorax sp. strains HH01 and HH02, originated from soil, and AmdP from Polymorphum gilvum found by a data base search. Altogether our findings give new insights into the class of AMDases and reveal many previously unknown enzyme candidates with high potential for bioindustrial processes. PMID:27610105

  15. Serine-202 is the putative precursor of the active site dehydroalanine of phenylalanine ammonia lyase. Site-directed mutagenesis studies on the enzyme from parsley (Petroselinum crispum L.).

    Science.gov (United States)

    Schuster, B; Rétey, J

    1994-08-01

    To investigate the possible role of serine as a precursor of dehydroalanine at the active site of phenylalanine ammonia lyase, two serines, conserved in all known PAL and histidase sequences, were changed to alanine by site-directed mutagenesis. The resulting mutant genes were subcloned into the expression vector pT7.7 and the gene products were assayed for PAL activity. Mutant PALMutS209A showed the same catalytic property as wild-type PAL, whereas mutant PALMutS202A was devoid of catalytic activity, indicating that serine-202 is the most likely precursor of the active site dehydroalanine.

  16. 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.

  17. Molecular cloning, characterization and expression of the phenylalanine ammonia-lyase gene from Juglans regia.

    Science.gov (United States)

    Xu, Feng; Deng, Guang; Cheng, Shuiyuan; Zhang, Weiwei; Huang, Xiaohua; Li, Linling; Cheng, Hua; Rong, Xiaofeng; Li, Jinbao

    2012-01-01

    Phenylalanine ammonia-lyase (PAL) is the first key enzyme of the phenypropanoid pathway. A full-length cDNA of PAL gene was isolated from Juglans regia for the first time, and designated as JrPAL. The full-length cDNA of the JrPAL gene contained a 1935bp open reading frame encoding a 645-amino-acid protein with a calculated molecular weight of about 70.4 kD and isoelectric point (pI) of 6.7. The deduced JrPAL protein showed high identities with other plant PALs. Molecular modeling of JrPAL showed that the 3D model of JrPAL was similar to that of PAL protein from Petroselinum crispum (PcPAL), implying that JrPAL may have similar functions with PcPAL. Phylogenetic tree analysis revealed that JrPAL shared the same evolutionary ancestor of other PALs and had a closer relationship with other angiosperm species. Transcription analysis revealed that JrPAL was expressed in all tested tissues including roots, stems, and leaves, with the highest transcription level being found in roots. Expression profiling analyses by real-time PCR revealed that JrPAL expression was induced by a variety of abiotic and biotic stresses, including UV-B, wounding, cold, abscisic acid and salicylic acid.

  18. Characterization of a Functional Role of the Bradyrhizobium japonicum Isocitrate Lyase in Desiccation Tolerance

    Directory of Open Access Journals (Sweden)

    Jeong-Min Jeon

    2015-07-01

    Full Text Available Bradyrhizobium japonicum is a nitrogen-fixing symbiont of soybean. In previous studies, transcriptomic profiling of B. japonicum USDA110, grown under various environmental conditions, revealed the highly induced gene aceA, encoding isocitrate lyase (ICL. The ICL catalyzes the conversion of isocitrate to succinate and glyoxylate in the glyoxylate bypass of the TCA cycle. Here, we evaluated the functional role of B. japonicum ICL under desiccation-induced stress conditions. We purified AceA (molecular mass = 65 kDa from B. japonicum USDA110, using a His-tag and Ni-NTA column approach, and confirmed its ICL enzyme activity. The aceA mutant showed higher sensitivity to desiccation stress (27% relative humidity (RH, compared to the wild type. ICL activity of the wild type strain increased approximately 2.5-fold upon exposure to 27% RH for 24 h. The aceA mutant also showed an increased susceptibility to salt stress. Gene expression analysis of aceA using qRT-PCR revealed a 148-fold induction by desiccation, while other genes involved in the glyoxylate pathway were not differentially expressed in this condition. Transcriptome analyses revealed that stress-related genes, such as chaperones, were upregulated in the wild-type under desiccating conditions, even though fold induction was not dramatic (ca. 1.5–2.5-fold.

  19. Characterization of a Functional Role of the Bradyrhizobium japonicum Isocitrate Lyase in Desiccation Tolerance.

    Science.gov (United States)

    Jeon, Jeong-Min; Lee, Hae-In; Sadowsky, Michael J; Sugawara, Masayuki; Chang, Woo-Suk

    2015-07-22

    Bradyrhizobium japonicum is a nitrogen-fixing symbiont of soybean. In previous studies, transcriptomic profiling of B. japonicum USDA110, grown under various environmental conditions, revealed the highly induced gene aceA, encoding isocitrate lyase (ICL). The ICL catalyzes the conversion of isocitrate to succinate and glyoxylate in the glyoxylate bypass of the TCA cycle. Here, we evaluated the functional role of B. japonicum ICL under desiccation-induced stress conditions. We purified AceA (molecular mass = 65 kDa) from B. japonicum USDA110, using a His-tag and Ni-NTA column approach, and confirmed its ICL enzyme activity. The aceA mutant showed higher sensitivity to desiccation stress (27% relative humidity (RH)), compared to the wild type. ICL activity of the wild type strain increased approximately 2.5-fold upon exposure to 27% RH for 24 h. The aceA mutant also showed an increased susceptibility to salt stress. Gene expression analysis of aceA using qRT-PCR revealed a 148-fold induction by desiccation, while other genes involved in the glyoxylate pathway were not differentially expressed in this condition. Transcriptome analyses revealed that stress-related genes, such as chaperones, were upregulated in the wild-type under desiccating conditions, even though fold induction was not dramatic (ca. 1.5-2.5-fold).

  20. Characterization of a New Cold-Adapted and Salt-Activated Polysaccharide Lyase Family 7 Alginate Lyase from Pseudoalteromonas sp. SM0524

    Science.gov (United States)

    Chen, Xiu-Lan; Dong, Sheng; Xu, Fei; Dong, Fang; Li, Ping-Yi; Zhang, Xi-Ying; Zhou, Bai-Cheng; Zhang, Yu-Zhong; Xie, Bin-Bin

    2016-01-01

    Marine bacterial alginate lyases play a role in marine alginate degradation and carbon cycling. Although a large number of alginate lyases have been characterized, reports on alginate lyases with special characteristics are still rather less. Here, a gene alyPM encoding an alginate lyase of polysaccharide lyase family 7 (PL7) was cloned from marine Pseudoalteromonas sp. SM0524 and expressed in Escherichia coli. AlyPM shows 41% sequence identity to characterized alginate lyases, indicating that AlyPM is a new PL7 enzyme. The optimal pH for AlyPM activity was 8.5. AlyPM showed the highest activity at 30°C and remained 19% of the highest activity at 5°C. AlyPM was unstable at temperatures above 30°C and had a low Tm of 37°C. These data indicate that AlyPM is a cold-adapted enzyme. Moreover, AlyPM is a salt-activated enzyme. AlyPM activity in 0.5–1.2 M NaCl was sixfolds higher than that in 0 M NaCl, probably caused by a significant increase in substrate affinity, because the Km of AlyPM in 0.5 M NaCl decreased more than 20-folds than that in 0 M NaCl. AlyPM preferably degraded polymannuronate and mainly released dimers and trimers. These data indicate that AlyPM is a new PL7 endo-alginate lyase with special characteristics. PMID:27486451

  1. Hits identified in library screening demonstrate selective CYP17A1 lyase inhibition.

    Science.gov (United States)

    Krug, Sebastian J; Hu, Qingzhong; Hartmann, Rolf W

    2013-03-01

    A screening of structurally different steroid hormone synthesis inhibitors was performed in order to find a starting point for the development of a new inhibitor of the bifunctional steroidogenic enzyme CYP17A1. Emphasis was placed on determination of selectivity between the two catalytic steps, namely 17α-hydroxylase and C(17,20)-lyase. For that purpose a new inhibition assay has been developed. Hits identified within this novel assay demonstrated selective inhibition of CYP17A1 lyase activity, and thus mark the basis for the development of selective C(17,20)-lyase inhibitors for the treatment of prostate cancer.

  2. A fluorescent substrate for carbon-phosphorus lyase: towards the pathway for organophosphonate metabolism in bacteria

    DEFF Research Database (Denmark)

    He, Shu-Mei; Lou, Yan; Hove-Jensen, Bjarne

    2009-01-01

    Many species of bacteria can use naturally occurring organophosphonates as a source of metabolic phosphate by cleaving the carbon-phosphorus bond with a multi-enzyme pathway collectively called carbon-phosphorus lyase (CP-lyase). Very little is known about the fate of organophosphonates entering...... this pathway. In order to detect metabolic intermediates we have synthesized a fluorescently labelled organophosphonate and show that this is a viable substrate for the CP-lyase pathway in Escherichia coli and that the expected product of CP-bond cleavage is formed. The in vivo competence of one potential...

  3. Thermodynamics of Enzyme-Catalyzed Reactions: Part 4. Lyases

    Science.gov (United States)

    Goldberg, Robert N.; Tewari, Yadu B.

    1995-09-01

    Equilibrium constants and enthalpy changes for reactions catalyzed by the lyase class of enzymes have been compiled. For each reaction the following information is given: the reference for the data; the reaction studied; the name of the enzyme used and its Enzyme Commission number; the method of measurement; the conditions of measurement (temperature, pH, ionic strength, and the buffer(s) and cofactor(s) used); the data and an evaluation of it; and, sometimes, commentary on the data and on any corrections which have been applied to it or any calculations for which the data have been used. The data from 106 references have been examined and evaluated. Chemical Abstract Service registry numbers are given for the substances involved in these various reactions. There is a cross reference between the substances and the Enzyme Commission numbers of the enzymes used to catalyze the reactions in which the substances participate.

  4. Tissue and method specificities of phenylalanine ammonia-lyase assay.

    Science.gov (United States)

    Kováčik, Jozef; Klejdus, Bořivoj

    2012-09-01

    A large number of studies have estimated phenylalanine ammonia-lyase (PAL) activity because it strongly reacts to various stimuli. Activity of this enzyme has been assayed mainly by means of spectrophotometry, but the precision of this method is poorly known. We compared assays of PAL activity using spectrophotometry and high performance liquid chromatography (HPLC) in two species (Matricaria chamomilla and Arabidopsis thaliana). Additionally, copper-exposed M. chamomilla plants and buffer with additive were also tested. Our data indicate that spectrophotometry both overestimates (leaves of M. chamomilla) and underestimates (leaves and roots of A. thaliana) PAL activity in comparison with HPLC, suggesting interference of UV-absorbing metabolites. HPLC also showed more accurate detection of cinnamic acid in Cu-exposed chamomile roots. Addition of dithiothreitol to the extraction buffer enhanced PAL activity but reduced proteins, indicating an artificial negative effect. A comparison of PAL activity in selected species is also provided.

  5. Structural Snapshots of Heparin Depolymerization by Heparin Lyase I

    Energy Technology Data Exchange (ETDEWEB)

    Han, Young-Hyun; Garron, Marie-Line; Kim, Hye-Yeon; Kim, Wan-Seok; Zhang, Zhenqing; Ryu, Kyeong-Seok; Shaya, David; Xiao, Zhongping; Cheong, Chaejoon; Kim, Yeong Shik; Linhardt, Robert J.; Jeon, Young Ho; Cygler, Miroslaw; (SNU); (Korea BSI); (McGill); (UST-Korea); (Rensselaer)

    2010-01-12

    Heparin lyase I (heparinase I) specifically depolymerizes heparin, cleaving the glycosidic linkage next to iduronic acid. Here, we show the crystal structures of heparinase I from Bacteroides thetaiotaomicron at various stages of the reaction with heparin oligosaccharides before and just after cleavage and product disaccharide. The heparinase I structure is comprised of a {beta}-jellyroll domain harboring a long and deep substrate binding groove and an unusual thumb-resembling extension. This thumb, decorated with many basic residues, is of particular importance in activity especially on short heparin oligosaccharides. Unexpected structural similarity of the active site to that of heparinase II with an ({alpha}/{alpha}){sub 6} fold is observed. Mutational studies and kinetic analysis of this enzyme provide insights into the catalytic mechanism, the substrate recognition, and processivity.

  6. Purification of L-glutamate-dependent citrate lyase from Clostridium sphenoides and electron microscopic analysis of citrate lyase isolated from Rhodopseudomonas gelatinosa, Streptococcus diacetilactis and C. sphenoides.

    Science.gov (United States)

    Antranikian, G; Klinner, C; Kümmel, A; Schwanitz, D; Zimmermann, T; Mayer, F; Gottschalk, G

    1982-08-01

    Citrate lyase from Clostridium sphenoides was purified 72-fold with a yield of 11%. In contrast to citrate lyase from other sources the activity of this enzyme was strictly dependent on the presence of L-glutamate. The purified enzyme was only stable in the presence of 150 mM L-glutamate or 7 mM L-glutamate plus glycerol, sucrose or bovine serum albumin. Changes of the L-glutamate pool and of enzyme activity in growing cells of C. sphenoides indicated that citrate lyase activity in this organism was regulated by the intracellular L-glutamate concentration. Citrate lyase isolated from C. sphenoides, Rhodopseudomonas gelatinosa and Streptococcus diacetilactis was investigated by electron microscopy using the negative staining technique. Three different projections of enzyme molecules were observed: 'star' form, 'ring' form and 'triangle' form. In samples from R. gelatinosa and S. diacetilactis, star and ring forms occurred in a ratio of about 1:9. Using the enzyme from S. diacetilactis it was demonstrated that this ratio could be altered in favour of the star form by the addition of citrate or tricarballylate. The triangle form was observed in less than 1% of all evaluated molecules and may represent a transition form. In lyase samples from C. sphenoides there existed a correlation between enzyme activity and the proportion of stars and rings at varying concentrations of L-glutamate.

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

    Directory of Open Access Journals (Sweden)

    Nicolae DRAGOŞ

    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 schoenoprasum. 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 ssp. montanum and Allium cepa, while the highest similarity is 77.5%, between Allium senescens ssp. montanum and Allium fistulosum. Leucine is the most common amino acid in all four alliinases, while cysteine is also more frequent than in other enzymes, suggesting a high stability of the molecules due to the possible disulfide bonds.

  8. 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.

  9. Volunteer Conservation Action Data Reveals Large-Scale and Long-Term Negative Population Trends of a Widespread Amphibian, the Common Toad (Bufo bufo)

    Science.gov (United States)

    Petrovan, Silviu O.

    2016-01-01

    Rare and threatened species are the most frequent focus of conservation science and action. With the ongoing shift from single-species conservation towards the preservation of ecosystem services, there is a greater need to understand abundance trends of common species because declines in common species can disproportionately impact ecosystems function. We used volunteer-collected data in two European countries, the United Kingdom (UK) and Switzerland, since the 1970s to assess national and regional trends for one of Europe’s most abundant amphibian species, the common toad (Bufo bufo). Millions of toads were moved by volunteers across roads during this period in an effort to protect them from road traffic. For Switzerland, we additionally estimated trends for the common frog (Rana temporaria), a similarly widespread and common amphibian species. We used state-space models to account for variability in detection and effort and included only populations with at least 5 years of data; 153 populations for the UK and 141 for Switzerland. Common toads declined continuously in each decade in both countries since the 1980s. Given the declines, this common species almost qualifies for International Union for the Conservation of Nature (IUCN) red-listing over this period despite volunteer conservation efforts. Reasons for the declines and wider impacts remain unknown. By contrast, common frog populations were stable or increasing in Switzerland, although there was evidence of declines after 2003. “Toads on Roads” schemes are vital citizen conservation action projects, and the data from such projects can be used for large scale trend estimations of widespread amphibians. We highlight the need for increased research into the status of common amphibian species in addition to conservation efforts focusing on rare and threatened species. PMID:27706154

  10. QM/MM investigation of the reaction rates of substrates of 2,3-dimethylmalate lyase: A catabolic protein isolated from Aspergillus niger.

    Science.gov (United States)

    Chotpatiwetchkul, Warot; Jongkon, Nathjanan; Hannongbua, Supa; Gleeson, M Paul

    2016-07-01

    Aspergillus niger is an industrially important microorganism used in the production of citric acid. It is a common cause of food spoilage and represents a health issue for patients with compromised immune systems. Recent studies on Aspergillus niger have revealed details on the isocitrate lyase (ICL) superfamily and its role in catabolism, including (2R, 3S)-dimethylmalate lyase (DMML). Members of this and related lyase super families are of considerable interest as potential treatments for bacterial and fungal infections, including Tuberculosis. In our efforts to better understand this class of protein, we investigate the catalytic mechanism of DMML, studying five different substrates and two different active site metals configurations using molecular dynamics (MD) and hybrid quantum mechanics/molecular mechanics (QM/MM) calculations. We show that the predicted barriers to reaction for the substrates show good agreement with the experimental kcat values. This results help to confirm the validity of the proposed mechanism and open up the possibility of developing novel mechanism based inhibitors specifically for this target.

  11. Genetics Home Reference: 17 alpha-hydroxylase/17,20-lyase deficiency

    Science.gov (United States)

    ... hypertension), low levels of potassium in the blood (hypokalemia), and abnormal sexual development. The severity of the ... these salt-regulating hormones leads to hypertension and hypokalemia. Loss of 17,20-lyase activity impairs sex ...

  12. 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

  13. 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

  14. Comparative sequence and structure analysis reveals the conservation and diversity of nucleotide positions and their associated tertiary interactions in the riboswitches.

    Directory of Open Access Journals (Sweden)

    Sri D Appasamy

    Full Text Available The tertiary motifs in complex RNA molecules play vital roles to either stabilize the formation of RNA 3D structure or to provide important biological functionality to the molecule. In order to better understand the roles of these tertiary motifs in riboswitches, we examined 11 representative riboswitch PDB structures for potential agreement of both motif occurrences and conservations. A total of 61 unique tertiary interactions were found in the reference structures. In addition to the expected common A-minor motifs and base-triples mainly involved in linking distant regions the riboswitch structures three highly conserved variants of A-minor interactions called G-minors were found in the SAM-I and FMN riboswitches where they appear to be involved in the recognition of the respective ligand's functional groups. From our structural survey as well as corresponding structure and sequence alignments, the agreement between motif occurrences and conservations are very prominent across the representative riboswitches. Our analysis provide evidence that some of these tertiary interactions are essential components to form the structure where their sequence positions are conserved despite a high degree of diversity in other parts of the respective riboswitches sequences. This is indicative of a vital role for these tertiary interactions in determining the specific biological function of riboswitch.

  15. 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...

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

    Directory of Open Access Journals (Sweden)

    Trikka Fotini A

    2005-12-01

    Full Text Available 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-amylase's signal peptide was fused to the N-terminal of an heterologously expressed pectin lyase in E. coli BL21 [DE3]. Thus pectin lyase secretion was achieved into the extracellular growth medium. E. coli cells harboring the recombinant plasmid heterologously express pectin lyase to around 22% of the total cellular proteins, as it was estimated by SDS-PAGE and image analysis. IPTG induces the heterologously expressed enzyme, which is initially distributed extracellularly (7 hour and later on at the periplasmic (9 hours or cytosolic fraction (20 hours. No pectin lyase activity was found in the membranes fraction and in the inclusion bodies. Encapsulation of the recombinant strains of E. coli in alginate or alginate/silica beads 1:5 showed that pectin lyase could degrade effectively its substrate, for at least ten operational cycles. Conclusion Secretion of an heterologously overexpressed pectin lyase in E. coli BL21 [DE3] was achieved in this study. For this purpose the signal peptide of α-amylase from B. subtilis was fused to the N-terminal domain of pectin lyase. Encapsulated E. coli BL21 [DE3] cells harboring pET29c/exPNL were used successfully for pectin degradation up to ten operational cycles indicating that under special conditions this might have biotechnological implementations.

  17. Alginate Lyase Exhibits Catalysis-Independent Biofilm Dispersion and Antibiotic Synergy

    OpenAIRE

    Lamppa, John W.; Karl E Griswold

    2013-01-01

    More than 2 decades of study support the hypothesis that alginate lyases are promising therapeutic candidates for treating mucoid Pseudomonas aeruginosa infections. In particular, the enzymes' ability to degrade alginate, a key component of mucoid biofilm matrix, has been the presumed mechanism by which they disrupt biofilms and enhance antibiotic efficacy. The systematic studies reported here show that, in an in vitro model, alginate lyase dispersion of P. aeruginosa biofilms and enzyme syne...

  18. Characterization of AlgMsp, an Alginate Lyase from Microbulbifer sp. 6532A

    OpenAIRE

    Swift, Steven M.; Hudgens, Jeffrey W.; Heselpoth, Ryan D.; Bales, Patrick M.; Daniel C. Nelson

    2014-01-01

    Alginate is a polysaccharide produced by certain seaweeds and bacteria that consists of mannuronic acid and guluronic acid residues. Seaweed alginate is used in food and industrial chemical processes, while the biosynthesis of bacterial alginate is associated with pathogenic Pseudomonas aeruginosa. Alginate lyases cleave this polysaccharide into short oligo-uronates and thus have the potential to be utilized for both industrial and medicinal applications. An alginate lyase gene, algMsp, from ...

  19. Cloning of a Putative Pectate Lyase Gene Expressed in the Subventral Esophageal Glands of Heterodera glycines.

    Science.gov (United States)

    De Boer, J M; Davis, E L; Hussey, R S; Popeijus, H; Smant, G; Baum, T J

    2002-03-01

    We report the cloning of a Heterodera glycines cDNA that has 72% identity at the amino acid level to a pectate lyase from Globodera rostochiensis. In situ hybridizations showed that the corresponding gene (Hg-pel-1) is expressed in the subventral esophageal gland cells of second-stage juveniles. The deduced amino acid sequence of the H. glycines cDNA shows homology to class III pectate lyases of bacterial and fungal origin.

  20. Characterization of AlgMsp, an alginate lyase from Microbulbifer sp. 6532A.

    Science.gov (United States)

    Swift, Steven M; Hudgens, Jeffrey W; Heselpoth, Ryan D; Bales, Patrick M; Nelson, Daniel C

    2014-01-01

    Alginate is a polysaccharide produced by certain seaweeds and bacteria that consists of mannuronic acid and guluronic acid residues. Seaweed alginate is used in food and industrial chemical processes, while the biosynthesis of bacterial alginate is associated with pathogenic Pseudomonas aeruginosa. Alginate lyases cleave this polysaccharide into short oligo-uronates and thus have the potential to be utilized for both industrial and medicinal applications. An alginate lyase gene, algMsp, from Microbulbifer sp. 6532A, was synthesized as an E.coli codon-optimized clone. The resulting 37 kDa recombinant protein, AlgMsp, was expressed, purified and characterized. The alginate lyase displayed highest activity at pH 8 and 0.2 M NaCl. Activity of the alginate lyase was greatest at 50°C; however the enzyme was not stable over time when incubated at 50°C. The alginate lyase was still highly active at 25°C and displayed little or no loss of activity after 24 hours at 25°C. The activity of AlgMsp was not dependent on the presence of divalent cations. Comparing activity of the lyase against polymannuronic acid and polyguluronic acid substrates showed a higher turnover rate for polymannuronic acid. However, AlgMSP exhibited greater catalytic efficiency with the polyguluronic acid substrate. Prolonged AlgMsp-mediated degradation of alginate produced dimer, trimer, tetramer, and pentamer oligo-uronates.

  1. Characterization of AlgMsp, an alginate lyase from Microbulbifer sp. 6532A.

    Directory of Open Access Journals (Sweden)

    Steven M Swift

    Full Text Available Alginate is a polysaccharide produced by certain seaweeds and bacteria that consists of mannuronic acid and guluronic acid residues. Seaweed alginate is used in food and industrial chemical processes, while the biosynthesis of bacterial alginate is associated with pathogenic Pseudomonas aeruginosa. Alginate lyases cleave this polysaccharide into short oligo-uronates and thus have the potential to be utilized for both industrial and medicinal applications. An alginate lyase gene, algMsp, from Microbulbifer sp. 6532A, was synthesized as an E.coli codon-optimized clone. The resulting 37 kDa recombinant protein, AlgMsp, was expressed, purified and characterized. The alginate lyase displayed highest activity at pH 8 and 0.2 M NaCl. Activity of the alginate lyase was greatest at 50°C; however the enzyme was not stable over time when incubated at 50°C. The alginate lyase was still highly active at 25°C and displayed little or no loss of activity after 24 hours at 25°C. The activity of AlgMsp was not dependent on the presence of divalent cations. Comparing activity of the lyase against polymannuronic acid and polyguluronic acid substrates showed a higher turnover rate for polymannuronic acid. However, AlgMSP exhibited greater catalytic efficiency with the polyguluronic acid substrate. Prolonged AlgMsp-mediated degradation of alginate produced dimer, trimer, tetramer, and pentamer oligo-uronates.

  2. The first myriapod genome sequence reveals conservative arthropod gene content and genome organisation in the centipede Strigamia maritima

    DEFF Research Database (Denmark)

    Chipman, Ariel D.; Ferrier, David E.K.; Brena, Carlo;

    2014-01-01

    many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air......Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We...... present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates...

  3. NMR determination of lysine pKa values in the Pol lambda lyase domain: mechanistic implications.

    Science.gov (United States)

    Gao, Guanghua; DeRose, Eugene F; Kirby, Thomas W; London, Robert E

    2006-02-14

    increase in the Hill coefficients observed in the complex is consistent with the screening of the interacting lysine residues by the DNA. The pKa of K312 residue increased to 10.58 in the complex, probably due to salt bridge formation with the 5'-phosphate group of the DNA. The pKa values obtained for the lyase domain of Pol lambda in the present study are consistent with recent crystallographic studies of Pol beta complexed with 5-phosphorylated abasic sugar analogues in nicked DNA which reveal an open site with no obvious interactions that would significantly depress the pK value for the active site lysine residue. It is suggested that due to the heterogeneity of the damaged DNA substrates with which Pol lambda as well as other related polymerases may be required to bind, the unexpectedly poor optimization of the lyase catalytic site may reflect a compromise of flexibility with catalytic efficiency.

  4. Crystal Structures of Two Novel Dye-Decolorizing Peroxidases Reveal a Beta-Bar Fold With a Conserved Heme-Binding Motif

    Energy Technology Data Exchange (ETDEWEB)

    Zubieta, C.; Krishna, S.S.; Kapoor, M.; Kozbial, P.; McMullan, D.; Axelrod, H.L.; Miller, M.D.; Abdubek, P.; Ambing, E.; Astakhova, T.; Carlton, D.; Chiu, H.J.; Clayton, T.; Deller, M.C.; Duan, L.; Elsliger, M.A.; Feuerhelm, J.; Grzechnik, S.K.; Hale, J.; Hampton, E.; Han, G.W.; /JCSG /SLAC, SSRL /Burnham Inst. Med. Res. /UC, San Diego /Scripps Res. Inst. /Novartis Res. Found.

    2007-10-31

    BtDyP from Bacteroides thetaiotaomicron (strain VPI-5482) and TyrA from Shewanella oneidensis are dye-decolorizing peroxidases (DyPs), members of a new family of heme-dependent peroxidases recently identified in fungi and bacteria. Here, we report the crystal structures of BtDyP and TyrA at 1.6 and 2.7 Angstroms, respectively. BtDyP assembles into a hexamer, while TyrA assembles into a dimer; the dimerization interface is conserved between the two proteins. Each monomer exhibits a two-domain, {alpha}+{beta} ferredoxin-like fold. A site for heme binding was identified computationally, and modeling of a heme into the proposed active site allowed for identification of residues likely to be functionally important. Structural and sequence comparisons with other DyPs demonstrate a conservation of putative heme-binding residues, including an absolutely conserved histidine. Isothermal titration calorimetry experiments confirm heme binding, but with a stoichiometry of 0.3:1 (heme:protein).

  5. Characterization of an Eukaryotic PL-7 Alginate Lyase in the Marine Red Alga Pyropia yezoensis.

    Science.gov (United States)

    Inoue, Akira; Mashino, Chieco; Uji, Toshiki; Saga, Naotsune; Mikami, Koji; Ojima, Takao

    2015-08-01

    Alginate lyases belonging to polysaccharide lyase family-7 (PL-7) are the most well studied on their structures and functions among whole alginate lyases. However, all characterized PL-7 alginate lyases are from prokaryotic bacteria cells. Here we report the first identification of eukaryotic PL-7 alginate lyase from marine red alga Pyropia yezoensis. The cDNA encoding an alginate lyase PyAly was cloned and was used for the construction of recombinant PyAly (rPyAly) expression system in Escherichia coli. Purified rPyAly was assayed to identify its enzymatic properties. Its expression pattern in P. yessoensis was also investigated. PyAly is likely a secreted protein consisting of an N-terminal signal peptide of 25 residues and a catalytic domain of 216 residues. The amino-acid sequence of the catalytic domain showed 19-29% identities to those of bacterial characterized alginate lyases classified into family PL-7. Recombinant PyAly protein, rPyAly, which was produced with E. coli BL21(DE3) by cold-inducible expression system, drastically decreased the viscosity of alginate solution in the early stage of reaction. The most preferable substrate for rPyAly was the poly(M) of alginate with an optimal temperature and pH at 35(o)C and 8.0, respectively. After reaction, unsaturated tri- and tetra-saccharides were produced from poly(M) as major end products. These enzymatic properties indicated that PyAly is an endolytic alginate lyase belonging to PL-7. Moreover, we found that the PyAly gene is split into 4 exons with 3 introns. PyAly was also specifically expressed in the gametophytic haplopid stage. This study demonstrates that PyAly in marine red alga P. yezoensis is a novel PL-7 alginate lyase with an endolytic manner. PyAly is a gametophyte-specifically expressed protein and its structural gene is composed of four exons and three introns. Thus, PyAly is the first enzymatically characterized eukaryotic PL-7 alginate lyase.

  6. Mesoporous phenylalanine ammonia lyase microspheres with improved stability through calcium carbonate templating.

    Science.gov (United States)

    Cui, Jiandong; Zhao, Yamin; Tan, Zhilei; Zhong, Cheng; Han, Peipei; Jia, Shiru

    2017-05-01

    Cross-linked enzyme aggregates (CLEAs) have recently emerged as a promising method for enzyme immobilization due to its simplicity and low cost. However, a lack of good size and morphological control over the as-prepared CLEAs has limited their practical applications in some cases. Here, monodisperse spherical CLEAs of phenylalanine ammonia lyase (PAL microspheres) were prepared based on CaCO3 microtemplates. The preparation procedure involves filling porous CaCO3 microtemplates with the protein by salt precipitation, glutaraldehyde crosslinking, and dissolution of the microtemplates. The formulation of CaCO3 templates with controlled size was studied in detail. Characterization of the prepared PAL microspheres was investigated. The results showed that the PAL microspheres with high immobilization efficiency (79%) exhibited excellent stability, including increased tolerance to proteolysis, low pH, and denaturants, and excellent mechanical properties. For example, free PAL almost lost all activity after they were incubated in the presence of trypsin for 2min, whereas PAL microspheres still retained 95% of their initial activity. Moreover, scanning electron microscope, transmission electron microscope, and N2 adsorption-desorption isotherms revealed that the resultant PAL microspheres possessed good monodispersity and mesoporous structure instead of the amorphous clusters of conventional CLEAs with few pores. Compared with conventional CLEAs, the monodisperse PAL microspheres with mesoporous make them more potentially useful for biomedical and biotechnological applications.

  7. Multiple tandem duplication of the phenylalanine ammonia-lyase genes in Cucumis sativus L.

    Science.gov (United States)

    Shang, Qing-Mao; Li, Liang; Dong, Chun-Juan

    2012-10-01

    Phenylalanine ammonia-lyase (PAL) is the first entry enzyme of the phenylpropanoid pathway, and therefore plays a key role in both plant development and stress defense. In many plants, PAL is encoded by a multi-gene family, and each member is differentially regulated in response to environmental stimuli. In the present study, we report that PAL in cucumber (Cucumis sativus L.) is encoded for by a family of seven genes (designated as CsPAL1-7). All seven CsPALs are arranged in tandem in two duplication blocks, which are located on chromosomes 4 and 6, respectively. The cDNA and protein sequences of the CsPALs share an overall high identity to each other. Homology modeling reveals similarities in their protein structures, besides several slight differences, implying the different activities in conversion of phenylalanine. Phylogenic analysis places CsPAL1-7 in a separate cluster rather than clustering with other plant PALs. Analyses of expression profiles in different cucumber tissues or in response to various stress or plant hormone treatments indicate that CsPAL1-7 play redundant, but divergent roles in cucumber development and stress response. This is consistent with our finding that CsPALs possess overlapping but different cis-elements in their promoter regions. Finally, several duplication events are discussed to explain the evolution of the cucumber PAL genes.

  8. 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 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.

  9. Production and characterization of a plant alpha-hydroxynitrile lyase in Escherichia coli.

    Science.gov (United States)

    Hughes, J; Lakey, J H; Hughes, M A

    1997-02-01

    The coding sequence of the cyanogenic alpha-hydroxynitrile lyase gene of Manihot esculenta Crantz (cassava) was cloned in the plasmid vector pMal-c2 and expressed in Escherichia coli strain JM105. DNA sequencing showed that the recombinant plasmid contained the same sequence as the cDNA clone pHNL10. Peptide sequencing of the recombinant protein showed that the N-terminus was heterogeneous, with either four or six additional amino acid residues compared with the native protein. Circular dichroism spectra indicated similar secondary structure contents for both proteins. Enzyme assays showed that specific activity of native and recombinant proteins were 0.24 and 0.26 mmol CN(-)/mg/min, respectively; that both proteins had optimal activity at 40 degrees C and pH 5.5; and that both proteins were inhibited by the serine protease inhibitor phenyl-methane sulfonyl flouride (PMSF). Isoelectric focusing of native and recombinant protein revealed multiple isoforms for both proteins; the recombinant protein had a more basic mean isoelectric point (pl) (5.1) than the native protein (4.5).

  10. PECTATE LYASE-LIKE10 is associated with pollen wall development in Brassica campestris.

    Science.gov (United States)

    Jiang, Jingjing; Yao, Lina; Yu, Youjian; Lv, Meiling; Miao, Ying; Cao, Jiashu

    2014-11-01

    PECTATE LYASE-LIKE10 (PLL10) was previously identified as one of the differentially expressed genes both in microspores during the late pollen developmental stages and in pistils during the fertilization process in Chinese cabbage (Brassica campestris ssp. chinensis). Here, antisense-RNA was used to study the functions of BcPLL10 in Chinese cabbage. Abnormal pollen was identified in the transgenic lines (bcpll10-4, -5, and -6). In fertilization experiments, fewer seeds were harvested when the antisense-RNA lines were used as pollen donor. In vivo and in vitro pollen germination assays less germinated pollen tubes were observed in bcpll10 lines. Scanning electron microscopy observation verified that the tryphine materials were over accumulated around the pollen surface and sticked them together in bcpll10. Moreover, transmission electron microscopy observation revealed that the internal endintine was overdeveloped and predominantly occupied the intine, and disturbed the normal proportional distribution of the two layers in the non-germinal furrow region; and no obvious demarcation existed between them in the germinal furrow region in the bcpll10 pollen. Collectively, this study presented a novel PLL gene that played an important role during the pollen wall development in B. campestris, which may also possess potential importance for male sterility usage in agriculture.

  11. 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.

  12. 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

    2014-01-01

    Rhamnogalacturonan I lyases (RGI lyases) (EC 4.2.2.-) catalyze cleavage of α-1,4 bonds between rhamnose and galacturonic acid in the backbone of pectins by β-elimination. In the present study, targeted improvement of the thermostability of a PL family 11 RGI lyase from Bacillus licheniformis (DSM......, were obtained due to additive stabilizing effects of single amino acid mutations (E434L, G55V, and G326E) compared to the wild type. The crystal structure of the B. licheniformis wild-type RGI lyase was also determined; the structural analysis corroborated that especially mutation of charged amino...

  13. 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.

  14. Optimization of Culturing Condition and Medium Composition for the Production of Alginate Lyase by a Marine Vibrio sp. YKW-34

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    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℃. 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.

  15. Bioactivation of cysteine conjugates of 1-nitropyrene oxides by cysteine conjugate beta-lyase purified from Peptostreptococcus magnus.

    OpenAIRE

    Kataoka, K; Kinouchi, T; Akimoto, S; Ohnishi, Y

    1995-01-01

    To determine the role of cysteine conjugate beta-lyase (beta-lyase) in the metabolism of mutagenic nitropolycyclic aromatic hydrocarbons, we determined the effect of beta-lyase on the mutagenicities and DNA binding of cysteine conjugates of 4,5-epoxy-4,5-dihydro-1-nitropyrene (1-NP 4,5-oxide) and 9,10-epoxy-9,10-dihydro-1-nitropyrene (1-NP 9,10-oxide), which are detoxified metabolites of the mutagenic compound 1-nitropyrene. We purified beta-lyase from Peptostreptococcus magnus GAI0663, since...

  16. 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.

  17. Mechanism of Hg-C Protonolysis in the Organomercurial Lyase MerB

    Energy Technology Data Exchange (ETDEWEB)

    Parks, Jerry M [ORNL; Guo, Hong [ORNL; Liang, Liyuan [ORNL; Miller, Susan M [ORNL; Summers, Anne O [ORNL; Smith, Jeremy C [ORNL

    2009-01-01

    Demethylation is a key reaction in global mercury cycling. The bacterial organomercurial lyase, MerB, catalyzes the demethylation of a wide range of organomercurials via Hg-C protonolysis. Two strictly conserved cysteine residues in the active site are required for catalysis, but the source of the catalytic proton and the detailed reaction mechanism have not been determined. Here, the two major proposed reaction mechanisms of MerB are investigated and compared using hybrid density functional theory calculations. A model of the active site was constructed from an X-ray crystal structure of the Hg(II)-bound MerB product complex. Stationary point structures and energies characterized for the Hg-C protonolysis of methylmercury rule out the direct protonation mechanism in which a cysteine residue delivers the catalytic proton directly to the organic leaving group. Instead, the calculations support a two-step mechanism in which Cys96 or Cys159 first donates a proton to Asp99, enabling coordination of two thiolates with R-Hg(II). At the rate-limiting transition state, Asp99 protonates the nascent carbanion in a trigonal planar, bis thiol-ligated R-Hg(II) species to cleave the Hg-C bond and release the hydrocarbon product. Reactions with two other substrates, vinylmercury and cis-2-butenyl-2-mercury, were also modeled, and the computed activation barriers for all three organomercurial substrates reproduce the trend in the experimentally observed enzymatic reaction rates. Analysis of atomic charges in the rate-limiting transition state structure using Natural Population Analysis shows that MerB lowers the activation free energy in the Hg-C protonolysis reaction by redistributing electron density into the leaving group and away from the catalytic proton.

  18. Comparative Developmental Transcriptomics Reveals Rewiring of a Highly Conserved Gene Regulatory Network during a Major Life History Switch in the Sea Urchin Genus Heliocidaris.

    Science.gov (United States)

    Israel, Jennifer W; Martik, Megan L; Byrne, Maria; Raff, Elizabeth C; Raff, Rudolf A; McClay, David R; Wray, Gregory A

    2016-03-01

    The ecologically significant shift in developmental strategy from planktotrophic (feeding) to lecithotrophic (nonfeeding) development in the sea urchin genus Heliocidaris is one of the most comprehensively studied life history transitions in any animal. Although the evolution of lecithotrophy involved substantial changes to larval development and morphology, it is not known to what extent changes in gene expression underlie the developmental differences between species, nor do we understand how these changes evolved within the context of the well-defined gene regulatory network (GRN) underlying sea urchin development. To address these questions, we used RNA-seq to measure expression dynamics across development in three species: the lecithotroph Heliocidaris erythrogramma, the closely related planktotroph H. tuberculata, and an outgroup planktotroph Lytechinus variegatus. Using well-established statistical methods, we developed a novel framework for identifying, quantifying, and polarizing evolutionary changes in gene expression profiles across the transcriptome and within the GRN. We found that major changes in gene expression profiles were more numerous during the evolution of lecithotrophy than during the persistence of planktotrophy, and that genes with derived expression profiles in the lecithotroph displayed specific characteristics as a group that are consistent with the dramatically altered developmental program in this species. Compared to the transcriptome, changes in gene expression profiles within the GRN were even more pronounced in the lecithotroph. We found evidence for conservation and likely divergence of particular GRN regulatory interactions in the lecithotroph, as well as significant changes in the expression of genes with known roles in larval skeletogenesis. We further use coexpression analysis to identify genes of unknown function that may contribute to both conserved and derived developmental traits between species. Collectively, our results

  19. ATP citrate lyase inhibitors as novel cancer therapeutic agents.

    Science.gov (United States)

    Zu, Xu-Yu; Zhang, Qing-Hai; Liu, Jiang-Hua; Cao, Ren-Xian; Zhong, Jing; Yi, Guang-Hui; Quan, Zhi-Hua; Pizzorno, Giuseppe

    2012-05-01

    ATP citrate lyase (ACL or ACLY) is an extra-mitochondrial enzyme widely distributed in various human and animal tissues. ACL links glucose and lipid metabolism by catalyzing the formation of acetyl-CoA and oxaloacetate from citrate produced by glycolysis in the presence of ATP and CoA. ACL is aberrantly expressed in many immortalized cells and tumors, such as breast, liver, colon, lung and prostate cancers, and is correlated reversely with tumor stage and differentiation, serving as a negative prognostic marker. ACL is an upstream enzyme of the long chain fatty acid synthesis, providing acetyl-CoA as an essential component of the fatty acid synthesis. Therefore, ACL is a key enzyme of cellular lipogenesis and potent target for cancer therapy. As a hypolipidemic strategy of metabolic syndrome and cancer treatment, many small chemicals targeting ACL have been designed and developed. This review article provides an update for the research and development of ACL inhibitors with a focus on their patent status, offering a new insight into their potential application.

  20. Cystathionine γ-lyase deficiency mediates neurodegeneration in Huntington's disease.

    Science.gov (United States)

    Paul, Bindu D; Sbodio, Juan I; Xu, Risheng; Vandiver, M Scott; Cha, Jiyoung Y; Snowman, Adele M; Snyder, Solomon H

    2014-05-01

    Huntington's disease is an autosomal dominant disease associated with a mutation in the gene encoding huntingtin (Htt) leading to expanded polyglutamine repeats of mutant Htt (mHtt) that elicit oxidative stress, neurotoxicity, and motor and behavioural changes. Huntington's disease is characterized by highly selective and profound damage to the corpus striatum, which regulates motor function. Striatal selectivity of Huntington's disease may reflect the striatally selective small G protein Rhes binding to mHtt and enhancing its neurotoxicity. Specific molecular mechanisms by which mHtt elicits neurodegeneration have been hard to determine. Here we show a major depletion of cystathionine γ-lyase (CSE), the biosynthetic enzyme for cysteine, in Huntington's disease tissues, which may mediate Huntington's disease pathophysiology. The defect occurs at the transcriptional level and seems to reflect influences of mHtt on specificity protein 1, a transcriptional activator for CSE. Consistent with the notion of loss of CSE as a pathogenic mechanism, supplementation with cysteine reverses abnormalities in cultures of Huntington's disease tissues and in intact mouse models of Huntington's disease, suggesting therapeutic potential.

  1. 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.

  2. Enhancement of cell viability and alkaline polygalacturonate lyase production by sorbitol co-feeding with methanol in Pichia pastoris fermentation.

    Science.gov (United States)

    Wang, Zhihao; Wang, Yun; Zhang, Dongxu; Li, Jianghua; Hua, Zhaozhe; Du, Guocheng; Chen, Jian

    2010-02-01

    Alkaline polygalacturonate lyase (PGL) production by Pichia pastoris GS115 was used as a model to study the mechanism and strategy for enhancing heterologous protein production. In order to enhance cell viability and volumetric recombinant protein productivity, sorbitol, which had been confirmed to be a non-repressive carbon source, was added together with methanol during the induction phase. The resultant PGL activity was up to 1593 U mL(-1), which was enhanced 1.85-fold compared to the control (863 U mL(-1)) cultured with sorbitol added at a constant rate of 3.6 g h(-1)L(-1) after an induction period of 100 h. Further results revealed that an appropriate sorbitol co-feeding strategy not only decreased the cell mortality to 8.8% (the control is about 23.1%) in the end of fermentation, but also reduced the proteolytic degradation of PGL.

  3. The crystal structure of human IRE1 luminal domain reveals a conserved dimerization interface required for activation of the unfolded protein response

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Jiahai; Liu, Chuan Yin; Back, Sung Hoon; Clark, Robert L.; Peisach, Daniel; Xu, Zhaohui; Kaufman, Randal J. (Michigan)

    2010-03-08

    The unfolded protein response (UPR) is an evolutionarily conserved mechanism by which all eukaryotic cells adapt to the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Inositol-requiring kinase 1 (IRE1) and PKR-related ER kinase (PERK) are two type I transmembrane ER-localized protein kinase receptors that signal the UPR through a process that involves homodimerization and autophosphorylation. To elucidate the molecular basis of the ER transmembrane signaling event, we determined the x-ray crystal structure of the luminal domain of human IRE1{alpha}. The monomer of the luminal domain comprises a unique fold of a triangular assembly of {beta}-sheet clusters. Structural analysis identified an extensive dimerization interface stabilized by hydrogen bonds and hydrophobic interactions. Dimerization creates an MHC-like groove at the interface. However, because this groove is too narrow for peptide binding and the purified luminal domain forms high-affinity dimers in vitro, peptide binding to this groove is not required for dimerization. Consistent with our structural observations, mutations that disrupt the dimerization interface produced IRE1{alpha} molecules that failed to either dimerize or activate the UPR upon ER stress. In addition, mutations in a structurally homologous region within PERK also prevented dimerization. Our structural, biochemical, and functional studies in vivo altogether demonstrate that IRE1 and PERK have conserved a common molecular interface necessary and sufficient for dimerization and UPR signaling.

  4. Mutational analysis of the yeast RNA helicase Sub2p reveals conserved domains required for growth, mRNA export, and genomic stability

    Science.gov (United States)

    Saguez, Cyril; Gonzales, Fernando A.; Schmid, Manfred; Bøggild, Andreas; Latrick, Chrysa M.; Malagon, Francisco; Putnam, Andrea; Sanderson, Lee; Jankowsky, Eckhard; Brodersen, Ditlev E.; Jensen, Torben Heick

    2013-01-01

    Sub2p/UAP56 is a highly conserved DEAD-box RNA helicase involved in the packaging and nuclear export of mRNA/protein particles (mRNPs). In Saccharomyces cerevisiae, Sub2p is recruited to active chromatin by the pentameric THO complex and incorporated into the larger transcription–export (TREX) complex. Sub2p also plays a role in the maintenance of genome integrity as its inactivation causes severe transcription-dependent recombination of DNA. Despite the central role of Sub2p in early mRNP biology, little is known about its function. Here, we report the presence of an N-terminal motif (NTM) conserved specifically in the Sub2p branch of RNA helicases. Mutation of the NTM causes nuclear accumulation of poly(A)+ RNA and impaired growth without affecting core helicase functions. Thus, the NTM functions as an autonomous unit. Moreover, two sub2 mutants, that are deficient in ATP binding, act in a trans-dominant negative fashion for growth and induce high recombination rates in vivo. Although wild-type Sub2p is prevented access to transcribed loci in such a background, this does not mechanistically explain the phenotype. PMID:23962665

  5. The polychrome works “Prayer of Tobias and Sarah” and “The Archangel Raphael reveals himself to Tobi and his son Tobias”, inside the Cathedral of Cosenza: diagnostic investigations and considerations on the conditions of conservation

    Directory of Open Access Journals (Sweden)

    Gianluca Nava

    2008-04-01

    Full Text Available The oil paintings and “Prayer of Tobias and Sarah” and “The Archangel Raphael reveals himself to Tobi and his son Tobias”, executed by Francesco Bruno at the end of the XVIII century on oval Conservation Science in Cultural Heritage 210 shaped canvas, were investigated by integrated physica-chemical and analytical methodologies in order to obtain scientific data capable of elucidating the state of conservation and the painting technique. Optical (OM and electronic (SEM-EDS microscopy, micro-FT-IR spectroscopy, were applied on some microfragments whilst the two whole paintings were analyzed by the non invasive IR reflectografy technique. The reflectography evidenced some pentimenti of author on the canvas “Prayer of Tobias and Sarah”, while invasive analysis made it possible to locate the stratigraphic sequence of each canvas and to characterise the constituent materials.

  6. 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.

  7. Genome-wide characterisation of the Gcn5 histone acetyltransferase in budding yeast during stress adaptation reveals evolutionarily conserved and diverged roles

    Directory of Open Access Journals (Sweden)

    Brodin David

    2010-03-01

    Full Text Available Abstract Background Gcn5 is a transcriptional coactivator with histone acetyltransferase activity that is conserved with regard to structure as well as its histone substrates throughout the eukaryotes. Gene regulatory networks within cells are thought to be evolutionarily diverged. The use of evolutionarily divergent yeast species, such as S. cerevisiae and S. pombe, which can be studied under similar environmental conditions, provides an opportunity to examine the interface between conserved regulatory components and their cellular applications in different organisms. Results We show that Gcn5 is important for a common set of stress responses in evolutionarily diverged yeast species and that the activity of the conserved histone acetyltransferase domain is required. We define a group of KCl stress response genes in S. cerevisiae that are specifically dependent on Gcn5. Gcn5 is localised to many Gcn5-dependent genes including Gcn5 repressed targets such as FLO8. Gcn5 regulates divergent sets of KCl responsive genes in S. cerevisiae and S. pombe. Genome-wide localization studies showed a tendency for redistribution of Gcn5 during KCl stress adaptation in S. cerevisiae from short genes to the transcribed regions of long genes. An analogous redistribution was not observed in S. pombe. Conclusions Gcn5 is required for the regulation of divergent sets of KCl stress-response genes in S. cerevisiae and S. pombe even though it is required a common group of stress responses, including the response to KCl. Genes that are physically associated with Gcn5 require its activity for their repression or activation during stress adaptation, providing support for a role of Gcn5 as a corepressor as well as a coactivator. The tendency of Gcn5 to re-localise to the transcribed regions of long genes during KCl stress adaptation suggests that Gcn5 plays a specific role in the expression of long genes under adaptive conditions, perhaps by regulating transcriptional

  8. Functional analysis of COP1 and SPA orthologs from Physcomitrella and rice during photomorphogenesis of transgenic Arabidopsis reveals distinct evolutionary conservation.

    Science.gov (United States)

    Ranjan, Aashish; Dickopf, Stephen; Ullrich, Kristian K; Rensing, Stefan A; Hoecker, Ute

    2014-07-01

    Plants have evolved light sensing mechanisms to optimally adapt their growth and development to the ambient light environment. The COP1/SPA complex is a key negative regulator of light signaling in the well-studied dicot Arabidopsis thaliana. COP1 and members of the four SPA proteins are part of an E3 ubiquitin ligase that acts in darkness to ubiquitinate several transcription factors involved in light responses, thereby targeting them for degradation by the proteasome. While COP1 is also found in humans, SPA proteins appear specific to plants. Here, we have functionally addressed evolutionary conservation of COP1 and SPA orthologs from the moss Physcomitrella, the monocot rice and the dicot Arabidopsis. To this end, we analyzed the activities of COP1- and SPA-like proteins from Physcomitrella patens and rice when expressed in Arabidopsis. Expression of rice COP1 and Physcomitrella COP1 protein sequences predominantly complemented all phenotypic aspects of the viable, hypomorphic cop1-4 mutant and the null, seedling-lethal cop1-5 mutant of Arabidopsis: rice COP1 fully rescued the constitutive-photomorphogenesis phenotype in darkness and the leaf expansion defect of cop1 mutants, while it partially restored normal photoperiodic flowering in cop1. Physcomitrella COP1 partially restored normal seedling growth and flowering time, while it fully restored normal leaf expansion in the cop1 mutants. In contrast, expression of a SPA ortholog from Physcomitrella (PpSPAb) in Arabidopsis spa mutants did not rescue any facet of the spa mutant phenotype, suggesting that the PpSPAb protein is not functionally conserved or that the Arabidopsis function evolved after the split of mosses and seed plants. The SPA1 ortholog from rice (OsSPA1) rescued the spa mutant phenotype in dark-grown seedlings, but did not complement any spa mutant phenotype in light-grown seedlings or in adult plants. Our results show that COP1 protein sequences from Physcomitrella, rice and Arabidopsis have

  9. Fate mapping in embryos of Neoceratodus forsteri reveals cranial neural crest participation in tooth development is conserved from lungfish to tetrapods.

    Science.gov (United States)

    Kundrát, Martin; Joss, Jean M P; Smith, Moya M

    2008-01-01

    Experimental evidence that the neural crest participates in tooth development in any osteichthyan fish has so far been lacking. Using vital dye cell-lineage tracking, we demonstrate that trigeminal stream neural crest cells contribute to the dental papilla of developing teeth in the Australian lungfish. Trigeminal neural crest cells labeled before migration have been traced during the earliest stages of tooth development. Neural crest cells from a single midbrain locus were relocated as ectomesenchyme in all developing teeth of the lungfish regardless of their topographical position in the dentition. These cells remain at the dental papilla interface and become cells committed to dentine production. Our findings provide the first cell-lineage evidence that cranial neural crest is fated to ectomesenchyme for tooth development and dentine production in the living sister-group to tetrapods. This shows that cranial neural crest contribution to teeth is conserved from this node on the tetrapod phylogeny.

  10. Analysis of the grape MYB R2R3 subfamily reveals expanded wine quality-related clades and conserved gene structure organization across Vitis and Arabidopsis genomes

    Directory of Open Access Journals (Sweden)

    Arce-Johnson Patricio

    2008-07-01

    Full Text Available Abstract Background The MYB superfamily constitutes the most abundant group of transcription factors described in plants. Members control processes such as epidermal cell differentiation, stomatal aperture, flavonoid synthesis, cold and drought tolerance and pathogen resistance. No genome-wide characterization of this family has been conducted in a woody species such as grapevine. In addition, previous analysis of the recently released grape genome sequence suggested expansion events of several gene families involved in wine quality. Results We describe and classify 108 members of the grape R2R3 MYB gene subfamily in terms of their genomic gene structures and similarity to their putative Arabidopsis thaliana orthologues. Seven gene models were derived and analyzed in terms of gene expression and their DNA binding domain structures. Despite low overall sequence homology in the C-terminus of all proteins, even in those with similar functions across Arabidopsis and Vitis, highly conserved motif sequences and exon lengths were found. The grape epidermal cell fate clade is expanded when compared with the Arabidopsis and rice MYB subfamilies. Two anthocyanin MYBA related clusters were identified in chromosomes 2 and 14, one of which includes the previously described grape colour locus. Tannin related loci were also detected with eight candidate homologues in chromosomes 4, 9 and 11. Conclusion This genome wide transcription factor analysis in Vitis suggests that clade-specific grape R2R3 MYB genes are expanded while other MYB genes could be well conserved compared to Arabidopsis. MYB gene abundance, homology and orientation within particular loci also suggests that expanded MYB clades conferring quality attributes of grapes and wines, such as colour and astringency, could possess redundant, overlapping and cooperative functions.

  11. Replacement of the endogenous starch debranching enzymes ISA1 and ISA2 of Arabidopsis with the rice orthologs reveals a degree of functional conservation during starch synthesis.

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    Sebastian Streb

    Full Text Available This study tested the interchangeability of enzymes in starch metabolism between dicotyledonous and monocotyledonous plant species. Amylopectin--a branched glucose polymer--is the major component of starch and is responsible for its semi-crystalline property. Plants synthesize starch with distinct amylopectin structures, varying between species and tissues. The structure determines starch properties, an important characteristic for cooking and nutrition, and for the industrial uses of starch. Amylopectin synthesis involves at least three enzyme classes: starch synthases, branching enzymes and debranching enzymes. For all three classes, several enzyme isoforms have been identified. However, it is not clear which enzyme(s are responsible for the large diversity of amylopectin structures. Here, we tested whether the specificities of the debranching enzymes (ISA1 and ISA2 are major determinants of species-dependent differences in amylopectin structure by replacing the dicotyledonous Arabidopsis isoamylases (AtISA1 and AtISA2 with the monocotyledonous rice (Oryza sativa isoforms. We demonstrate that the ISA1 and ISA2 are sufficiently well conserved between these species to form heteromultimeric chimeric Arabidopsis/rice isoamylase enzymes. Furthermore, we were able to reconstitute the endosperm-specific rice OsISA1 homomultimeric complex in Arabidopsis isa1isa2 mutants. This homomultimer was able to facilitate normal rates of starch synthesis. The resulting amylopectin structure had small but significant differences in comparison to wild-type Arabidopsis amylopectin. This suggests that ISA1 and ISA2 have a conserved function between plant species with a major role in facilitating the crystallization of pre-amylopectin synthesized by starch synthases and branching enzymes, but also influencing the final structure of amylopectin.

  12. A conserved role for the NAM/miR164 developmental module reveals a common mechanism underlying carpel margin fusion in monocarpous and syncarpous eurosids.

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    Aurelie Chantal Marie Vialette-Guiraud

    2016-01-01

    Full Text Available The majority of angiosperms are syncarpous- their gynoecium is composed of two or more fused carpels. In Arabidopsis thaliana, this fusion is regulated through the balance of expression between CUP SHAPED COTYLEDON (CUC genes, which are orthologues of the Petunia hybrida transcription factor NO APICAL MERISTEM (NAM, and their post-transcriptional regulator miR164. Accordingly, the expression of a miR164-insensitive form of Ar. thaliana CUC2 causes a radical breakdown of carpel fusion. Here, we investigate the role of the NAM/miR164 genetic module in carpel closure in monocarpous plants. We show that the disruption of this module in monocarpous flowers of Ar. thaliana aux1-22 mutants causes a failure of carpel closure, similar to the failure of carpel fusion observed in the wild-type genetic background. This observation suggested that closely related mechanisms may bring about carpel closure and carpel fusion, at least in Ar. thaliana. We therefore tested whether these mechanisms were conserved in a eurosid species that is monocarpous in its wild-type form. We observed that expression of MtNAM, the NAM ortholog in the monocarpous eurosid Medicago truncatula, decreases during carpel margin fusion, suggesting a role for the NAM/miR164 module in this process. We transformed M. truncatula with a miR164-resistant form of MtNAM and observed, among other phenotypes, incomplete carpel closure in the resulting transformants. These data confirm the underlying mechanistic similarity between carpel closure and carpel fusion which we observed in Ar. thaliana. Our observations suggest that the role of the NAM/miR164 module in the fusion of carpel margins has been conserved at least since the most recent common ancestor of the eurosid clade, and open the possibility that a similar mechanism may have been responsible for carpel closure at much earlier stages of angiosperm evolution. We combine our results with studies of early-diverging angiosperms to speculate

  13. 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...

  14. The bifunctional role of LiuE from Pseudomonas aeruginosa, displays additionally HIHG-CoA lyase enzymatic activity.

    Science.gov (United States)

    Chávez-Avilés, Mauricio; Díaz-Pérez, Alma Laura; Campos-García, Jesús

    2010-04-01

    Pseudomonas aeruginosa is able to utilize leucine/isovalerate and acyclic terpenes as sole carbon sources. Key enzymes which play an important role in these catabolic pathways are 3-hydroxy-3-methylglutaryl-coenzyme A (CoA) lyase (EC 4.1.3.4; HMG-CoA lyase) and the 3-hydroxy-3-isohexenylglutaryl-CoA lyase (EC 4.1.2.26; HIHG-CoA lyase), respectively. HMG-CoA lyase is encoded by the liuE gene while the gene for HIHG-CoA lyase remains unidentified. A mutant in the liuE gene was unable to utilize both leucine/isovalerate and acyclic terpenes indicates an involvement of liuE in both catabolic pathways (Chávez-Avilés et al. 2009, FEMS Microbiol Lett 296:117-123). The LiuE protein was purified as a His-tagged recombinant protein and in addition to show HMG-CoA lyase activity (Chávez-Avilés et al. 2009, FEMS Microbiol Lett 296:117-123), also displays HIHG-CoA lyase activity, indicating a bifunctional role in both the leucine/isovalerate and acyclic terpenes catabolic pathways.

  15. Identification of amino acid residues critical for catalysis and stability in Aspergillus niger family 1 pectin lyase A

    NARCIS (Netherlands)

    Sanchez-Torres, P.; Visser, J.; Benen, J.A.E.

    2003-01-01

    Site-directed-mutagenesis studies were performed on family 1 pectin lyase A (PL1A) from Aspergillus niger to gain insight into the reaction mechanism for the pectin lyase-catalysed beta-elimination cleavage of methylesterified polygalacturonic acid and to stabilize the enzyme at slightly basic pH. O

  16. 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...

  17. Identification of amino acid residues critical for catalysis and stability in Aspergillus niger family 1 pectin lyase A

    NARCIS (Netherlands)

    Sanchez-Torres, P.; Visser, J.; Benen, J.A.E.

    2003-01-01

    Site-directed-mutagenesis studies were performed on family 1 pectin lyase A (PL1A) from Aspergillus niger to gain insight into the reaction mechanism for the pectin lyase-catalysed beta-elimination cleavage of methylesterified polygalacturonic acid and to stabilize the enzyme at slightly basic pH.

  18. Conserved regulation of the Hansenula polymorpha MOX promoter in Saccharomyces cerevisiae reveals insights in the transcriptional activation by Adr1p.

    Science.gov (United States)

    Pereira, G G; Hollenberg, C P

    1996-05-15

    The Hansenula polymorpha MOX gene encodes a peroxisomal enzyme that catalyzes the first step of the highly specialized methanol metabolism. MOX is strongly transcribed in cells growing in methanol and completely repressed in glucose. We show here that the MOX promoter confers a glucose-repressible expression upon a lacZ reporter gene in Saccharomyces cerevisiae, an unrelated yeast species that lacks the methanol metabolism. Repression was mediated by a 200-bp region of the MOX promoter, termed MOX-B, and was counteracted by Adr1p, a transcription factor involved in the derepression of S. cerevisiae genes encoding peroxisomal proteins, the class to which MOX belongs. Binding of Adr1p to MOX-B was demonstrated by gel retardation and DNaseI-footprinting, and Adr1p was shown to interact with a DNA region containing only a half of the putative Adr1p consensus binding site. Our findings suggest that Adr1p is a conserved regulator for genes encoding peroxisomal proteins at least in other yeast species, and that its interaction with the DNA is dependent on the promoter context.

  19. Functional analysis of the conserved transcriptional regulator CfWor1 in Cladosporium fulvum reveals diverse roles in the virulence of plant pathogenic fungi.

    Science.gov (United States)

    Okmen, Bilal; Collemare, Jérôme; Griffiths, Scott; van der Burgt, Ate; Cox, Russell; de Wit, Pierre J G M

    2014-04-01

    Fungal Wor1-like proteins are conserved transcriptional regulators that are reported to regulate the virulence of several plant pathogenic fungi by affecting the expression of virulence genes. Here, we report the functional analysis of CfWor1, the homologue of Wor1 in Cladosporium fulvum. Δcfwor1 mutants produce sclerotium-like structures and rough hyphae, which are covered with a black extracellular matrix. These mutants do not sporulate and are no longer virulent on tomato. A CE.CfWor1 transformant that constitutively expresses CfWor1 produces fewer spores with altered morphology and is also reduced in virulence. RNA-seq and RT-qrtPCR analyses suggest that reduced virulence of Δcfwor1 mutants is due to global downregulation of transcription, translation and mitochondrial respiratory chain. The reduced virulence of the CE.CfWor1 transformant is likely due to downregulation of effector genes. Complementation of a non-virulent Δfosge1 (Wor1-homologue) mutant of Fusarium oxysporum f. sp. lycopersici with CfWor1 restored expression of the SIX effector genes in this fungus, but not its virulence. Chimeric proteins of CfWor1/FoSge1 also only partially restored defects of the Δfosge1 mutant, suggesting that these transcriptional regulators have functionally diverged. Altogether, our results suggest that CfWor1 primarily regulates development of C. fulvum, which indirectly affects the expression of a subset of virulence genes.

  20. The hot pepper (Capsicum annuum microRNA transcriptome reveals novel and conserved targets: a foundation for understanding MicroRNA functional roles in hot pepper.

    Directory of Open Access Journals (Sweden)

    Dong-Gyu Hwang

    Full Text Available MicroRNAs (miRNAs are a class of non-coding RNAs approximately 21 nt in length which play important roles in regulating gene expression in plants. Although many miRNA studies have focused on a few model plants, miRNAs and their target genes remain largely unknown in hot pepper (Capsicum annuum, one of the most important crops cultivated worldwide. Here, we employed high-throughput sequencing technology to identify miRNAs in pepper extensively from 10 different libraries, including leaf, stem, root, flower, and six developmental stage fruits. Based on a bioinformatics pipeline, we successfully identified 29 and 35 families of conserved and novel miRNAs, respectively. Northern blot analysis was used to validate further the expression of representative miRNAs and to analyze their tissue-specific or developmental stage-specific expression patterns. Moreover, we computationally predicted miRNA targets, many of which were experimentally confirmed using 5' rapid amplification of cDNA ends analysis. One of the validated novel targets of miR-396 was a domain rearranged methyltransferase, the major de novo methylation enzyme, involved in RNA-directed DNA methylation in plants. This work provides the first reliable draft of the pepper miRNA transcriptome. It offers an expanded picture of pepper miRNAs in relation to other plants, providing a basis for understanding the functional roles of miRNAs in pepper.

  1. The expression and function of the achaete-scute genes in Tribolium castaneum reveals conservation and variation in neural pattern formation and cell fate specification

    Science.gov (United States)

    Wheeler, Scott R.; Carrico, Michelle L.; Wilson, Beth A.; Brown, Susan J.; Skeath, James B.

    2003-01-01

    The study of achaete-scute (ac/sc) genes has recently become a paradigm to understand the evolution and development of the arthropod nervous system. We describe the identification and characterization of the ac/sc genes in the coleopteran insect species Tribolium castaneum. We have identified two Tribolium ac/sc genes - achaete-scute homolog (Tc-ASH) a proneural gene and asense (Tc-ase) a neural precursor gene that reside in a gene complex. Focusing on the embryonic central nervous system we find that Tc-ASH is expressed in all neural precursors and the proneural clusters from which they segregate. Through RNAi and misexpression studies we show that Tc-ASH is necessary for neural precursor formation in Tribolium and sufficient for neural precursor formation in Drosophila. Comparison of the function of the Drosophila and Tribolium proneural ac/sc genes suggests that in the Drosophila lineage these genes have maintained their ancestral function in neural precursor formation and have acquired a new role in the fate specification of individual neural precursors. Furthermore, we find that Tc-ase is expressed in all neural precursors suggesting an important and conserved role for asense genes in insect nervous system development. Our analysis of the Tribolium ac/sc genes indicates significant plasticity in gene number, expression and function, and implicates these modifications in the evolution of arthropod neural development.

  2. Crystal structure, SAXS and kinetic mechanism of hyperthermophilic ADP-dependent glucokinase from Thermococcus litoralis reveal a conserved mechanism for catalysis.

    Directory of Open Access Journals (Sweden)

    Jaime Andrés Rivas-Pardo

    Full Text Available ADP-dependent glucokinases represent a unique family of kinases that belong to the ribokinase superfamily, being present mainly in hyperthermophilic archaea. For these enzymes there is no agreement about the magnitude of the structural transitions associated with ligand binding and whether they are meaningful to the function of the enzyme. We used the ADP-dependent glucokinase from Thermococcus litoralis as a model to investigate the conformational changes observed in X-ray crystallographic structures upon substrate binding and to compare them with those determined in solution in order to understand their interplay with the glucokinase function. Initial velocity studies indicate that catalysis follows a sequential ordered mechanism that correlates with the structural transitions experienced by the enzyme in solution and in the crystal state. The combined data allowed us to resolve the open-closed conformational transition that accounts for the complete reaction cycle and to identify the corresponding clusters of aminoacids residues responsible for it. These results provide molecular bases for a general mechanism conserved across the ADP-dependent kinase family.

  3. Three-dimensional reconstructions of the bacteriophage CUS-3 virion reveal a conserved coat protein I-domain but a distinct tailspike receptor-binding domain

    Energy Technology Data Exchange (ETDEWEB)

    Parent, Kristin N., E-mail: kparent@msu.edu [Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378 (United States); Tang, Jinghua; Cardone, Giovanni [Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378 (United States); Gilcrease, Eddie B. [University of Utah School of Medicine, Division of Microbiology and Immunology, Department of Pathology, Salt Lake City, UT 84112 (United States); Janssen, Mandy E.; Olson, Norman H. [Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378 (United States); Casjens, Sherwood R., E-mail: sherwood.casjens@path.utah.edu [University of Utah School of Medicine, Division of Microbiology and Immunology, Department of Pathology, Salt Lake City, UT 84112 (United States); Baker, Timothy S., E-mail: tsb@ucsd.edu [Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378 (United States); University of California, San Diego, Division of Biological Sciences, La Jolla, CA, 92093 (United States)

    2014-09-15

    CUS-3 is a short-tailed, dsDNA bacteriophage that infects serotype K1 Escherichia coli. We report icosahedrally averaged and asymmetric, three-dimensional, cryo-electron microscopic reconstructions of the CUS-3 virion. Its coat protein structure adopts the “HK97-fold” shared by other tailed phages and is quite similar to that in phages P22 and Sf6 despite only weak amino acid sequence similarity. In addition, these coat proteins share a unique extra external domain (“I-domain”), suggesting that the group of P22-like phages has evolved over a very long time period without acquiring a new coat protein gene from another phage group. On the other hand, the morphology of the CUS-3 tailspike differs significantly from that of P22 or Sf6, but is similar to the tailspike of phage K1F, a member of the extremely distantly related T7 group of phages. We conclude that CUS-3 obtained its tailspike gene from a distantly related phage quite recently. - Highlights: • Asymmetric and symmetric three-dimensional reconstructions of phage CUS-3 are presented. • CUS-3 major capsid protein has a conserved I-domain, which is found in all three categories of “P22-like phage”. • CUS-3 has very different tailspike receptor binding domain from those of P22 and Sf6. • The CUS-3 tailspike likely was acquired by horizontal gene transfer.

  4. Alginate lyases from alginate-degrading Vibrio splendidus 12B01 are endolytic.

    Science.gov (United States)

    Badur, Ahmet H; Jagtap, Sujit Sadashiv; Yalamanchili, Geethika; Lee, Jung-Kul; Zhao, Huimin; Rao, Christopher V

    2015-03-01

    Alginate lyases are enzymes that degrade alginate through β-elimination of the glycosidic bond into smaller oligomers. We investigated the alginate lyases from Vibrio splendidus 12B01, a marine bacterioplankton species that can grow on alginate as its sole carbon source. We identified, purified, and characterized four polysaccharide lyase family 7 alginates lyases, AlyA, AlyB, AlyD, and AlyE, from V. splendidus 12B01. The four lyases were found to have optimal activity between pH 7.5 and 8.5 and at 20 to 25°C, consistent with their use in a marine environment. AlyA, AlyB, AlyD, and AlyE were found to exhibit a turnover number (kcat) for alginate of 0.60 ± 0.02 s(-1), 3.7 ± 0.3 s(-1), 4.5 ± 0.5 s(-1), and 7.1 ± 0.2 s(-1), respectively. The Km values of AlyA, AlyB, AlyD, and AlyE toward alginate were 36 ± 7 μM, 22 ± 5 μM, 60 ± 2 μM, and 123 ± 6 μM, respectively. AlyA and AlyB were found principally to cleave the β-1,4 bonds between β-d-mannuronate and α-l-guluronate and subunits; AlyD and AlyE were found to principally cleave the α-1,4 bonds involving α-l-guluronate subunits. The four alginate lyases degrade alginate into longer chains of oligomers.

  5. Alginate Lyases from Alginate-Degrading Vibrio splendidus 12B01 Are Endolytic

    Science.gov (United States)

    Badur, Ahmet H.; Jagtap, Sujit Sadashiv; Yalamanchili, Geethika; Lee, Jung-Kul; Zhao, Huimin

    2015-01-01

    Alginate lyases are enzymes that degrade alginate through β-elimination of the glycosidic bond into smaller oligomers. We investigated the alginate lyases from Vibrio splendidus 12B01, a marine bacterioplankton species that can grow on alginate as its sole carbon source. We identified, purified, and characterized four polysaccharide lyase family 7 alginates lyases, AlyA, AlyB, AlyD, and AlyE, from V. splendidus 12B01. The four lyases were found to have optimal activity between pH 7.5 and 8.5 and at 20 to 25°C, consistent with their use in a marine environment. AlyA, AlyB, AlyD, and AlyE were found to exhibit a turnover number (kcat) for alginate of 0.60 ± 0.02 s−1, 3.7 ± 0.3 s−1, 4.5 ± 0.5 s−1, and 7.1 ± 0.2 s−1, respectively. The Km values of AlyA, AlyB, AlyD, and AlyE toward alginate were 36 ± 7 μM, 22 ± 5 μM, 60 ± 2 μM, and 123 ± 6 μM, respectively. AlyA and AlyB were found principally to cleave the β-1,4 bonds between β-d-mannuronate and α-l-guluronate and subunits; AlyD and AlyE were found to principally cleave the α-1,4 bonds involving α-l-guluronate subunits. The four alginate lyases degrade alginate into longer chains of oligomers. PMID:25556193

  6. Inhibition of N-acetylneuraminate lyase by N-acetyl-4-oxo-D-neuraminic acid.

    Science.gov (United States)

    Gross, H J; Brossmer, R

    1988-05-09

    We show that the 4-oxo analogue of N-acetyl-D-neuraminic acid strongly inhibits N-acetylneuraminate lyase (NeuAc aldolase, EC 4.1.3.3) from Clostridum perfringens (Ki = 0.025 mM) and Escherichia coli (Ki = 0.15 mM). In each case the inhibition was competitive. N-Acetyl-D-neuraminic acid; N-Acetylneuraminate lyase; N-Acetyl-D-neuraminic acid analog; 5-Acetamido-3,5-dideoxy-beta-D-manno-non-2,4-diulosonic acid; 2-Deoxy-2,3-didehydro-N-acetyl-4-oxo-neuraminic acid; Competitive inhibitor.

  7. Structural Determinants Responsible for Substrate Recognition and Mode of Action in Family 11 Polysaccharide Lyases*

    OpenAIRE

    Ochiai, Akihito; Itoh, Takafumi; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

    2009-01-01

    A saprophytic Bacillus subtilis secretes two types of rhamnogalacturonan (RG) lyases, endotype YesW and exotype YesX, which are responsible for an initial cleavage of the RG type I (RG-I) region of plant cell wall pectin. Polysaccharide lyase family 11 YesW and YesX with a significant sequence identity (67.8%) cleave glycoside bonds between rhamnose and galacturonic acid residues in RG-I through a β-elimination reaction. Here we show the structural determinants for sub...

  8. 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

  9. A rapid, sensitive, simple plate assay for detection of microbial alginate lyase activity.

    Science.gov (United States)

    Sawant, Shailesh S; Salunke, Bipinchandra K; Kim, Beom Soo

    2015-09-01

    Screening of microorganisms capable of producing alginate lyase enzyme is commonly carried out by investigating their abilities to grow on alginate-containing solid media plates and occurrence of a clearance zone after flooding the plates with agents such as 10% (w/v) cetyl pyridinium chloride (CPC), which can form complexes with alginate. Although the CPC method is good, advantageous, and routinely used, the agar in the media interferes with the action of CPC, which makes judgment about clearance zones very difficult. In addition, this method takes a minimum of 30 min to obtain the zone of hydrolysis after flooding and the hydrolyzed area is not sharply discernible. An improved plate assay is reported herein for the detection of extracellular alginate lyase production by microorganisms. In this method, alginate-containing agar plates are flooded with Gram's iodine instead of CPC. Gram's iodine forms a bluish black complex with alginate but not with hydrolyzed alginate, giving sharp, distinct zones around the alginate lyase producing microbial colonies within 2-3 min. Gram's iodine method was found to be more effective than the CPC method in terms of visualization and measurement of zone size. The alginate-lyase-activity area indicated using the Gram's iodine method was found to be larger than that indicated by the CPC method. Both methods (CPC and Gram's iodine) showed the largest alginate lyase activity area for Saccharophagus degradans (ATCC 43961) followed by Microbulbifer mangrovi (KCTC 23483), Bacillus cereus (KF801505) and Paracoccus sp. LL1 (KP288668) grown on minimal sea salt medium. The rate of growth and metabolite production in alginate-containing minimal sea salt liquid medium, followed trends similar to that of the zone activity areas for the four bacteria under study. These results suggested that the assay developed in this study of Gram's iodine could be useful to predict the potential of microorganisms to produce alginate lyase. The method also

  10. Structural and functional studies of titin's fn3 modules reveal conserved surface patterns and binding to myosin S1--a possible role in the Frank-Starling mechanism of the heart.

    Science.gov (United States)

    Muhle-Goll, C; Habeck, M; Cazorla, O; Nilges, M; Labeit, S; Granzier, H

    2001-10-19

    The A-band part of titin, a striated-muscle specific protein spanning from the Z-line to the M-line, mainly consists of a well-ordered super-repeat array of immunoglobulin-like and fibronectin-type III (fn3)-like domains. Since it has been suspected that the fn3 domains might represent titin's binding sites to myosin, we have developed structural models for all of titin's 132 fn3-like domains. A subset of eight experimentally determined fn3 structures from a range of proteins, including titin itself, was used as homology templates. After grouping the models according to their position within the super-repeat segment of the central A-band titin region, we analyzed the models with respect to side-chain conservation. This showed that conserved residues form an extensive surface pattern predominantly at one side of the domains, whereas domains outside the central C-zone super-repeat region show generally less conserved surfaces. Since the conserved surface residues may function as protein-binding sites, we experimentally studied the binding properties of expressed multi-domain fn3 fragments. This revealed that fn3 fragments specifically bind to the sub-fragment 1 of myosin. We also measured the effect of fn3 fragments on the contractile properties of single cardiac myocytes. At sub-maximal Ca(2+) concentrations, fn3 fragments significantly enhance active tension. This effect is most pronounced at short sarcomere length, and as a result the length-dependence of Ca(2+) activation is reduced. A model of how titin's fn3-like domains may influence actomyosin interaction is proposed.

  11. Profiling of proteolytic enzymes in the gut of the tick Ixodes ricinus reveals an evolutionarily conserved network of aspartic and cysteine peptidases

    Directory of Open Access Journals (Sweden)

    Mareš Michael

    2008-03-01

    Full Text Available Abstract Background Ticks are vectors for a variety of viral, bacterial and parasitic diseases in human and domestic animals. To survive and reproduce ticks feed on host blood, yet our understanding of the intestinal proteolytic machinery used to derive absorbable nutrients from the blood meal is poor. Intestinal digestive processes are limiting factors for pathogen transmission since the tick gut presents the primary site of infection. Moreover, digestive enzymes may find practical application as anti-tick vaccine targets. Results Using the hard tick, Ixodes ricinus, we performed a functional activity scan of the peptidase complement in gut tissue extracts that demonstrated the presence of five types of peptidases of the cysteine and aspartic classes. We followed up with genetic screens of gut-derived cDNA to identify and clone genes encoding the cysteine peptidases cathepsins B, L and C, an asparaginyl endopeptidase (legumain, and the aspartic peptidase, cathepsin D. By RT-PCR, expression of asparaginyl endopeptidase and cathepsins B and D was restricted to gut tissue and to those developmental stages feeding on blood. Conclusion Overall, our results demonstrate the presence of a network of cysteine and aspartic peptidases that conceivably operates to digest host blood proteins in a concerted manner. Significantly, the peptidase components of this digestive network are orthologous to those described in other parasites, including nematodes and flatworms. Accordingly, the present data and those available for other tick species support the notion of an evolutionary conservation of a cysteine/aspartic peptidase system for digestion that includes ticks, but differs from that of insects relying on serine peptidases.

  12. Metabolic flux ratio analysis and multi-objective optimization revealed a globally conserved and coordinated metabolic response of E. coli to paraquat-induced oxidative stress.

    Science.gov (United States)

    Shen, Tie; Rui, Bin; Zhou, Hong; Zhang, Ximing; Yi, Yin; Wen, Han; Zheng, Haoran; Wu, Jihui; Shi, Yunyu

    2013-01-27

    . coli to paraquat-induced oxidative stress is globally conserved and coordinated.

  13. Cross-disease comparison of amyotrophic lateral sclerosis and spinal muscular atrophy reveals conservation of selective vulnerability but differential neuromuscular junction pathology.

    Science.gov (United States)

    Comley, Laura H; Nijssen, Jik; Frost-Nylen, Johanna; Hedlund, Eva

    2016-05-01

    Neuromuscular junctions are primary pathological targets in the lethal motor neuron diseases spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Synaptic pathology and denervation of target muscle fibers has been reported prior to the appearance of clinical symptoms in mouse models of both diseases, suggesting that neuromuscular junctions are highly vulnerable from the very early stages, and are a key target for therapeutic intervention. Here we examined neuromuscular pathology longitudinally in three clinically relevant muscle groups in mouse models of ALS and SMA in order to assess their relative vulnerabilities. We show for the first time that neuromuscular junctions of the extraocular muscles (responsible for the control of eye movement) were resistant to degeneration in endstage SMA mice, as well as in late symptomatic ALS mice. Tongue muscle neuromuscular junctions were also spared in both animal models. Conversely, neuromuscular junctions of the lumbrical muscles of the hind-paw were vulnerable in both SMA and ALS, with a loss of neuronal innervation and shrinkage of motor endplates in both diseases. Thus, the pattern of selective vulnerability was conserved across these two models of motor neuron disease. However, the first evidence of neuromuscular pathology occurred at different timepoints of disease progression, with much earlier evidence of presynaptic involvement in ALS, progressing to changes on the postsynaptic side. Conversely, in SMA changes appeared concomitantly at the neuromuscular junction, suggesting that mechanisms of neuromuscular disruption are distinct in these diseases. J. Comp. Neurol. 524:1424-1442, 2016. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

  14. Cross‐disease comparison of amyotrophic lateral sclerosis and spinal muscular atrophy reveals conservation of selective vulnerability but differential neuromuscular junction pathology

    Science.gov (United States)

    Nijssen, Jik; Frost‐Nylen, Johanna

    2015-01-01

    Neuromuscular junctions are primary pathological targets in the lethal motor neuron diseases spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Synaptic pathology and denervation of target muscle fibers has been reported prior to the appearance of clinical symptoms in mouse models of both diseases, suggesting that neuromuscular junctions are highly vulnerable from the very early stages, and are a key target for therapeutic intervention. Here we examined neuromuscular pathology longitudinally in three clinically relevant muscle groups in mouse models of ALS and SMA in order to assess their relative vulnerabilities. We show for the first time that neuromuscular junctions of the extraocular muscles (responsible for the control of eye movement) were resistant to degeneration in endstage SMA mice, as well as in late symptomatic ALS mice. Tongue muscle neuromuscular junctions were also spared in both animal models. Conversely, neuromuscular junctions of the lumbrical muscles of the hind‐paw were vulnerable in both SMA and ALS, with a loss of neuronal innervation and shrinkage of motor endplates in both diseases. Thus, the pattern of selective vulnerability was conserved across these two models of motor neuron disease. However, the first evidence of neuromuscular pathology occurred at different timepoints of disease progression, with much earlier evidence of presynaptic involvement in ALS, progressing to changes on the postsynaptic side. Conversely, in SMA changes appeared concomitantly at the neuromuscular junction, suggesting that mechanisms of neuromuscular disruption are distinct in these diseases. J. Comp. Neurol. 524:1424–1442, 2016. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc. PMID:26502195

  15. Novel low abundance and transient RNAs in yeast revealed by tiling microarrays and ultra high-throughput sequencing are not conserved across closely related yeast species.

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    Albert Lee

    2008-12-01

    Full Text Available A complete description of the transcriptome of an organism is crucial for a comprehensive understanding of how it functions and how its transcriptional networks are controlled, and may provide insights into the organism's evolution. Despite the status of Saccharomyces cerevisiae as arguably the most well-studied model eukaryote, we still do not have a full catalog or understanding of all its genes. In order to interrogate the transcriptome of S. cerevisiae for low abundance or rapidly turned over transcripts, we deleted elements of the RNA degradation machinery with the goal of preferentially increasing the relative abundance of such transcripts. We then used high-resolution tiling microarrays and ultra high-throughput sequencing (UHTS to identify, map, and validate unannotated transcripts that are more abundant in the RNA degradation mutants relative to wild-type cells. We identified 365 currently unannotated transcripts, the majority presumably representing low abundance or short-lived RNAs, of which 185 are previously unknown and unique to this study. It is likely that many of these are cryptic unstable transcripts (CUTs, which are rapidly degraded and whose function(s within the cell are still unclear, while others may be novel functional transcripts. Of the 185 transcripts we identified as novel to our study, greater than 80 percent come from regions of the genome that have lower conservation scores amongst closely related yeast species than 85 percent of the verified ORFs in S. cerevisiae. Such regions of the genome have typically been less well-studied, and by definition transcripts from these regions will distinguish S. cerevisiae from these closely related species.

  16. A genome-wide screen for regulators of TORC1 in response to amino acid starvation reveals a conserved Npr2/3 complex.

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    Taavi K Neklesa

    2009-06-01

    Full Text Available TORC1 is a central regulator of cell growth in response to amino acid availability, yet little is known about how it is regulated. Here, we performed a reverse genetic screen in yeast for genes necessary to inactivate TORC1. The screen consisted of monitoring the expression of a TORC1 sensitive GFP-based transcriptional reporter in all yeast deletion strains using flow cytometry. We find that in response to amino acid starvation, but not to carbon starvation or rapamycin treatment, cells lacking NPR2 and NPR3 fail to fully (1 activate transcription factors Gln3/Gat1, (2 dephosphorylate TORC1 effector Npr1, and (3 repress ribosomal protein gene expression. Both mutants show proliferation defects only in media containing a low quality nitrogen source, such as proline or ammonia, whereas no defects are evident when cells are grown in the presence of glutamine or peptone mixture. Proliferation defects in npr2Delta and npr3Delta cells can be completely rescued by artificially inhibiting TORC1 by rapamycin, demonstrating that overactive TORC1 in both strains prevents their ability to adapt to an environment containing a low quality nitrogen source. A biochemical purification of each demonstrates that Npr2 and Npr3 form a heterodimer, and this interaction is evolutionarily conserved since the human homologs of NPR2 and NPR3 (NPRL2 and NPRL3, respectively also co-immunoprecipitate. We conclude that, in yeast, the Npr2/3 complex mediates an amino acid starvation signal to TORC1.

  17. Genetic diversity of and differentiation among five populations of blunt snout bream (Megalobrama amblycephala revealed by SRAP markers: implications for conservation and management.

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    Wei Ji

    Full Text Available The blunt snout bream (Megalobrama amblycephala is an important freshwater aquaculture fish throughout China. Because of widespread introductions of this species to many regions, the genetic diversity of wild and natural populations is now threatened. In the present study, SRAP (sequence-related amplified polymorphism markers were used to assess genetic diversity of blunt snout bream. Three natural populations (Liangzi Lake, Poyang Lake and Yuni Lake, one cultured population (Nanxian and one genetic strain ('Pujiang No. 1' of blunt snout bream were screened with 88 SRAP primer combinations, of which 13 primer pairs produced stable and reproducible amplification patterns. In total, 172 bands were produced, of which 132 bands were polymorphic. Nei's gene diversity (h and Shannon's information index (I values provided evidence of differences in genetic diversity among the five populations (Poyang Lake>Liangzi Lake>Nanxian>'Pujiang No. 1'>Yuni Lake. Based on cluster analysis conducted on genetic distance values, the five blunt snout bream populations were divided into three groups, Poyang Lake and Liangzi Lake (natural populations, Nanxian and 'Pujiang No. 1' (cultured population and genetically selected strain, and Yuni Lake (natural population. Significant genetic differentiation was found among the five populations using analysis of molecular variance (AMOVA, with more genetic divergence existing among populations (55.49%, than within populations (44.51%. This molecular marker technique is a simple and efficient method to quantify genetic diversity within and among fish populations, and is employed here to help manage and conserve germplasm variability of blunt snout bream and to support the ongoing selective breeding programme for this fish.

  18. 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.

  19. Pyruvate formate lyase acts as a formate supplier for metabolic processes during anaerobiosis in Staphylococcus aureus.

    Science.gov (United States)

    Leibig, Martina; Liebeke, Manuel; Mader, Diana; Lalk, Michael; Peschel, Andreas; Götz, Friedrich

    2011-02-01

    Previous studies demonstrated an upregulation of pyruvate formate lyase (Pfl) and NAD-dependent formate dehydrogenase (Fdh) in Staphylococcus aureus biofilms. To investigate their physiological role, we constructed fdh and pfl deletion mutants (Δfdh and Δpfl). Although formate dehydrogenase activity in the fdh mutant was lost, it showed little phenotypic alterations under oxygen-limited conditions. In contrast, the pfl mutant displayed pleiotropic effects and revealed the importance of formate production for anabolic metabolism. In the pfl mutant, no formate was produced, glucose consumption was delayed, and ethanol production was decreased, whereas acetate and lactate production were unaffected. All metabolic alterations could be restored by addition of formate or complementation of the Δpfl mutant. In compensation reactions, serine and threonine were consumed better by the Δpfl mutant than by the wild type, suggesting that their catabolism contributes to the refilling of formyl-tetrahydrofolate, which acts as a donor of formyl groups in, e.g., purine and protein biosynthesis. This notion was supported by reduced production of formylated peptides by the Δpfl mutant compared to that of the parental strain, as demonstrated by weaker formyl-peptide receptor 1 (FPR1)-mediated activation of leukocytes with the mutant. FPR1 stimulation could also be restored either by addition of formate or by complementation of the mutation. Furthermore, arginine consumption and arc operon transcription were increased in the Δpfl mutant. Unlike what occurred with the investigated anaerobic conditions, a biofilm is distinguished by nutrient, oxygen, and pH gradients, and we thus assume that Pfl plays a significant role in the anaerobic layer of a biofilm. Fdh might be critical in (micro)aerobic layers, as formate oxidation is correlated with the generation of NADH/H(+), whose regeneration requires respiration.

  20. Off-road sampling reveals a different grassland bird community than roadside sampling: implications for survey design and estimates to guide conservation

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    Troy I. Wellicome

    2014-06-01

    Full Text Available Grassland bird species continue to decline steeply across North America. Road-based surveys such as the North American Breeding Bird Survey (BBS are often used to estimate trends and population sizes and to build species distribution models for grassland birds, although roadside survey counts may introduce bias in estimates because of differences in habitats along roadsides and in off-road surveys. We tested for differences in land cover composition and in the avian community on 21 roadside-based survey routes and in an equal number of adjacent off-road walking routes in the grasslands of southern Alberta, Canada. Off-road routes (n = 225 point counts had more native grassland and short shrubs and less fallow land and road area than the roadside routes (n = 225 point counts. Consequently, 17 of the 39 bird species differed between the two route types in frequency of occurrence and relative abundance, measured using an indicator species analysis. Six species, including five obligate grassland species, were more prevalent at off-road sites; they included four species listed under the Canadian federal Species At Risk Act or listed by the Committee on the Status of Endangered Wildlife in Canada: Sprague's Pipit (Anthus spragueii, Baird's Sparrow (Ammodramus bairdii, the Chestnut-collared Longspur (Calcarius ornatus, and McCown's Longspur (Rhynchophanes mccownii. The six species were as much as four times more abundant on off-road sites. Species more prevalent along roadside routes included common species and those typical of farmland and other human-modified habitats, e.g., the European Starling (Sturnus vulgaris, the Black-billed Magpie (Pica hudsonia, and the House Sparrow (Passer domesticus. Differences in avian community composition between roadside and off-road surveys suggest that the use of BBS data when generating population estimates or distribution models may overestimate certain common species and underestimate others of conservation

  1. Evolutionary and Functional Analysis of Old World Primate TRIM5 Reveals the Ancient Emergence of Primate Lentiviruses and Convergent Evolution Targeting a Conserved Capsid Interface.

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    Kevin R McCarthy

    2015-08-01

    Full Text Available The widespread distribution of lentiviruses among African primates, and the lack of severe pathogenesis in many of these natural reservoirs, are taken as evidence for long-term co-evolution between the simian immunodeficiency viruses (SIVs and their primate hosts. Evidence for positive selection acting on antiviral restriction factors is consistent with virus-host interactions spanning millions of years of primate evolution. However, many restriction mechanisms are not virus-specific, and selection cannot be unambiguously attributed to any one type of virus. We hypothesized that the restriction factor TRIM5, because of its unique specificity for retrovirus capsids, should accumulate adaptive changes in a virus-specific fashion, and therefore, that phylogenetic reconstruction of TRIM5 evolution in African primates should reveal selection by lentiviruses closely related to modern SIVs. We analyzed complete TRIM5 coding sequences of 22 Old World primates and identified a tightly-spaced cluster of branch-specific adaptions appearing in the Cercopithecinae lineage after divergence from the Colobinae around 16 million years ago. Functional assays of both extant TRIM5 orthologs and reconstructed ancestral TRIM5 proteins revealed that this cluster of adaptations in TRIM5 specifically resulted in the ability to restrict Cercopithecine lentiviruses, but had no effect (positive or negative on restriction of other retroviruses, including lentiviruses of non-Cercopithecine primates. The correlation between lineage-specific adaptations and ability to restrict viruses endemic to the same hosts supports the hypothesis that lentiviruses closely related to modern SIVs were present in Africa and infecting the ancestors of Cercopithecine primates as far back as 16 million years ago, and provides insight into the evolution of TRIM5 specificity.

  2. 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.

  3. Structure of a conserved hypothetical protein SA1388 from S. aureus reveals a capped hexameric toroid with two PII domain lids and a dinuclear metal center

    Energy Technology Data Exchange (ETDEWEB)

    Saikatendu, Kumar Singh; Zhang, Xuejun; Kinch, Lisa; Leybourne, Matthew; Grishin, Nick V.; Zhang, Hong (Texas-D); (U. of Texas-SMED)

    2009-01-26

    The protein encoded by the SA1388 gene from Staphylococcus aureus was chosen for structure determination to elucidate its domain organization and confirm our earlier remote homology based prediction that it housed a nitrogen regulatory PII protein-like domain. SA1388 was predicted to contain a central PII-like domain and two flanking regions, which together belong to the NIF3-like protein family. Proteins like SA1388 remain a poorly studied group and their structural characterization could guide future investigations aimed at understanding their function. The structure of SA1388 has been solved to 2.0{angstrom} resolution by single wavelength anomalous dispersion phasing method using selenium anomalous signals. It reveals a canonical NIF3-like fold containing two domains with a PII-like domain inserted in the middle of the polypeptide. The N and C terminal halves of the NIF3-like domains are involved in dimerization, while the PII domain forms trimeric contacts with symmetry related monomers. Overall, the NIF3-like domains of SA1388 are organized as a hexameric toroid similar to its homologs, E. coli ybgI and the hypothetical protein SP1609 from Streptococcus pneumoniae. The openings on either side of the toroid are partially covered by trimeric 'lids' formed by the PII domains. The junction of the two NIF3 domains has two zinc ions bound at what appears to be a histidine rich active site. A well-defined electron density corresponding to an endogenously bound ligand of unknown identity is observed in close proximity to the metal site. SA1388 is the third member of the NIF3-like family of proteins to be structurally characterized, the other two also being hypothetical proteins of unknown function. The structure of SA1388 confirms our earlier prediction that the inserted domain that separates the two NIF3 domains adopts a PII-like fold and reveals an overall capped toroidal arrangement for the protein hexamer. The six PII-like domains form two trimeric

  4. Structure of a conserved hypothetical protein SA1388 from S. aureus reveals a capped hexameric toroid with two PII domain lids and a dinuclear metal center

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    Leybourne Matthew

    2006-12-01

    Full Text Available Abstract Background The protein encoded by the SA1388 gene from Staphylococcus aureus was chosen for structure determination to elucidate its domain organization and confirm our earlier remote homology based prediction that it housed a nitrogen regulatory PII protein-like domain. SA1388 was predicted to contain a central PII-like domain and two flanking regions, which together belong to the NIF3-like protein family. Proteins like SA1388 remain a poorly studied group and their structural characterization could guide future investigations aimed at understanding their function. Results The structure of SA1388 has been solved to 2.0Å resolution by single wavelength anomalous dispersion phasing method using selenium anomalous signals. It reveals a canonical NIF3-like fold containing two domains with a PII-like domain inserted in the middle of the polypeptide. The N and C terminal halves of the NIF3-like domains are involved in dimerization, while the PII domain forms trimeric contacts with symmetry related monomers. Overall, the NIF3-like domains of SA1388 are organized as a hexameric toroid similar to its homologs, E. coli ybgI and the hypothetical protein SP1609 from Streptococcus pneumoniae. The openings on either side of the toroid are partially covered by trimeric "lids" formed by the PII domains. The junction of the two NIF3 domains has two zinc ions bound at what appears to be a histidine rich active site. A well-defined electron density corresponding to an endogenously bound ligand of unknown identity is observed in close proximity to the metal site. Conclusion SA1388 is the third member of the NIF3-like family of proteins to be structurally characterized, the other two also being hypothetical proteins of unknown function. The structure of SA1388 confirms our earlier prediction that the inserted domain that separates the two NIF3 domains adopts a PII-like fold and reveals an overall capped toroidal arrangement for the protein hexamer. The

  5. RNA-sequencing analysis of TCDD-induced responses in zebrafish liver reveals high relatedness to in vivo mammalian models and conserved biological pathways.

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    Zhi-Hua Li

    Full Text Available TCDD is one of the most persistent environmental toxicants in biological systems and its effect through aryl hydrocarbon receptor (AhR has been well characterized. However, the information on TCDD-induced toxicity in other molecular pathways is rather limited. To fully understand molecular toxicity of TCDD in an in vivo animal model, adult zebrafish were exposed to TCDD at 10 nM for 96 h and the livers were sampled for RNA-sequencing based transcriptomic profiling. A total of 1,058 differently expressed genes were identified based on fold-change>2 and TPM (transcripts per million >10. Among the top 20 up-regulated genes, 10 novel responsive genes were identified and verified by RT-qPCR analysis on independent samples. Transcriptomic analysis indicated several deregulated pathways associated with cell cycle, endocrine disruptors, signal transduction and immune systems. Comparative analyses of TCDD-induced transcriptomic changes between fish and mammalian models revealed that proteomic pathway is consistently up-regulated while calcium signaling pathway and several immune-related pathways are generally down-regulated. Finally, our study also suggested that zebrafish model showed greater similarity to in vivo mammalian models than in vitro models. Our study indicated that the zebrafish is a valuable in vivo model in toxicogenomic analyses for understanding molecular toxicity of environmental toxicants relevant to human health. The expression profiles associated with TCDD could be useful for monitoring environmental dioxin and dioxin-like contamination.

  6. Nuclear Magnetic Resonance Solution Structures of Lacticin Q and Aureocin A53 Reveal a Structural Motif Conserved among Leaderless Bacteriocins with Broad-Spectrum Activity.

    Science.gov (United States)

    Acedo, Jeella Z; van Belkum, Marco J; Lohans, Christopher T; Towle, Kaitlyn M; Miskolzie, Mark; Vederas, John C

    2016-02-02

    Lacticin Q (LnqQ) and aureocin A53 (AucA) are leaderless bacteriocins from Lactococcus lactis QU5 and Staphylococcus aureus A53, respectively. These bacteriocins are characterized by the absence of an N-terminal leader sequence and are active against a broad range of Gram-positive bacteria. LnqQ and AucA consist of 53 and 51 amino acids, respectively, and have 47% identical sequences. In this study, their three-dimensional structures were elucidated using solution nuclear magnetic resonance and were shown to consist of four α-helices that assume a very similar compact, globular overall fold (root-mean-square deviation of 1.7 Å) with a highly cationic surface and a hydrophobic core. The structures of LnqQ and AucA resemble the shorter two-component leaderless bacteriocins, enterocins 7A and 7B, despite having low levels of sequence identity. Homology modeling revealed that the observed structural motif may be shared among leaderless bacteriocins with broad-spectrum activity against Gram-positive organisms. The elucidated structures of LnqQ and AucA also exhibit some resemblance to circular bacteriocins. Despite their similar overall fold, inhibition studies showed that LnqQ and AucA have different antimicrobial potency against the Gram-positive strains tested, suggesting that sequence disparities play a crucial role in their mechanisms of action.

  7. Royal jelly-like protein localization reveals differences in hypopharyngeal glands buildup and conserved expression pattern in brains of bumblebees and honeybees

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    Štefan Albert

    2014-03-01

    Full Text Available Royal jelly proteins (MRJPs of the honeybee bear several open questions. One of them is their expression in tissues other than the hypopharyngeal glands (HGs, the site of royal jelly production. The sole MRJP-like gene of the bumblebee, Bombus terrestris (BtRJPL, represents a pre-diversification stage of the MRJP gene evolution in bees. Here we investigate the expression of BtRJPL in the HGs and the brain of bumblebees. Comparison of the HGs of bumblebees and honeybees revealed striking differences in their morphology with respect to sex- and caste-specific appearance, number of cells per acinus, and filamentous actin (F-actin rings. At the cellular level, we found a temporary F-actin-covered meshwork in the secretory cells, which suggests a role for actin in the biogenesis of the end apparatus in HGs. Using immunohistochemical localization, we show that BtRJPL is expressed in the bumblebee brain, predominantly in the Kenyon cells of the mushroom bodies, the site of sensory integration in insects, and in the optic lobes. Our data suggest that a dual gland-brain function preceded the multiplication of MRJPs in the honeybee lineage. In the course of the honeybee evolution, HGs dramatically changed their morphology in order to serve a food-producing function.

  8. Activity and Enantioselectivity of the Hydroxynitrile Lyase MeHNL in Dry Organic Solvents

    NARCIS (Netherlands)

    Hanefeld, U.; Paravidino, M.; Sorgedrager, M.; Orru, R.V.A.

    2010-01-01

    Water concentration affects both the enantioselectivity and activity of enzymes in dry organic media. Its influence has been investigated using the hydrocyanation of benzaldehyde catalyzed by hydroxynitrile lyase cross-linked enzyme aggregate (MeHNL-CLEA) as a model reaction. The enzyme displayed hi

  9. Volatile sulphur compounds-forming abilities of lactic acid bacteria: C-S lyase activities.

    Science.gov (United States)

    Bustos, Irene; Martínez-Bartolomé, Miguel A; Achemchem, Fouad; Peláez, Carmen; Requena, Teresa; Martínez-Cuesta, M Carmen

    2011-08-01

    Volatile sulphur compounds (VSCs) are of prime importance in the overall aroma of cheese and make a significant contribution to their typical flavours. Thus, the control of VSCs formation offers considerable potential for industrial applications. Here, lactic acid bacteria (LAB) from different ecological origins were screened for their abilities to produce VSCs from L-methionine. From the data presented, VSC-forming abilities were shown to be strain-specific and were correlated with the C-S lyase enzymatic activities determined using different approaches. High VSCs formation were detected for those strains that were also shown to possess high thiol-producing abilities (determined either by agar plate or spectrophotometry assays). Moreover, differences in C-S lyase activities were shown to correspond with the enzymatic potential of the strains as determined by in situ gel visualization. Therefore, the assessment of the C-S lyase enzymatic potential, by means of either of these techniques, could be used as a valuable approach for the selection of LAB strains with high VSC-producing abilities thus, representing an effective way to enhance cheese sulphur aroma compounds synthesis. In this regard, this study highlights the flavour forming potential of the Streptococcus thermophilus STY-31, that therefore could be used as a starter culture in cheese manufacture. Furthermore, although C-S lyases are involved in both biosynthetic and catabolic pathways, an association between methionine and cysteine auxotrophy of the selected strains and their VSCs-producing abilities could not be found.

  10. Biochemical and structural characterization of a novel bacterial manganese-dependent hydroxynitrile lyase.

    NARCIS (Netherlands)

    Hajnal, I.; Lyskowski, A.; Hanefeld, U.; Gruber, K.; Schwab, H.; Steiner, K.

    2013-01-01

    Hydroxynitrile lyases (HNLs), which catalyse the decomposition of cyanohydrins, are found mainly in plants. In vitro, they are able to catalyse the synthesis of enantiopure cyanohydrins, which are versatile building blocks in the chemical industry. Recently, HNLs have also been discovered in bacteri

  11. 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

  12. Purification and characterization of alginate lyase from locally isolated marine Pseudomonas stutzeri MSEA04.

    Science.gov (United States)

    Beltagy, Ehab A; El-Borai, Aliaa; Lewiz, Marina; ElAssar, Samy A

    2016-09-01

    An alginate lyase with high specific enzyme activity was purified from Pseudomonas stutzeri MSEA04, isolated from marine brown algae. The alginate lyase was purified by precipitation with ammonium sulphate, acetone and ethanol individually. 70% ethanol fraction showed maximum specific activity (133.3 U/mg). This fraction was re-purified by anion exchange chromatography DEAE- Cellulose A-52. The loaded protein was separated into 3 peaks. The second protein peak was the major one which contained 48.2% of the total protein recovered and 79.4% of the total recovered activity. The collected fractions of this peak were subjected to further purification by re-chromatography on Sephadex G-100. Alginate lyase activity was fractionated in the Sephadex column into one major peak, and the specific activity of this fraction reached 116 U/mg. The optimal substrate concentration, pH and temperature for alginate lyase activity were 8 mg/ml, pH 7.5 and 37 °C, respectively. While, Km and Vmax values were 1.07 mg alginate/ ml and 128.2 U/mg protein, respectively. The enzyme was partially stable below 50 °C, and the activity of the enzyme was strongly enhanced by K(+), and strongly inhibited by Ba(+2), Cd(+2), Fe(+2) and Zn(+2). The purified enzyme yielded a single band on SDS-PAGE with molecular weight (40.0 kDa).

  13. The roles of active site residues in the catalytic mechanism of methylaspartate ammonia-lyase

    NARCIS (Netherlands)

    Raj, Hans; Poelarends, Gerrit J

    2013-01-01

    Methylaspartate ammonia-lyase (MAL; EC 4.3.1.2) catalyzes the reversible addition of ammonia to mesaconate to yield l-threo-(2S,3S)-3-methylaspartate and l-erythro-(2S,3R)-3-methylaspartate as products. In the proposed minimal mechanism for MAL of Clostridium tetanomorphum, Lys-331 acts as the (S)-s

  14. 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

  15. Crystal structure and characterization of a novel L-serine ammonia-lyase from Rhizomucor miehei.

    Science.gov (United States)

    Qin, Zhen; Yan, Qiaojuan; Ma, Qingjun; Jiang, Zhengqiang

    2015-10-23

    L-serine ammonia-lyase, as a member of the β-family of pyridoxal-5'-phosphate (PLP) dependent enzymes, catalyzes the conversion of L-serine (L-threonine) to pyruvate (α-ketobutyrate) and ammonia. The crystal structure of L-serine ammonia-lyase from Rhizomucor miehei (RmSDH) was solved at 1.76 Å resolution by X-ray diffraction method. The overall structure of RmSDH had the characteristic β-family PLP dependent enzyme fold. It consisted of two distinct domains, both of which show the typical open twisted α/β structure. A PLP cofactor was located in the crevice between the two domains, which was attached to Lys52 by a Schiff-base linkage. Unique residue substitutions (Gly78, Pro79, Ser146, Ser147 and Thr312) were discovered at the catalytic site of RmSDH by comparison of structures of RmSDH and other reported eukaryotic L-serine ammonia-lyases. Optimal pH and temperature of the purified RmSDH were 7.5 and 40 °C, respectively. It was stable in the pH range of 7.0-9.0 and at temperatures below 40 °C. This is the first crystal structure of a fungal L-serine ammonia-lyase. It will be useful to study the catalytic mechanism of β-elimination enzymes and will provide a basis for further enzyme engineering.

  16. Overexpression of the plg1 gene encoding pectin lyase in Penicillium griseoroseum.

    Science.gov (United States)

    Cardoso, Patrícia Gomes; Ribeiro, João Batista; Teixeira, Janaina Aparecida; de Queiroz, Marisa Vieira; de Araújo, Elza Fernandes

    2008-03-01

    The pectin lyase (PL) is an industrially important enzyme since it is used for maceration and clarification in the process of fruit juice production in food industries. In order to increase the yields of pectin lyase we cloned the plg1 (pectin lyase 1) from Penicillium griseoroseum gene under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) and the terminator region of the tryptophan synthetase (trpC) gene from Aspergillus nidulans (plasmid pAN52-Plg1) and transformed this construct into the P. griseoroseum strain PG63. One of the pAN52-Plg1 multi-copy transformants (strain 105) grown in culture medium containing glucose or sugar cane juice showed PL activities of 4,804 or 5,202 U ml(-1) respectively, which represented 57- and 132-fold increases. In addition, the apparent specific activity of PL produced by this strain was much higher than the one observed for a commercial pectinase preparation. Evaluation of the extracellular proteins in the culture supernatant of strain 105 by SDS-PAGE showed the presence of a clear and strong band of approximately 40 kDa that probably corresponds to PL. The enzyme yields reported here demonstrate that the system we developed is able to express pectin lyase at levels comparable to, or exceeding, previously reported data.

  17. Activity and Enantioselectivity of the Hydroxynitrile Lyase MeHNL in Dry Organic Solvents

    NARCIS (Netherlands)

    Hanefeld, U.; Paravidino, M.; Sorgedrager, M.; Orru, R.V.A.

    2010-01-01

    Water concentration affects both the enantioselectivity and activity of enzymes in dry organic media. Its influence has been investigated using the hydrocyanation of benzaldehyde catalyzed by hydroxynitrile lyase cross-linked enzyme aggregate (MeHNL-CLEA) as a model reaction. The enzyme displayed

  18. Characterization of the N-linked glycosylation site of recombinant pectate lyase

    NARCIS (Netherlands)

    Colangelo, J.; Licon, V.; Benen, J.A.E.; Visser, J.; Bergmann, C.; Orlando, R.

    1999-01-01

    Recombinant pectate lyase from Aspergillus niger was overexpressed in Aspergillus nidulans. The two recombinant proteins produced differed in molecular mass by 1200 Da, which suggested that the larger molecular weight protein was glycosylated. The deduced amino acid sequence was searched for potenti

  19. Sugar- and nitrogen-dependent regulation of an Amanita muscaria phenylalanine ammonium lyase gene.

    Science.gov (United States)

    Nehls, U; Ecke, M; Hampp, R

    1999-03-01

    The cDNA of a key enzyme of secondary metabolism, phenylalanine ammonium lyase, was identified for an ectomycorrhizal fungus by differential screening of a mycorrhizal library. The gene was highly expressed in hyphae grown at low external monosaccharide concentrations, but its expression was 30-fold reduced at elevated concentrations. Gene repression was regulated by hexokinase.

  20. Production of Alginate Oligosaccharides (AOS as Prebiotic Ingredients through by Alginate lyase enzyme

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    Fahriza Sri Afni

    2017-04-01

    Full Text Available Prebiotics is indigestible foods that can not be digested but can stimulate the growth and activity of bacteria in the digestive tract effecting human health. Alginate oligosaccharides (AOS can be used as a source of prebiotic. That compounds can be produced enzymatically by cutting long chain alginates using alginate lyase. The aim of this study was to produce alginate lyase enzyme then producing Alginate oligosaccharides (AOS as a prebiotic ingredients. The alginate lyase enzyme can be produced from Bacillus megaterium bacteria using a discontinuous fermentor. The enzyme was  optimum temperature of 45°C and an optimum pH of 7.0. Alginate oligosaccharides production was performed with the addition of different enzyme concentrations 25, 50, 75, and 100 U. The result of the addition of enzyme (25, 50,75 U showed that the value of polymerization degrees (DP were between 4-5. However, the addition of enzyme (100 U was in the range of  DP 3-4. Bacterial probiotic growth test results of Bifidobacteria and Lactobacillus showed that 1% added AOS media were able to increase the growth of probiotic bacteria compared to themedia without addition of AOS. The addition Alginate lyase activity of 50 U in AOS production is the best treatment of both probiotic bacteria.

  1. Activity and Enantioselectivity of the Hydroxynitrile Lyase MeHNL in Dry Organic Solvents

    NARCIS (Netherlands)

    Hanefeld, U.; Paravidino, M.; Sorgedrager, M.; Orru, R.V.A.

    2010-01-01

    Water concentration affects both the enantioselectivity and activity of enzymes in dry organic media. Its influence has been investigated using the hydrocyanation of benzaldehyde catalyzed by hydroxynitrile lyase cross-linked enzyme aggregate (MeHNL-CLEA) as a model reaction. The enzyme displayed hi

  2. Hematopoietic sphingosine 1-phosphate lyase deficiency decreases atherosclerotic lesion development in LDL-receptor deficient mice.

    Directory of Open Access Journals (Sweden)

    Martine Bot

    Full Text Available AIMS: Altered sphingosine 1-phosphate (S1P homeostasis and signaling is implicated in various inflammatory diseases including atherosclerosis. As S1P levels are tightly controlled by S1P lyase, we investigated the impact of hematopoietic S1P lyase (Sgpl1(-/- deficiency on leukocyte subsets relevant to atherosclerosis. METHODS AND RESULTS: LDL receptor deficient mice that were transplanted with Sgpl1(-/- bone marrow showed disrupted S1P gradients translating into lymphopenia and abrogated lymphocyte mitogenic and cytokine response as compared to controls. Remarkably however, Sgpl1(-/- chimeras displayed mild monocytosis, due to impeded stromal retention and myelopoiesis, and plasma cytokine and macrophage expression patterns, that were largely compatible with classical macrophage activation. Collectively these two phenotypic features of Sgpl1 deficiency culminated in diminished atherogenic response. CONCLUSIONS: Here we not only firmly establish the critical role of hematopoietic S1P lyase in controlling S1P levels and T cell trafficking in blood and lymphoid tissue, but also identify leukocyte Sgpl1 as critical factor in monocyte macrophage differentiation and function. Its, partly counterbalancing, pro- and anti-inflammatory activity spectrum imply that intervention in S1P lyase function in inflammatory disorders such as atherosclerosis should be considered with caution.

  3. Structural basis for the entrance into the phenylpropanoid metabolism catalyzed by phenylalanine ammonia-lyase.

    Science.gov (United States)

    Ritter, Holger; Schulz, Georg E

    2004-12-01

    Because of its key role in secondary phenylpropanoid metabolism, Phe ammonia-lyase is one of the most extensively studied plant enzymes. To provide a basis for detailed structure-function studies, the enzyme from parsley (Petroselinum crispum) was crystallized, and the structure was elucidated at 1.7-A resolution. It contains the unusual electrophilic 4-methylidene-imidazole-5-one group, which is derived from a tripeptide segment in two autocatalytic dehydration reactions. The enzyme resembles His ammonia-lyase from the general His degradation pathway but contains 207 additional residues, mainly in an N-terminal extension rigidifying a domain interface and in an inserted alpha-helical domain restricting the access to the active center. Presumably, Phe ammonia-lyase developed from His ammonia-lyase when fungi and plants diverged from the other kingdoms. A pathway of the catalyzed reaction is proposed in agreement with established biochemical data. The inactivation of the enzyme by a nucleophile is described in detail.

  4. Diversity of RuBisCO and ATP citrate lyase genes in soda lake sediments

    NARCIS (Netherlands)

    Kovaleva, O.L.; Tourova, T.P.; Muyzer, G.; Kolganova, T.V.; Sorokin, D.Y.

    2011-01-01

    Sediments from six soda lakes of the Kulunda Steppe (Altai, Russia) and from hypersaline alkaline lakes of Wadi Natrun (Egypt) were analyzed for the presence of cbb and aclB genes encoding key enzymes Ci assimilation (RuBisCO in Calvin-Benson and ATP citrate lyase in rTCA cycles, respectively). The

  5. Structural (betaalpha)8 TIM barrel model of 3-hydroxy-3-methylglutaryl-coenzyme A lyase.

    Science.gov (United States)

    Casals, Núria; Gómez-Puertas, Paulino; Pié, Juan; Mir, Cecilia; Roca, Ramón; Puisac, Beatriz; Aledo, Rosa; Clotet, Josep; Menao, Sebastián; Serra, Dolors; Asins, Guillermina; Till, Jacqueline; Elias-Jones, Alun C; Cresto, Juan C; Chamoles, Nestor A; Abdenur, Jose E; Mayatepek, Ertan; Besley, Guy; Valencia, Alfonso; Hegardt, Fausto G

    2003-08-01

    This study describes three novel homozygous missense mutations (S75R, S201Y, and D204N) in the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase gene, which caused 3-hydroxy-3-methylglutaric aciduria in patients from Germany, England, and Argentina. Expression studies in Escherichia coli show that S75R and S201Y substitutions completely abolished the HMG-CoA lyase activity, whereas D204N reduced catalytic efficiency to 6.6% of the wild type. We also propose a three-dimensional model for human HMG-CoA lyase containing a (betaalpha)8 (TIM) barrel structure. The model is supported by the similarity with analogous TIM barrel structures of functionally related proteins, by the localization of catalytic amino acids at the active site, and by the coincidence between the shape of the substrate (HMG-CoA) and the predicted inner cavity. The three novel mutations explain the lack of HMG-CoA lyase activity on the basis of the proposed structure: in S75R and S201Y because the new amino acid residues occlude the substrate cavity, and in D204N because the mutation alters the electrochemical environment of the active site. We also report the localization of all missense mutations reported to date and show that these mutations are located in the beta-sheets around the substrate cavity.

  6. The role of amino acid residues in the active site of L-methionine γ-lyase from Pseudomonas putida.

    Science.gov (United States)

    Fukumoto, Mitsuki; Kudou, Daizou; Murano, Shouko; Shiba, Tomoo; Sato, Dan; Tamura, Takashi; Harada, Shigeharu; Inagaki, Kenji

    2012-01-01

    Cys116, Lys240*, and Asp241* (asterisks indicate residues from the second subunit of the active dimer) at the active site of L-methionine γ-lyase of Pseudomonas putida (MGL_Pp) are highly conserved among heterologous MGLs. In a previous study, we found that substitution of Cys116 for His led to a drastic increase in activity toward L-cysteine and a decrease in that toward L-methionine. In this study, we examined some properties of the C116H mutant by kinetic analysis and 3D structural analysis. We assumed that substitution of Cys116 for His broke the original hydrogen-bond network and that this induced a significant effect of Tyr114 as a general acid catalyst, possibly due to the narrow space in the active site. The C116H mutant acquired a novel β-elimination activity and lead a drastic conformation change in the histidine residue at position 116 by binding the substrate, suggesting that this His residue affects the reaction specificity of C116H. Furthermore, we suggest that Lys240* is important for substrate recognition and structural stability and that Asp241* is also involved in substrate specificity in the elimination reaction. Based on this, we suggest that the hydrogen-bond network among Cys116, Lys240*, and Asp241* contributes to substrate specificity that is, to L-methionine recognition at the active site in MGL_Pp.

  7. Structural and Biochemical Characterization of a Copper-Binding Mutant of the Organomercurial Lyase MerB: Insight into the Key Role of the Active Site Aspartic Acid in Hg-Carbon Bond Cleavage and Metal Binding Specificity.

    Science.gov (United States)

    Wahba, Haytham M; Lecoq, Lauriane; Stevenson, Michael; Mansour, Ahmed; Cappadocia, Laurent; Lafrance-Vanasse, Julien; Wilkinson, Kevin J; Sygusch, Jurgen; Wilcox, Dean E; Omichinski, James G

    2016-02-23

    In bacterial resistance to mercury, the organomercurial lyase (MerB) plays a key role in the detoxification pathway through its ability to cleave Hg-carbon bonds. Two cysteines (C96 and C159; Escherichia coli MerB numbering) and an aspartic acid (D99) have been identified as the key catalytic residues, and these three residues are conserved in all but four known MerB variants, where the aspartic acid is replaced with a serine. To understand the role of the active site serine, we characterized the structure and metal binding properties of an E. coli MerB mutant with a serine substituted for D99 (MerB D99S) as well as one of the native MerB variants containing a serine residue in the active site (Bacillus megaterium MerB2). Surprisingly, the MerB D99S protein copurified with a bound metal that was determined to be Cu(II) from UV-vis absorption, inductively coupled plasma mass spectrometry, nuclear magnetic resonance, and electron paramagnetic resonance studies. X-ray structural studies revealed that the Cu(II) is bound to the active site cysteine residues of MerB D99S, but that it is displaced following the addition of either an organomercurial substrate or an ionic mercury product. In contrast, the B. megaterium MerB2 protein does not copurify with copper, but the structure of the B. megaterium MerB2-Hg complex is highly similar to the structure of the MerB D99S-Hg complexes. These results demonstrate that the active site aspartic acid is crucial for both the enzymatic activity and metal binding specificity of MerB proteins and suggest a possible functional relationship between MerB and its only known structural homologue, the copper-binding protein NosL.

  8. Structural And Biochemical Characterization of the Therapeutic A. Variabilis Phenylalanine Ammonia Lyase

    Energy Technology Data Exchange (ETDEWEB)

    Wang, L.; Gamez, A.; Archer, H.; Abola, E.E.; Sarkissian, C.N.; Fitzpatrick, P.; Wendt, D.; Zhang, Y.; Vellard, M.; Bliesath, J.; Bell, S.; Lemont, J.; Scriver, C.R.; Stevens, R.C.

    2009-05-26

    We have recently observed promising success in a mouse model for treating the metabolic disorder phenylketonuria with phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides and Anabaena variabilis. Both molecules, however, required further optimization in order to overcome problems with protease susceptibility, thermal stability, and aggregation. Previously, we optimized PAL from R. toruloides, and in this case we reduced aggregation of the A. variabilis PAL by mutating two surface cysteine residues (C503 and C565) to serines. Additionally, we report the structural and biochemical characterization of the A. variabilis PAL C503S/C565S double mutant and carefully compare this molecule with the R. toruloides engineered PAL molecule. Unlike previously published PAL structures, significant electron density is observed for the two active-site loops in the A. variabilis C503S/C565S double mutant, yielding a complete view of the active site. Docking studies and N-hydroxysuccinimide-biotin binding studies support a proposed mechanism in which the amino group of the phenylalanine substrate is attacked directly by the 4-methylidene-imidazole-5-one prosthetic group. We propose a helix-to-loop conformational switch in the helices flanking the inner active-site loop that regulates accessibility of the active site. Differences in loop stability among PAL homologs may explain the observed variation in enzyme efficiency, despite the highly conserved structure of the active site. A. variabilis C503S/C565S PAL is shown to be both more thermally stable and more resistant to proteolytic cleavage than R. toruloides PAL. Additional increases in thermal stability and protease resistance upon ligand binding may be due to enhanced interactions among the residues of the active site, possibly locking the active-site structure in place and stabilizing the tetramer. Examination of the A. variabilis C503S/C565S PAL structure, combined with analysis of its physical properties, provides

  9. Comparative proteome analysis reveals conserved and specific adaptation patterns of Staphylococcus aureus after internalization by different types of human non-professional phagocytic host cells.

    Science.gov (United States)

    Surmann, Kristin; Michalik, Stephan; Hildebrandt, Petra; Gierok, Philipp; Depke, Maren; Brinkmann, Lars; Bernhardt, Jörg; Salazar, Manuela G; Sun, Zhi; Shteynberg, David; Kusebauch, Ulrike; Moritz, Robert L; Wollscheid, Bernd; Lalk, Michael; Völker, Uwe; Schmidt, Frank

    2014-01-01

    Staphylococcus aureus is a human pathogen that can cause a wide range of diseases. Although formerly regarded as extracellular pathogen, it has been shown that S. aureus can also be internalized by host cells and persist within these cells. In the present study, we comparatively analyzed survival and physiological adaptation of S. aureus HG001 after internalization by two human lung epithelial cell lines (S9 and A549), and human embryonic kidney cells (HEK 293). Combining enrichment of bacteria from host-pathogen assays by cell sorting and quantitation of the pathogen's proteome by mass spectrometry we characterized S. aureus adaptation during the initial phase between 2.5 h and 6.5 h post-infection. Starting with about 2 × 10(6) bacteria, roughly 1450 S. aureus proteins, including virulence factors and metabolic enzymes were identified by spectral comparison and classical database searches. Most of the bacterial adaptation reactions, such as decreased levels of ribosomal proteins and metabolic enzymes or increased amounts of proteins involved in arginine and lysine biosynthesis, enzymes coding for terminal oxidases and stress responsive proteins or activation of the sigma factor SigB were observed after internalization into any of the three cell lines studied. However, differences were noted in central carbon metabolism including regulation of fermentation and threonine degradation. Since these differences coincided with different intracellular growth behavior, complementary profiling of the metabolome of the different non-infected host cell types was performed. This revealed similar levels of intracellular glucose but host cell specific differences in the amounts of amino acids such as glycine, threonine or glutamate. With this comparative study we provide an impression of the common and specific features of the adaptation of S. aureus HG001 to specific host cell environments as a starting point for follow-up studies with different strain isolates and regulatory

  10. Comparative proteome analysis reveals conserved and specific adaptation patterns of Staphylococcus aureus after internalization by different types of human non-professional phagocytic host cells

    Directory of Open Access Journals (Sweden)

    Kristin eSurmann

    2014-08-01

    Full Text Available Staphylococcus aureus is a human pathogen that can cause a wide range of diseases. Although formerly regarded as extracellular pathogen, it has been shown that S. aureus can also be internalized by host cells and persist within these cells. In the present study, we comparatively analyzed survival and physiological adaptation of S. aureus HG001 after internalization by two human lung epithelial cell lines (S9 and A549, and human embryonic kidney cells (HEK 293. Combining enrichment of bacteria from host-pathogen assays by cell sorting and quantitation of the pathogen´s proteome by mass spectrometry we characterized S. aureus adaptation during the initial phase between 2.5 h and 6.5 h post-infection. Starting with about 2x106 bacteria, roughly 1,450 S. aureus proteins, including virulence factors and metabolic enzymes were identified by spectral comparison and classical database searches. Most of the bacterial adaptation reactions, such as decreases in levels of ribosomal proteins and metabolic enzymes or increases in amounts of proteins involved in arginine and lysine biosynthesis, coding for terminal oxidases and stress responsive genes or activation of the sigma factor SigB were observed after internalization into any of the three cell lines studied. However, differences were noted in central carbon metabolism including regulation of fermentation and threonine degradation. Since these differences coincided with different intracellular growth behavior, complementary profiling of the metabolome of the different non-infected host cell types was performed. This revealed similar levels of intracellular glucose but host cell specific differences in the amounts of amino acids such as glycine, threonine or glutamate. With this comparative study we provide an impression of the common and specific features of the adaptation of S. aureus HG001 to specific host cell environments as a starting point for follow-up studies with different strain isolates and

  11. Cloning and characterization of a novel oligoalginate lyase from a newly isolated bacterium Sphingomonas sp. MJ-3.

    Science.gov (United States)

    Park, Hwan Hee; Kam, Natania; Lee, Eun Yeol; Kim, Hee Sook

    2012-04-01

    A bacterium possessing alginate-degrading activity was isolated from marine brown seaweed soup liquefied by salted and fermented anchovy. The isolated strain was designated as Sphingomonas sp. MJ-3 based on the analyses of 16S ribosomal DNA sequences, 16S-23S internal transcribed spacer region sequences, biochemical characteristics, and cellular fatty acid composition. A novel alginate lyase gene was cloned from genomic DNA library and then expressed in Escherichia coli. When the deduced amino acid sequence was compared with the sequences on the databases, interestingly, the cloned gene product was predicted to consist of AlgL (alginate lyase L)-like and heparinase-like protein domain. The MJ-3 alginate lyase gene shared below 27.0% sequence identity with exolytic alginate lyase of Sphingomonas sp. A1. The optimal pH and temperature for the recombinant MJ-3 alginate lyase were 6.5 and 50°C, respectively. The final degradation products of alginate oligosaccharides were analyzed by electrospray ionization mass spectrometry and proved to be alginate monosaccharides. Based on the results, the recombinant alginate lyase from Sphingomonas sp. MJ-3 is regarded as an oligoalginate lyase that can degrade oligoalginate and alginate into alginate monosaccharides.

  12. Molecular Cloning, Characterization and Expression of the Phenylalanine Ammonia-Lyase Gene from Juglans regia

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    Feng Xu

    2012-06-01

    Full Text Available Phenylalanine ammonia-lyase (PAL is the first key enzyme of the phenypropanoid pathway. A full-length cDNA of PAL gene was isolated from Juglans regia for the first time, and designated as JrPAL. The full-length cDNA of the JrPAL gene contained a 1935bp open reading frame encoding a 645-amino-acid protein with a calculated molecular weight of about 70.4 kD and isoelectric point (pI of 6.7. The deduced JrPAL protein showed high identities with other plant PALs. Molecular modeling of JrPAL showed that the 3D model of JrPAL was similar to that of PAL protein from Petroselinum crispum (PcPAL, implying that JrPAL may have similar functions with PcPAL. Phylogenetic tree analysis revealed that JrPAL shared the same evolutionary ancestor of other PALs and had a closer relationship with other angiosperm species. Transcription analysis revealed that JrPAL was expressed in all tested tissues including roots, stems, and leaves, with the highest transcription level being found in roots. Expression profiling analyses by real-time PCR revealed that JrPAL expression was induced by a variety of abiotic and biotic stresses, including UV-B, wounding, cold, abscisic acid and salicylic acid.

  13. Conservative management.

    Science.gov (United States)

    Kruis, W; Leifeld, L; Pfützer, R

    2012-01-01

    Treatment of diverticulitis comprises at least two options: conservative or surgical management. There is a recent trend to limit surgical treatment of acute diverticulitis and to favor conservative management. This review addresses general aspects of conservative patient care with special focus on the treatment of patients with a first attack of diverticulitis. The presentation does not include a discussion of specific drugs which is given in other sections of this issue.

  14. Functional conservation and divergence of four ginger AP1/AGL9 MADS-box genes revealed by analysis of their expression and protein-protein interaction, and ectopic expression of AhFUL gene in Arabidopsis.

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    Xiumei Li

    Full Text Available Alpinia genus are known generally as ginger-lilies for showy flowers in the ginger family, Zingiberaceae, and their floral morphology diverges from typical monocotyledon flowers. However, little is known about the functions of ginger MADS-box genes in floral identity. In this study, four AP1/AGL9 MADS-box genes were cloned from Alpinia hainanensis, and protein-protein interactions (PPIs and roles of the four genes in floral homeotic conversion and in floral evolution are surveyed for the first time. AhFUL is clustered to the AP1 lineage, AhSEP4 and AhSEP3b to the SEP lineage, and AhAGL6-like to the AGL6 lineage. The four genes showed conserved and divergent expression patterns, and their encoded proteins were localized in the nucleus. Seven combinations of PPI (AhFUL-AhSEP4, AhFUL-AhAGL6-like, AhFUL-AhSEP3b, AhSEP4-AhAGL6-like, AhSEP4-AhSEP3b, AhAGL6-like-AhSEP3b, and AhSEP3b-AhSEP3b were detected, and the PPI patterns in the AP1/AGL9 lineage revealed that five of the 10 possible combinations are conserved and three are variable, while conclusions cannot yet be made regarding the other two. Ectopic expression of AhFUL in Arabidopsis thaliana led to early flowering and floral organ homeotic conversion to sepal-like or leaf-like. Therefore, we conclude that the four A. hainanensis AP1/AGL9 genes show functional conservation and divergence in the floral identity from other MADS-box genes.

  15. Pectate lyase pollen allergens: sensitization profiles and cross-reactivity pattern.

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    Ulrike Pichler

    Full Text Available Pollen released by allergenic members of the botanically unrelated families of Asteraceae and Cupressaceae represent potent elicitors of respiratory allergies in regions where these plants are present. As main allergen sources the Asteraceae species ragweed and mugwort, as well as the Cupressaceae species, cypress, mountain cedar, and Japanese cedar have been identified. The major allergens of all species belong to the pectate lyase enzyme family. Thus, we thought to investigate cross-reactivity pattern as well as sensitization capacities of pectate lyase pollen allergens in cohorts from distinct geographic regions.The clinically relevant pectate lyase pollen allergens Amb a 1, Art v 6, Cup a 1, Jun a 1, and Cry j 1 were purified from aqueous pollen extracts, and patients' sensitization pattern of cohorts from Austria, Canada, Italy, and Japan were determined by IgE ELISA and cross-inhibition experiments. Moreover, we performed microarray experiments and established a mouse model of sensitization.In ELISA and ELISA inhibition experiments specific sensitization pattern were discovered for each geographic region, which reflected the natural allergen exposure of the patients. We found significant cross-reactivity within Asteraceae and Cupressaceae pectate lyase pollen allergens, which was however limited between the orders. Animal experiments showed that immunization with Asteraceae allergens mainly induced antibodies reactive within the order, the same was observed for the Cupressaceae allergens. Cross-reactivity between orders was minimal. Moreover, Amb a 1, Art v 6, and Cry j 1 showed in general higher immunogenicity.We could cluster pectate lyase allergens in four categories, Amb a 1, Art v 6, Cup a 1/Jun a 1, and Cry j 1, respectively, at which each category has the potential to sensitize predisposed individuals. The sensitization pattern of different cohorts correlated with pollen exposure, which should be considered for future allergy

  16. Crystal structure and characterization of a novel L-serine ammonia-lyase from Rhizomucor miehei

    Energy Technology Data Exchange (ETDEWEB)

    Qin, Zhen [College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center of Food Nutrition and Human Health, China Agricultural University, Beijing 100083 (China); Yan, Qiaojuan [College of Engineering, China Agricultural University, Beijing 100083 (China); Ma, Qingjun [Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071 (China); Jiang, Zhengqiang, E-mail: zhqjiang@cau.edu.cn [College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center of Food Nutrition and Human Health, China Agricultural University, Beijing 100083 (China)

    2015-10-23

    L-serine ammonia-lyase, as a member of the β-family of pyridoxal-5′-phosphate (PLP) dependent enzymes, catalyzes the conversion of L-serine (L-threonine) to pyruvate (α-ketobutyrate) and ammonia. The crystal structure of L-serine ammonia-lyase from Rhizomucor miehei (RmSDH) was solved at 1.76 Å resolution by X-ray diffraction method. The overall structure of RmSDH had the characteristic β-family PLP dependent enzyme fold. It consisted of two distinct domains, both of which show the typical open twisted α/β structure. A PLP cofactor was located in the crevice between the two domains, which was attached to Lys52 by a Schiff-base linkage. Unique residue substitutions (Gly78, Pro79, Ser146, Ser147 and Thr312) were discovered at the catalytic site of RmSDH by comparison of structures of RmSDH and other reported eukaryotic L-serine ammonia-lyases. Optimal pH and temperature of the purified RmSDH were 7.5 and 40 °C, respectively. It was stable in the pH range of 7.0–9.0 and at temperatures below 40 °C. This is the first crystal structure of a fungal L-serine ammonia-lyase. It will be useful to study the catalytic mechanism of β-elimination enzymes and will provide a basis for further enzyme engineering. - Highlights: • The crystal structure of a fungal L-serine ammonia-lyase (RmSDH) was solved. • Five unique residue substitutions are found at the catalytic site of RmSDH. • RmSDH was expressed in Pichia. pastoris and biochemically characterized. • RmSDH has potential application in splitting D/L-serine.

  17. Purification, characterization and induction of L-phenylalanine ammonia-lyase in Phaseolus vulgaris.

    Science.gov (United States)

    da Cunha, A

    1988-12-01

    The enzyme L-phenylalanine ammonia-lyase was purified from leaves of Phaseolus vulgaris by Sephacryl S-200 gel filtration and Sepharose-4-B--succinyl-aminoethyl-L-phenylalanine affinity chromatography. L-Phenylalanine ammonia-lyase was specifically eluted from the affinity matrix with its substrate L-phenylalanine at 20-25 degrees C. The purified enzyme was shown to be homogeneous by gel electrophoresis both in presence and absence of SDS. Its Mr, determined by gel filtration and non-denaturing gel electrophoresis, was 320,000 +/- 9000 and 330,000 +/- 4000 respectively. After SDS electrophoresis only one band of Mr 83,000 +/- 4000 was detected, indicating that the enzyme is an oligomer containing four subunits. The pH optimum of enzyme activity was 8.8-9.2. Ampholyte isoelectrofocusing in polyacrylamide demonstrated the presence of a single charged species at pH 4.2. The homogeneous enzyme catalyzed the deamination of L-phenylalanine to trans-cinnamate but did not catalyze the transamination of L-phenylalanine to L-phenylpyruvate. The enzyme showed Km 1.25 mM for L-phenylalanine. Antibodies to homogeneous L-phenylalanine ammonia-lyase recognised specific epitopes on L-phenylalanine aminotransferase as demonstrated by immunoaffinity purification and immunoblotting. The induction of L-phenylalanine ammonia-lyase activity during phaseollin biosynthesis in the Phaseolus vulgaris--Colletotrichum lindemuthianum interaction was regulated by an increase in enzyme concentration resulting from an increase in de novo synthesis of L-phenylalanine ammonia-lyase protein.

  18. A role for glutamate-333 of Saccharomyces cerevisiae cystathionine γ-lyase as a determinant of specificity.

    Science.gov (United States)

    Hopwood, Emily M S; Ahmed, Duale; Aitken, Susan M

    2014-02-01

    Cystathionine γ-lyase (CGL) catalyzes the hydrolysis of l-cystathionine (l-Cth), producing l-cysteine (l-Cys), α-ketobutyrate and ammonia, in the second step of the reverse transsulfuration pathway, which converts l-homocysteine (l-Hcys) to l-Cys. Site-directed variants substituting residues E48 and E333 with alanine, aspartate and glutamine were characterized to probe the roles of these acidic residues, conserved in fungal and mammalian CGL sequences, in the active-site of CGL from Saccharomyces cerevisiae (yCGL). The pH optimum of variants containing the alanine or glutamine substitutions of E333 is increased by 0.4-1.2 pH units, likely due to repositioning of the cofactor and modification of the pKa of the pyridinium nitrogen. The pH profile of yCGL-E48A/E333A resembles that of Escherichia coli cystathionine β-lyase. The effect of substituting E48, E333 or both residues is the 1.3-3, 26-58 and 124-568-fold reduction, respectively, of the catalytic efficiency of l-Cth hydrolysis. The Km(l-Cth) of E333 substitution variants is increased ~17-fold, while Km(l-OAS) is within 2.5-fold of the wild-type enzyme, indicating that residue E333 interacts with the distal amine moiety of l-Cth, which is not present in the alternative substrate O-acetyl-l-serine. The catalytic efficiency of yCGL for α,γ-elimination of O-succinyl-l-homoserine (kcat/Km(l-OSHS)=7±2), which possesses a distal carboxylate, but lacks an amino group, is 300-fold lower than that of the physiological l-Cth substrate (kcat/Km(l-Cth)=2100±100) and 260-fold higher than that of l-Hcys (kcat/Km(l-Hcys)=0.027±0.005), which lacks both distal polar moieties. The results of this study suggest that the glutamate residue at position 333 is a determinant of specificity.

  19. Structure of the HopA1(21-102)-ShcA chaperone-effector complex of Pseudomonas syringae reveals conservation of a virulence factor binding motif from animal to plant pathogens.

    Science.gov (United States)

    Janjusevic, Radmila; Quezada, Cindy M; Small, Jennifer; Stebbins, C Erec

    2013-02-01

    Pseudomonas syringae injects numerous bacterial proteins into host plant cells through a type 3 secretion system (T3SS). One of the first such bacterial effectors discovered, HopA1, is a protein that has unknown functions in the host cell but possesses close homologs that trigger the plant hypersensitive response in resistant strains. Like the virulence factors in many bacterial pathogens of animals, HopA1 depends upon a cognate chaperone in order to be effectively translocated by the P. syringae T3SS. Herein, we report the crystal structure of a complex of HopA1(21-102) with its chaperone, ShcA, determined to 1.56-Å resolution. The structure reveals that three key features of the chaperone-effector interactions found in animal pathogens are preserved in the Gram-negative pathogens of plants, namely, (i) the interaction of the chaperone with a nonglobular polypeptide of the effector, (ii) an interaction centered on the so-called β-motif, and (iii) the presence of a conserved hydrophobic patch in the chaperone that recognizes the β-motif. Structure-based mutagenesis and biochemical studies have established that the β-motif is critical for the stability of this complex. Overall, these results show that the β-motif interactions are broadly conserved in bacterial pathogens utilizing T3SSs, spanning an interkingdom host range.

  20. Metagenome Sequence Analysis of Filamentous Microbial Communities Obtained from Geochemically Distinct Geothermal Channels Reveals Specialization of Three Aquificales Lineages

    Directory of Open Access Journals (Sweden)

    Cristina eTakacs-vesbach

    2013-05-01

    Full Text Available The Aquificales are thermophilic microorganisms that inhabit hydrothermal systems worldwide and are considered one of the earliest lineages of the domain Bacteria. We analyzed metagenome sequence obtained from six thermal ‘filamentous streamer’ communities (~40 Mbp per site, which targeted three different groups of Aquificales found in Yellowstone National Park (YNP. Unassembled metagenome sequence and PCR-amplified 16S rRNA gene libraries revealed that acidic, sulfidic sites were dominated by Hydrogenobaculum (Aquificaceae populations, whereas the circumneutral pH (6.5 - 7.8 sites containing dissolved sulfide were dominated by Sulfurihydrogenibium spp. (Hydrogenothermaceae. Thermocrinis (Aquificaceae populations were found primarily in the circumneutral sites with undetectable sulfide, and to a lesser extent in one sulfidic system at pH 8. Phylogenetic analysis of assembled sequence containing 16S rRNA genes as well as conserved protein-encoding genes revealed that the composition and function of these communities varied across geochemical conditions. Each Aquificales lineage contained genes for CO2 fixation by the reverse TCA cycle, but only the Sulfurihydrogenibium populations perform citrate cleavage using ATP citrate lyase (Acl. The Aquificaceae populations use an alternative pathway catalyzed by two separate enzymes, citryl CoA synthetase (Ccs and citryl CoA lyase (Ccl. All three Aquificales lineages contained evidence of aerobic respiration, albeit due to completely different types of heme Cu oxidases (subunit I involved in oxygen reduction. The distribution of Aquificales populations and differences among functional genes involved in energy generation and electron transport is consistent with the hypothesis that geochemical parameters (e.g., pH, sulfide, H2, O2 have resulted in niche specialization among members of the Aquificales.

  1. 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.

  2. Conformational Analysis of the Streptococcus pneumoniae Hyaluronate Lyase and Characterization of Its Hyaluronan-specific Carbohydrate-binding Module*

    Science.gov (United States)

    Suits, Michael D. L.; Pluvinage, Benjamin; Law, Adrienne; Liu, Yan; Palma, Angelina S.; Chai, Wengang; Feizi, Ten; Boraston, Alisdair B.

    2014-01-01

    For a subset of pathogenic microorganisms, including Streptococcus pneumoniae, the recognition and degradation of host hyaluronan contributes to bacterial spreading through the extracellular matrix and enhancing access to host cell surfaces. The hyaluronate lyase (Hyl) presented on the surface of S. pneumoniae performs this role. Using glycan microarray screening, affinity electrophoresis, and isothermal titration calorimetry we show that the N-terminal module of Hyl is a hyaluronan-specific carbohydrate-binding module (CBM) and the founding member of CBM family 70. The 1.2 Å resolution x-ray crystal structure of CBM70 revealed it to have a β-sandwich fold, similar to other CBMs. The electrostatic properties of the binding site, which was identified by site-directed mutagenesis, are distinct from other CBMs and complementary to its acidic ligand, hyaluronan. Dynamic light scattering and solution small angle x-ray scattering revealed the full-length Hyl protein to exist as a monomer/dimer mixture in solution. Through a detailed analysis of the small angle x-ray scattering data, we report the pseudoatomic solution structures of the monomer and dimer forms of the full-length multimodular Hyl. PMID:25100731

  3. Cysteine Conjugate β-Lyase Activity of Rat Erythrocytes and Formation of β-Lyase-Derived Globin Monoadducts and Cross-Links after in Vitro Exposure of Erythrocytes to S-(1,2-Dichlorovinyl)-L-cysteine

    OpenAIRE

    Barshteyn, Nella; Elfarra, Adnan A.

    2009-01-01

    S-(1,2-Dichlorovinyl)-L-cysteine (DCVC), a mutagenic and nephrotoxic metabolite of trichloroethylene can be bioactivated to reactive metabolites, S-(1,2-dichlorovinyl)-L-cysteine sulfoxide (DCVCS) or chlorothioketene and/or 2-chlorothionoacetyl chloride, by cysteine conjugate S-oxidase (S-oxidase) and cysteine conjugate β-lyase (β-lyase), respectively. Previously, we characterized reactivity of DCVCS with Hb upon incubation of erythrocytes with DCVCS and provided evidence for formation of dis...

  4. Square conservation systems and Hamiltonian systems

    Institute of Scientific and Technical Information of China (English)

    王斌; 曾庆存; 季仲贞

    1995-01-01

    The internal and external relationships between the square conservation scheme and the symplectic scheme are revealed by a careful study on the interrelation between the square conservation system and the Hamiltonian system in the linear situation, thus laying a theoretical basis for the application and extension of symplectic schemes to square conservations systems, and of those schemes with quadratic conservation properties to Hamiltonian systems.

  5. Reshaping conservation

    DEFF Research Database (Denmark)

    Funder, Mikkel; Danielsen, Finn; Ngaga, Yonika

    2013-01-01

    members strengthen the monitoring practices to their advantage, and to some extent move them beyond the reach of government agencies and conservation and development practitioners. This has led to outcomes that are of greater social and strategic value to communities than the original 'planned' benefits......, although the monitoring scheme has also to some extent become dominated by local 'conservation elites' who negotiate the terrain between the state and other community members. Our findings suggest that we need to move beyond simplistic assumptions of community strategies and incentives in participatory...... conservation and allow for more adaptive and politically explicit governance spaces in protected area management....

  6. Wildlife Conservation

    OpenAIRE

    Clive L. Spash; Aldred, Jonathan

    1998-01-01

    In this paper we consider how conservation has arisen as a key aspect of the reaction to human-initiated degradation and disappearance of ecosystems, wild lands. and wildlife. Concern over species extinction is given an historical perspective which shows the way in which pressure on wild and natural aspects of global ecology have changed in recent centuries. The role of conservation in the struggle to protect the environment is then analysed using underlying ethical arguments behind the econo...

  7. 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

    glyoxylate would increase, leading to excess formation of malate and succinate compared to the wild-type. However, metabolic network analysis showed that an increased icl expression did not result in an increased glyoxylate bypass flux. The analysis did show a global response with respect to gene expression......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......, leading to an increased flux through the oxidative part of the TCA cycle. Instead of an increased production of succinate and malate, a major increase in fumarate production was observed. The effect of malonate, a competitive inhibitor of succinate dehydrogenase (SDH), on the physiological behaviour...

  8. Syntheses of L-tyrosine-related amino acids by tyrosine phenol-lyase of Citrobacter intermedius.

    Science.gov (United States)

    Nagasawa, T; Utagawa, T; Goto, J; Kim, C J; Tani, Y; Kumagai, H; Yamada, H

    1981-06-01

    Degradation of tyrosine to phenol, pyruvate and ammonia by tyrosine phenol-lyase from Citrobacter intermedius (formerly named Escherichia intermedia) is readily reversible at high concentrations of pyruvate and ammonia. Spectrophotometric studies indicate that ammonia is the first substrate which interacts with bound pyridoxal 5'-phosphate. Kinetic results show that pyruvate is the second substrate bound, hence phenol must be the third. When an appropriate phenol derivative is substituted for phenol, the corresponding tyrosine analogue can be synthesized. 3-Fluoro-, 2-fluoro-, 3-chloro-, 2-chloro-, 3-bromo-, 2-bromo-, 2-iodo-, 3-methyl-, 2-methyl- and 2-methoxy-L-tyrosines have been synthesized by this reaction. By using various phenol derivatives or tyrosine analogues as substrates, the substrate specificity of tyrosine phenol-lyase is investigated and the situation of its active site is discussed.

  9. Inhibition of Candida albicans isocitrate lyase activity by sesterterpene sulfates from the tropical sponge Dysidea sp.

    Science.gov (United States)

    Lee, Dongha; Shin, Jongheon; Yoon, Kyung-Mi; Kim, Tae-Im; Lee, So-Hyoung; Lee, Hyi-Seung; Oh, Ki-Bong

    2008-10-15

    Seven sesterterpene sulfates (1-7) were isolated from the tropical sponge Dysidea sp. and their inhibitory activities against isocitrate lyase (ICL) from Candida albicans were evaluated. Among the isolated natural products compound 6 and 7 were found to be strong ICL inhibitors. The isolated compounds (1-7) also showed potent antibacterial effect against Bacillus subtilis and Proteus vulgaris, but did not display antifungal activity.

  10. Mechanism of benzaldehyde lyase studied via thiamin diphosphate-bound intermediates and kinetic isotope effects.

    Science.gov (United States)

    Chakraborty, Sumit; Nemeria, Natalia; Yep, Alejandra; McLeish, Michael J; Kenyon, George L; Jordan, Frank

    2008-03-25

    Direct spectroscopic observation of thiamin diphosphate-bound intermediates was achieved on the enzyme benzaldehyde lyase, which carries out reversible and highly enantiospecific conversion of ( R)-benzoin to benzaldehyde. The key enamine intermediate could be observed at lambda max 393 nm in the benzoin breakdown direction and in the decarboxylase reaction starting with benzoylformate. With benzaldehyde as substrate, no intermediates could be detected, only formation of benzoin at 314 nm. To probe the rate-limiting step in the direction of ( R)-benzoin synthesis, the (1)H/ (2)H kinetic isotope effect was determined for benzaldehyde labeled at the aldehyde position and found to be small (1.14 +/- 0.03), indicating that ionization of the C2alphaH from C2alpha-hydroxybenzylthiamin diphosphate is not rate limiting. Use of the alternate substrates benzoylformic and phenylpyruvic acids (motivated by the observation that while a carboligase, benzaldehyde lyase could also catalyze the slow decarboxylation of 2-oxo acids) enabled the observation of the substrate-thiamin covalent intermediate via the 1',4'-iminopyrimidine tautomer, characteristic of all intermediates with a tetrahedral C2 substituent on ThDP. The reaction of benzaldehyde lyase with the chromophoric substrate analogue ( E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid and its decarboxylated product ( E)-3-(pyridine-3-yl)acrylaldehyde enabled the detection of covalent adducts with both. Neither adduct underwent further reaction. An important finding of the studies is that all thiamin-related intermediates are in a chiral environment on benzaldehyde lyase as reflected by their circular dichroism signatures.

  11. 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.

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

    Science.gov (United States)

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

    2015-08-05

    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.

  13. Production of Diamino propionic acid ammonia lyase by a new strain of Salmonella typhimurium PU011

    Directory of Open Access Journals (Sweden)

    Shiva Kumar Vasanth V

    2002-03-01

    Full Text Available Abstract Background Seeds of the legume plant Lathyrus sativus, which is grown in arid and semi arid tropical regions, contain Diamino Propionic acid (DAP. DAP is a neurotoxin, which, when consumed, causes a disease called Lathyrism. Lathryrism may manifest as Neurolathyrism or Osteolathyrism, in which the nervous system, and bone formation respectively, are affected. DAP ammonia lyase is produced by a few microorganisms such as Salmonella typhi, Salmonella typhimurium and Pseudomonas, and is capable of detoxifying DAP. Results S. typhimurium PU011, a non-virulent bacterial strain isolated in our lab, was found to produce DAP ammonia lyase enzyme when grown in minimal medium containing DAP. There was a direct correlation between biomass yield and enzyme activity, until 16 h post inoculation in minimal medium containing DAP. Following ammonium sulphate precipitation and passing through Sephadex G100, CM-Sephadex and DEAE-Sephacel for crude enzyme extract preparation, about 68-fold enzyme purity was obtained. The purified enzyme gave maximum activity at pH 8.0 and was stable up to 45 degrees C. The Km value for the substrate was found to be 0.685mM, calculated from a Line Weaver Burk plot. Conclusion A new bacterial strain, S.typhimurium PU 011, which is capable of producing DAP ammonia lyase, was isolated.

  14. Characterization of phycoviolobilin phycoerythrocyanin-alpha 84-cystein-lyase-(isomerizing) from Mastigocladus laminosus.

    Science.gov (United States)

    Zhao, Kai-Hong; Wu, Dong; Wang, Lu; Zhou, Ming; Storf, Max; Bubenzer, Claudia; Strohmann, Brigitte; Scheer, Hugo

    2002-09-01

    Cofactor requirements and enzyme kinetics have been studied of the novel, dual-action enzyme, the isomerizing phycoviolobilin phycoerythrocyanin-alpha84-cystein-lyase(PVB-PEC-lyase) from Mastigocladus laminosus, which catalyses both the covalent attachment of phycocyanobilin to PecA, the apo-alpha-subunit of phycoerythrocyanin, and its isomerization to phycoviolobilin. Thiols and the divalent metals, Mg2+ or Mn2+, were required, and the reaction was aided by the detergent, Triton X-100. Phosphate buffer inhibits precipitation of the proteins present in the reconstitution mixture, but at the same time binds the required metal. Kinetic constants were obtained for both substrates, the chromophore (Km = 12-16 micro m, depending on [PecA], kcat approximately 1.2 x 10-4.s-1) and the apoprotein (Km = 2.4 micro m at 14 micro m PCB, kcat = 0.8 x 10-4.s-1). The kinetic analysis indicated that the reconstitution reaction proceeds by a sequential mechanism. By a combination of untagged and His-tagged subunits, evidence was obtained for a complex formation between PecE and PecF (subunits of PVB-PEC-lyase), and by experiments with single subunits for the prevalent function of PecE in binding and PecF in isomerizing the chromophore.

  15. Effect of cysteine on the inactivation of cystathionine gamma-lyase by D,L-propargylglycine.

    Directory of Open Access Journals (Sweden)

    Awata,Shiro

    1989-12-01

    Full Text Available In vivo inactivation of cystathionine gamma-lyase by D,L-propargylglycine, a suicide inhibitor, was found to be less profound in rat kidney than in the liver. We investigated the cause of this difference using rat tissues. We fractionated kidney extract to characterize the substance which protected enzyme, and found that cysteine exhibits protecting action. Addition of 0.3 mM L-cysteine to the incubation mixture containing dialyzed kidney supernatant and 0.5 mM D,L-propargylglycine resulted in the protection of cystathionine gamma-lyase from the inactivation by the inhibitor. The content of cysteine in the kidney was six-fold higher than that in the liver. Thus, we have concluded that one of the reasons why the in vivo inactivation of cystathionine gamma-lyase in rat kidney was less than that in the liver is the presence of a higher concentration of cysteine in the kidney. S-Carboxymethylcysteine, a cysteine derivative, exhibited a similar, but weaker, protective effect.

  16. Improvement of aromatic thiol release through the selection of yeasts with increased β-lyase activity.

    Science.gov (United States)

    Belda, Ignacio; Ruiz, Javier; Navascués, Eva; Marquina, Domingo; Santos, Antonio

    2016-05-16

    The development of a selective medium for the rapid differentiation of yeast species with increased aromatic thiol release activity has been achieved. The selective medium was based on the addition of S-methyl-l-cysteine (SMC) as β-lyase substrate. In this study, a panel of 245 strains of Saccharomyces cerevisiae strains was tested for their ability to grow on YCB-SMC medium. Yeast strains with an increased β-lyase activity grew rapidly because of their ability to release ammonium from SMC in comparison to others, and allowed for the easy isolation and differentiation of yeasts with promising properties in oenology, or another field, for aromatic thiol release. The selective medium was also helpful for the discrimination between those S. cerevisiae strains, which present a common 38-bp deletion in the IRC7 sequence (present in around 88% of the wild strains tested and are likely to be less functional for 4-mercapto-4-methylpentan-2-one (4MMP) production), and those S. cerevisiae strains homozygous for the full-length IRC7 allele. The medium was also helpful for the selection of non-Saccharomyces yeasts with increased β-lyase activity. Based on the same medium, a highly sensitive, reproducible and non-expensive GC-MS method for the evaluation of the potential volatile thiol release by different yeast isolates was developed.

  17. Rev1 is a base excision repair enzyme with 5′-deoxyribose phosphate lyase activity

    Science.gov (United States)

    Prasad, Rajendra; Poltoratsky, Vladimir; Hou, Esther W.; Wilson, Samuel H.

    2016-01-01

    Rev1 is a member of the Y-family of DNA polymerases and is known for its deoxycytidyl transferase activity that incorporates dCMP into DNA and its ability to function as a scaffold factor for other Y-family polymerases in translesion bypass events. Rev1 also is involved in mutagenic processes during somatic hypermutation of immunoglobulin genes. In light of the mutation pattern consistent with dCMP insertion observed earlier in mouse fibroblast cells treated with a base excision repair-inducing agent, we questioned whether Rev1 could also be involved in base excision repair (BER). Here, we uncovered a weak 5′-deoxyribose phosphate (5′-dRP) lyase activity in mouse Rev1 and demonstrated the enzyme can mediate BER in vitro. The full-length Rev1 protein and its catalytic core domain are similar in their ability to support BER in vitro. The dRP lyase activity in both of these proteins was confirmed by NaBH4 reduction of the Schiff base intermediate and kinetics studies. Limited proteolysis, mass spectrometry and deletion analysis localized the dRP lyase active site to the C-terminal segment of Rev1's catalytic core domain. These results suggest that Rev1 could serve as a backup polymerase in BER and could potentially contribute to AID-initiated antibody diversification through this activity. PMID:27683219

  18. Role of active-site residues Tyr55 and Tyr114 in catalysis and substrate specificity of Corynebacterium diphtheriae C-S lyase.

    Science.gov (United States)

    Astegno, Alessandra; Allegrini, Alessandra; Piccoli, Stefano; Giorgetti, Alejandro; Dominici, Paola

    2015-01-01

    In recent years, there has been increased interest in bacterial methionine biosynthesis enzymes as antimicrobial targets because of their pivotal role in cell metabolism. C-S lyase from Corynebacterium diphtheriae is a pyridoxal 5'-phosphate-dependent enzyme in the transsulfuration pathway that catalyzes the α,β-elimination of sulfur-containing amino acids, such as L-cystathionine, to generate ammonia, pyruvate, and homocysteine, the immediate precursor of L-methionine. In order to gain deeper insight into the functional and dynamic properties of the enzyme, mutants of two highly conserved active-site residues, Y55F and Y114F, were characterized by UV-visible absorbance, fluorescence, and CD spectroscopy in the absence and presence of substrates and substrate analogs, as well as by steady-state kinetic studies. Substitution of Tyr55 with Phe apparently causes a 130-fold decrease in K(d)(PLP) at pH 8.5 providing evidence that Tyr55 plays a role in cofactor binding. Moreover, spectral data show that the mutant accumulates the external aldimine intermediate suggesting that the absence of interaction between the hydroxyl moiety and PLP-binding residue Lys222 causes a decrease in the rate of substrate deprotonation. Mutation of Tyr114 with Phe slightly influences hydrolysis of L-cystathionine, and causes a change in substrate specificity towards L-serine and O-acetyl-L-serine compared to the wild type enzyme. These findings, together with computational data, provide useful insights in the substrate specificity of C-S lyase, which seems to be regulated by active-site architecture and by the specific conformation in which substrates are bound, and will aid in development of inhibitors.

  19. Characterization of smart auto-degradative hydrogel matrix containing alginate lyase to enhance levofloxacin delivery against bacterial biofilms.

    Science.gov (United States)

    Islan, German A; Dini, Cecilia; Bartel, Laura C; Bolzán, Alejandro D; Castro, Guillermo R

    2015-12-30

    The aim of the present work is the characterization of smart auto-degradable microspheres composed of calcium alginate/high methoxylated pectin containing an alginate lyase (AL) from Sphingobacterium multivorum and levofloxacin. Microspheres were prepared by ionotropic gelation containing AL in its inactive form at pH 4.0. Incubation of microspheres in Tris-HCl and PBS buffers at pH 7.40 allowed to establish the effect of ion-chelating phosphate on matrix erodability and suggested an intrinsically activation of AL by turning the pH close to neutrality. Scanning electron and optical microscopies revealed the presence of holes and surface changes in AL containing microspheres. Furthermore, texturometric parameters, DSC profiles and swelling properties were showing strong changes in microspheres properties. Encapsulation of levofloxacin into microspheres containing AL showed 70% efficiency and 35% enhancement of antimicrobial activity against Pseudomonas aeruginosa biofilm. Levofloxacin release from microspheres was not changed at acidic pH, but was modified at neutral pH in presence of AL. Advantageously, only gel matrix debris were detectable after overnight incubation, indicating an autodegradative gel process activated by the pH. Absence of matrix cytotoxicity and a reduction of the levofloxacin toxicity after encapsulation were observed in mammalian CHO-K1 cell cultures. These properties make the system a potent and versatile tool for antibiotic oral delivery targeted to intestine, enhancing the drug bioavailability to eradicate bacterial biofilm and avoiding possible intestinal obstructions.

  20. Synthesis of green note aroma compounds by biotransformation of fatty acids using yeast cells coexpressing lipoxygenase and hydroperoxide lyase.

    Science.gov (United States)

    Buchhaupt, Markus; Guder, Jan Christopher; Etschmann, Maria Magdalena Walburga; Schrader, Jens

    2012-01-01

    Green notes are substances that characterize the aroma of freshly cut grass, cucumbers, green apples, and foliage. In plants, they are synthesized by conversion of linolenic or linoleic acid via the enzymes lipoxygenase (LOX) and hydroperoxide lyase (HPL) to short-chained aldehydes. Current processes for production of natural green notes rely on plant homogenates as enzyme sources but are limited by low enzyme concentration and low specificity. In an alternative approach, soybean LOX2 and watermelon HPL were overexpressed in Saccharomyces cerevisiae. After optimization of the expression constructs, a yeast strain coexpressing LOX and HPL was applied in whole cell biotransformation experiments. Whereas addition of linolenic acid to growing cultures of this strain yielded no products, we were able to identify high green note concentrations when resting cells were used. The primary biotransformation product was 3(Z)-hexenal, a small amount of which isomerized to 2(E)-hexenal. Furthermore, both aldehydes were reduced to the corresponding green note alcohols by endogenous yeast alcohol dehydrogenase to some extent. As the cosolvent ethanol was the source of reducing equivalents for green note alcohol formation, the hexenal/hexenol ratio could be influenced by the use of alternative cosolvents. Further investigations to identify the underlying mechanism of the rather low biocatalyst stability revealed a high toxicity of linolenic acid to yeast cells. The whole cell catalyst containing LOX and HPL enzyme activity described here can be a promising approach towards a highly efficient microbial green note synthesis process.

  1. Characterization of the phenylalanine ammonia-lyase gene (SlPAL5) from tomato (Solanum lycopersicum L.).

    Science.gov (United States)

    Guo, Jia; Wang, Myeong-Hyeon

    2009-07-01

    Phylogenetic analysis based on the deduced amino acid sequence of phenylalanine ammonia-lyase gene (SlPAL5) cDNA from tomato (Solanum lycopersicum L.) revealed high sequence similarity to PAL genes in Nicotiana tabacum (92%), Ipomoea nil (87%), Manihot esculenta (84%), and Catharanthus roseus (84%). The SlPAL5 gene exists as multiple copies in the tomato plant, and its transcription was strongly expressed in old leaves and flowers. From 5 days post-anthesis to the onset of ripening, SlPAL5 expression decreased gradually but was maintained at a comparatively high level; SlPAL5 transcript expression was very low at the mature-green stage. SlPAL5 expression was significantly induced in response to NaCl, mannitol, and cold treatment; SlPAL5 expression decreased gradually after treatment with abscisic acid and H(2)O(2); SlPAL5 transcript decreased after exposure to methyl viologen for 3 h and increased after 6 h and maintained a stable expression level until 24 h, suggesting that the SlPAL5 gene may function in the response to abiotic stress.

  2. 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.

  3. A critical life-supporting role for cystathionine γ-lyase in the absence of dietary cysteine supply.

    Science.gov (United States)

    Mani, Sarathi; Yang, Guangdong; Wang, Rui

    2011-05-15

    This study examined the important relationship between cystathionine γ-lyase (CSE) functionality and cysteine supply for normal growth and life span. Mice with a targeted deletion of the CSE gene (CSE-KO) were fed a cysteine-limited diet and their growth and survival patterns as well as levels of cysteine, homocysteine, glutathione, and hydrogen sulfide (H2S) were measured. CSE-KO mice fed a cysteine-limited diet exhibited growth retardation; decreased levels of cysteine, glutathione, and H2S; and increased plasma homocysteine level. However, histological examinations of liver did not reveal any abnormality and plasma levels of aspartate aminotransferase, alanine aminotransferase, and albumin were normal in these animals. No CSE-KO mice survived after 12 weeks of feeding with the cysteine-limited diet. Supplementation of H2S to the CSE-KO mice failed to reverse the aforementioned abnormalities. On the other hand, supplementation of cysteine in the drinking water of the CSE-KO mice significantly increased plasma cysteine and glutathione levels. This eventually led to an increase in body weight and rescued the animals from death. In conclusion, CSE is critical for cysteine biosynthesis through the transsulfuration pathway and the combination of CSE deficiency and lack of dietary cysteine supply would threaten life sustainability.

  4. Biosynthesis of pseudoisoeugenols in tissue cultures of Pimpinella anisum. Phenylalanine ammonia lyase and cinnamic acid 4-hydroxylase activities.

    Science.gov (United States)

    Reichling, J; Kemmerer, B; Sauer-Gürth, H

    1995-07-28

    The genus Pimpinella contains pseudoisoeugenols, phenylpropanoids with a rare 2,5-dioxy substitution pattern on the phenyl ring. To study the biosynthesis of these compounds, we set up a leaf-differentiating tissue culture of Pimpinella anisum. These cultures mainly produce epoxy-pseudoisoeugenol-(2-methylbutyrate). To corroborate the biosynthetic pathway of epoxy-pseudoisoeugenol-(2-methylbutyrate) as proposed on the basis of investigations with 13C/14C-labelled precursors, the key steps of the pathway were investigated at an enzyme level. Experiments with cell-free homogenates clearly revealed that L-phenylalanine is converted to (E)-cinnamic acid by phenylalanine ammonia lyase and that (E)-cinnamic acid is converted to p-coumaric acid by cinnamic acid 4-hydroxylase. L-2-aminooxy-3-phenylpropionic acid, an analogue of L-phenylalanine, inhibited the incorporation of L-[3'-13C]phenylalanine into epoxy-pseudoisoeugenol-(2-methylbutyrate). Up to 2% of the precursor DL-[3'-13C]phenyllactate was incorporated into epoxy-pseudoisoeugenol-(2-methylbutyrate). Inhibition experiments with oxalacetic acid clearly showed that cinnamic acid is not formed by dehydration of phenyllactic acid in this leaf-differentiating tissue culture of P. anisum.

  5. Isolation of a novel alginate lyase-producing Bacillus litoralis strain and its potential to ferment Sargassum horneri for biofertilizer.

    Science.gov (United States)

    Wang, Mingpeng; Chen, Lei; Liu, Zhengyi; Zhang, Zhaojie; Qin, Song; Yan, Peisheng

    2016-12-01

    Algae have long been used to augment plant productivity through their beneficial effects. Alginate oligosaccharide is believed to be one of the important components to enhance growth and crop yield. In this study, we isolated and characterized a Bacillus litoralis strain, named Bacillus M3, from decayed kelps. We further demonstrated that the M3 strain could secrete alginate lyase to degrade alginate. The crude enzyme exhibited the highest activity (33.74 U/mg) at pH 7.0 and 50°C. The M3 strain was also able to ferment the brown alga Sargassum horneri. Fermentation results revealed that a fermentation period of 8-12 hr was the best harvest time with the highest level of alginate oligosaccharides. Plant growth assay showed that the seaweed fermentation extract had an obvious promotion effect on root and seedling growth of Lycopersicon eseulentum L. Our results suggest that fermentation extract of Sargassum horneri by the novel strain of Bacillus litoralis M3 has significant development potential for biofertilizer production and agriculture application. © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  6. Induction of L-phenylalanine ammonia-lyase during utilization of phenylalanine as a carbon or nitrogen source in Rhodotorula glutinis.

    OpenAIRE

    Marusich, W C; Jensen, R A; Zamir, L O

    1981-01-01

    Rhodotorula glutinis is a convenient source of L-phenylalanine ammonia-lyase, an enzyme that is useful as a biochemical reagent in the assay of L-phenylalanine. There have been previous descriptions of induced lyase production in complex medium where induction occurs late in exponential growth, suggesting a role in secondary metabolism such as is the case in higher plants. A higher specific activity of L-phenylalanine ammonia-lyase (sixfold higher than a complex medium) can be obtained during...

  7. Conservation endocrinology

    Science.gov (United States)

    McCormick, Stephen; Romero, Michael

    2017-01-01

    Endocrinologists can make significant contributions to conservation biology by helping to understand the mechanisms by which organisms cope with changing environments. Field endocrine techniques have advanced rapidly in recent years and can provide substantial information on the growth, stress, and reproductive status of individual animals, thereby providing insight into current and future responses of populations to changes in the environment. Environmental stressors and reproductive status can be detected nonlethally by measuring a number of endocrine-related endpoints, including steroids in plasma, living and nonliving tissue, urine, and feces. Information on the environmental or endocrine requirements of individual species for normal growth, development, and reproduction will provide critical information for species and ecosystem conservation. For many taxa, basic information on endocrinology is lacking, and advances in conservation endocrinology will require approaches that are both “basic” and “applied” and include integration of laboratory and field approaches.

  8. Molecular cloning, purification, and characterization of a novel polyMG-specific alginate lyase responsible for alginate MG block degradation in Stenotrophomas maltophilia KJ-2

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Su In; Kim, Hee Sook [Kyungsung Univ., Busan (Korea, Republic of). Dept. of Food Science and Biotechnology; Choi, Sung Hee; Lee, Eun Yeol [Kyung Hee Univ., Gyeonggi-do (Korea, Republic of). Dept. of Chemical Engineering

    2012-09-15

    A gene for a polyMG-specific alginate lyase possessing a novel structure was identified and cloned from Stenotrophomas maltophilia KJ-2 by using PCR with homologous nucleotide sequences-based primers. The recombinant alginate lyase consisting of 475 amino acids was purified on Ni-Sepharose column and exhibited the highest activity at pH 8 and 40 C. Interestingly, the recombinant alginate lyase was expected to have a similar catalytic active site of chondroitin B lyase but did not show chondroitin lyase activity. In the test of substrate specificity, the recombinant alginate lyase preferentially degraded the glycosidic bond of polyMG-block than polyM-block and polyG-block. The chemical structures of the degraded alginate oligosaccharides were elucidated to have mannuronate (M) at the reducing end on the basis of NMR analysis, supporting that KJ-2 polyMG-specific alginate lyase preferably degraded the glycosidic bond in M-G linkage than that in G-M linkage. The KJ-2 polyMG-specific alginate lyase can be used in combination with other alginate lyases for a synergistic saccharification of alginate. (orig.)

  9. Characterization of splice variants of the genes encoding human mitochondrial HMG-CoA lyase and HMG-CoA synthase, the main enzymes of the ketogenesis pathway.

    Science.gov (United States)

    Puisac, Beatriz; Ramos, Mónica; Arnedo, María; Menao, Sebastián; Gil-Rodríguez, María Concepción; Teresa-Rodrigo, María Esperanza; Pié, Angeles; de Karam, Juan Carlos; Wesselink, Jan-Jaap; Giménez, Ignacio; Ramos, Feliciano J; Casals, Nuria; Gómez-Puertas, Paulino; Hegardt, Fausto G; Pié, Juan

    2012-04-01

    The genes HMGCS2 and HMGCL encode the two main enzymes for ketone-body synthesis, mitochondrial HMG-CoA synthase and HMG-CoA lyase. Here, we identify and describe possible splice variants of these genes in human tissues. We detected an alternative transcript of HMGCS2 carrying a deletion of exon 4, and two alternative transcripts of HMGCL with deletions of exons 5 and 6, and exons 5, 6 and 7, respectively. All splice variants maintained the reading frame. However, Western blot studies and overexpression measurements in eukaryotic or prokaryotic cell models did not reveal HL or mHS protein variants. Both genes showed a similar distribution of the inactive variants in different tissues. Surprisingly, the highest percentages were found in tissues where almost no ketone bodies are synthesized: heart, skeletal muscle and brain. Our results suggest that alternative splicing might coordinately block the two main enzymes of ketogenesis in specific human tissues.

  10. Colorful Conservation

    Science.gov (United States)

    Skophammer, Karen

    2011-01-01

    Some people only think about conservation on Earth Day. Being in the "art business" however, this author is always conscious of the many products she thinks get wasted when they could be reused, recycled, and restored--especially in a school building and art room. In this article, she describes an art lesson that allows students to paint…

  11. [Conservation Units.

    Science.gov (United States)

    Texas Education Agency, Austin.

    Each of the six instructional units deals with one aspect of conservation: forests, water, rangeland, minerals (petroleum), and soil. The area of the elementary school curriculum with which each correlates is indicated. Lists of general and specific objectives are followed by suggested teaching procedures, including ideas for introducing the…

  12. [Conservation Units.

    Science.gov (United States)

    Texas Education Agency, Austin.

    Instructional units deal with each aspect of conservation: forests, wildlife, rangelands, water, minerals, and soil. The area of the secondary school curriculum with which each is correlated is indicated. Lists of general and specific objectives are followed by suggested teaching procedures, including ideas for introducing the topic, questions to…

  13. Cysteine S-conjugate β-lyases: important roles in the metabolism of naturally occurring sulfur and selenium-containing compounds, xenobiotics and anticancer agents.

    Science.gov (United States)

    Cooper, Arthur J L; Krasnikov, Boris F; Niatsetskaya, Zoya V; Pinto, John T; Callery, Patrick S; Villar, Maria T; Artigues, Antonio; Bruschi, Sam A

    2011-06-01

    Cysteine S-conjugate β-lyases are pyridoxal 5'-phosphate-containing enzymes that catalyze β-elimination reactions with cysteine S-conjugates that possess a good leaving group in the β-position. The end products are aminoacrylate and a sulfur-containing fragment. The aminoacrylate tautomerizes and hydrolyzes to pyruvate and ammonia. The mammalian cysteine S-conjugate β-lyases thus far identified are enzymes involved in amino acid metabolism that catalyze β-lyase reactions as non-physiological side reactions. Most are aminotransferases. In some cases the lyase is inactivated by reaction products. The cysteine S-conjugate β-lyases are of much interest to toxicologists because they play an important key role in the bioactivation (toxication) of halogenated alkenes, some of which are produced on an industrial scale and are environmental contaminants. The cysteine S-conjugate β-lyases have been reviewed in this journal previously (Cooper and Pinto in Amino Acids 30:1-15, 2006). Here, we focus on more recent findings regarding: (1) the identification of enzymes associated with high-M(r) cysteine S-conjugate β-lyases in the cytosolic and mitochondrial fractions of rat liver and kidney; (2) the mechanism of syncatalytic inactivation of rat liver mitochondrial aspartate aminotransferase by the nephrotoxic β-lyase substrate S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (the cysteine S-conjugate of tetrafluoroethylene); (3) toxicant channeling of reactive fragments from the active site of mitochondrial aspartate aminotransferase to susceptible proteins in the mitochondria; (4) the involvement of cysteine S-conjugate β-lyases in the metabolism/bioactivation of drugs and natural products; and (5) the role of cysteine S-conjugate β-lyases in the metabolism of selenocysteine Se-conjugates. This review emphasizes the fact that the cysteine S-conjugate β-lyases are biologically more important than hitherto appreciated.

  14. Ammonia lyases and aminomutases as biocatalysts for the synthesis of α-amino and β-amino acids.

    Science.gov (United States)

    Turner, Nicholas J

    2011-04-01

    Ammonia lyases catalyse the reversible addition of ammonia to cinnamic acid (1: R=H) and p-hydroxycinnamic (1: R=OH) to generate L-phenylalanine (2: R=H) and L-tyrosine (2: R=OH) respectively (Figure 1a). Both phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) are widely distributed in plants, fungi and prokaryotes. Recently there has been interest in the use of these enzymes for the synthesis of a broader range of L-arylalanines. Aminomutases catalyse a related reaction, namely the interconversion of α-amino acids to β-amino acids (Figure 1b). In the case of L-phenylalanine, this reaction is catalysed by phenylalanine aminomutase (PAM) and proceeds stereospecifically via the intermediate cinnamic acid to generate β-Phe 3. Ammonia lyases and aminomutases are related in sequence and structure and share the same active site cofactor 4-methylideneimidazole-5-one (MIO). There is currently interest in the possibility of using these biocatalysts to prepare a wide range of enantiomerically pure l-configured α-amino and β-amino acids. Recent reviews have focused on the mechanism of these MIO containing enzymes. The aim of this review is to review recent progress in the application of ammonia lyase and aminomutase enzymes to prepare enantiomerically pure α-amino and β-amino acids.

  15. Heron conservation

    Science.gov (United States)

    Kushlan, J.A.; Hafner, H.

    2000-01-01

    Herons are large, popular and, in many cases, spectacular birds found in wetlands world-wide, both tropical and temperate, natural and man-made. Some populations are very small and localized, some have decreased, some have expanded their ranges, and a few are pests of human activities. In the fifteen years since the publication of the latest monographic treatment of the family, The Herons Handbook, there has been a tremendous increase in our knowledge of heron status and conservation requirements, set against a backdrop of increasing concern about the future of the world?s wetland habitats. This book provides a comprehensive update following two distinct threads. The status and conservation needs of herons are first presented on a regional basis, in a series of chapters set at a continental or subcontinental scale. Over 200 biologists and heron conservationists have contributed to the data summarized here, and the very latest census and survey results provide the most up-to-date and detailed picture of heron populations currently available. Chapters discussing several critical issues in heron conservation follow, tending to focus on the international nature of the problems.

  16. Global alteration of the drug-binding pocket of human P-glycoprotein (ABCB1) by substitution of fifteen conserved residues reveals a negative correlation between substrate size and transport efficiency.

    Science.gov (United States)

    Vahedi, Shahrooz; Chufan, Eduardo E; Ambudkar, Suresh V

    2017-11-01

    P-glycoprotein (P-gp), an ATP-dependent efflux pump, is linked to the development of multidrug resistance in cancer cells. However, the drug-binding sites and translocation pathways of this transporter are not yet well-characterized. We recently demonstrated the important role of tyrosine residues in regulating P-gp ATP hydrolysis via hydrogen bond formations with high affinity modulators. Since tyrosine is both a hydrogen bond donor and acceptor, and non-covalent interactions are key in drug transport, in this study we investigated the global effect of enrichment of tyrosine residues in the drug-binding pocket on the drug binding and transport function of P-gp. By employing computational analysis, 15 conserved residues in the drug-binding pocket of human P-gp that interact with substrates were identified and then substituted with tyrosine, including 11 phenylalanine (F72, F303, F314, F336, F732, F759, F770, F938, F942, F983, F994), two leucine (L339, L975), one isoleucine (I306), and one methionine (M949). Characterization of the tyrosine-rich P-gp mutant in HeLa cells demonstrated that this major alteration in the drug-binding pocket by introducing fifteen additional tyrosine residues is well tolerated and has no measurable effect on total or cell surface expression of this mutant. Although the tyrosine-enriched mutant P-gp could transport small to moderate size (1000 Daltons) substrates such as NBD-cyclosporine A, Bodipy-paclitaxel and Bodipy-vinblastine was significantly decreased. This was further supported by the physico-chemical characterization of seventeen tested substrates, which revealed a negative correlation between drug transport and molecular size for the tyrosine-enriched P-gp mutant. Published by Elsevier Inc.

  17. Structure of the unique SEFIR domain from human interleukin 17 receptor A reveals a composite ligand-binding site containing a conserved α-helix for Act1 binding and IL-17 signaling

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Bing [Oklahoma State University, Stillwater, OK 74078 (United States); Liu, Caini; Qian, Wen [Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195 (United States); Han, Yue [Oklahoma State University, Stillwater, OK 74078 (United States); Li, Xiaoxia, E-mail: lix@ccf.org [Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195 (United States); Deng, Junpeng, E-mail: lix@ccf.org [Oklahoma State University, Stillwater, OK 74078 (United States)

    2014-05-01

    Crystal structure of the SEFIR domain from human IL-17 receptor A provides new insights into IL-17 signaling. Interleukin 17 (IL-17) cytokines play a crucial role in mediating inflammatory and autoimmune diseases. A unique intracellular signaling domain termed SEFIR is found within all IL-17 receptors (IL-17Rs) as well as the key adaptor protein Act1. SEFIR-mediated protein–protein interaction is a crucial step in IL-17 cytokine signaling. Here, the 2.3 Å resolution crystal structure of the SEFIR domain of IL-17RA, the most commonly shared receptor for IL-17 cytokine signaling, is reported. The structure includes the complete SEFIR domain and an additional α-helical C-terminal extension, which pack tightly together to form a compact unit. Structural comparison between the SEFIR domains of IL-17RA and IL-17RB reveals substantial differences in protein topology and folding. The uniquely long insertion between strand βC and helix αC in IL-17RA SEFIR is mostly well ordered, displaying a helix (αCC′{sub ins}) and a flexible loop (CC′). The DD′ loop in the IL-17RA SEFIR structure is much shorter; it rotates nearly 90° with respect to the counterpart in the IL-17RB SEFIR structure and shifts about 12 Å to accommodate the αCC′{sub ins} helix without forming any knots. Helix αC was identified as critical for its interaction with Act1 and IL-17-stimulated gene expression. The data suggest that the heterotypic SEFIR–SEFIR association via helix αC is a conserved and signature mechanism specific for IL-17 signaling. The structure also suggests that the downstream motif of IL-17RA SEFIR together with helix αC could provide a composite ligand-binding surface for recruiting Act1 during IL-17 signaling.

  18. Structural insights into catalysis by βC-S lyase from Streptococcus anginosus.

    Science.gov (United States)

    Kezuka, Yuichiro; Yoshida, Yasuo; Nonaka, Takamasa

    2012-10-01

    Hydrogen sulfide (H(2)S) is a causative agent of oral malodor and may play an important role in the pathogenicity of oral bacteria such as Streptococcus anginosus. In this microorganism, H(2)S production is associated with βC-S lyase (Lcd) encoded by lcd gene, which is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the α,β-elimination of sulfur-containing amino acids. When Lcd acts on L-cysteine, H(2)S is produced along with pyruvate and ammonia. To understand the H(2)S-producing mechanism of Lcd in detail, we determined the crystal structures of substrate-free Lcd (internal aldimine form) and two reaction intermediate complexes (external aldimine and α-aminoacrylate forms). The formation of intermediates induced little changes in the overall structure of the enzyme and in the active site residues, with the exception of Lys234, a PLP-binding residue. Structural and mutational analyses highlighted the importance of the active site residues Tyr60, Tyr119, and Arg365. In particular, Tyr119 forms a hydrogen bond with the side chain oxygen atom of L-serine, a substrate analog, in the external aldimine form suggesting its role in the recognition of the sulfur atom of the true substrate (L-cysteine). Tyr119 also plays a role in fixing the PLP cofactor at the proper position during catalysis through binding with its side chain. Finally, we partly modified the catalytic mechanism known for cystalysin, a βC-S lyase from Treponema denticola, and proposed an improved mechanism, which seems to be common to the βC-S lyases from oral bacteria.

  19. Strict reaction and substrate specificity of AGXT2L1, the human O-phosphoethanolamine phospho-lyase.

    Science.gov (United States)

    Schiroli, Davide; Cirrincione, Simona; Donini, Stefano; Peracchi, Alessio

    2013-07-01

    Dysregulated expression of the AGXT2L1 gene has been associated to neuropsychiatric disorders. Recently the gene product was shown to possess O-phosphoethanolamine phospho-lyase activity. We here analyze the specificity of AGXT2L1 in terms of both reaction and substrate. We show that the enzyme, despite having evolved from a transaminase ancestor, is at least 500-fold more active as a lyase than as an aminotransferase. Furthermore, the lyase reaction is very selective for O-phosphoethanolamine, strongly discriminating against closely related compounds, and we dissect the factors that contribute to such narrow substrate specificity. Overall, AGXT2L1 function appears to be rigidly confined to phospholipid metabolism, which is altered in neuropsychiatric disturbances. Copyright © 2013 International Union of Biochemistry and Molecular Biology, Inc.

  20. Molecular cloning and characterization of l-methionine γ-lyase from Streptomyces avermitilis.

    Science.gov (United States)

    Kudou, Daizou; Yasuda, Eri; Hirai, Yoshiyuki; Tamura, Takashi; Inagaki, Kenji

    2015-10-01

    A pyridoxal 5'-phosphate-dependent methionine γ-lyase (MGL) was cloned from Streptomyces avermitilis catalyzed the degradation of methionine to α-ketobutyrate, methanethiol, and ammonia. The sav7062 gene (1,242 bp) was corresponded to 413 amino acid residues with a molecular mass of 42,994 Da. The deduced amino acid sequence showed a high degree of similarity to those of other MGL enzymes. The sav7062 gene was overexpressed in Escherichia coli. The enzyme was purified to homogeneity and exhibited the MGL catalytic activities. We cloned the enzyme that has the MGL activity in Streptomyces for the first time.

  1. 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...... base is substituted with alanine (R279A). We discover that the three central subsites (- 1, + 1, and +2) have a profound preference for galacturonate but that the distal subsites call accommodate methylated galactUronate. h Is reasonable to assume therefore that pectate Iyase call cleave pectin...

  2. Effect of cysteine on the inactivation of cystathionine gamma-lyase by D,L-propargylglycine.

    OpenAIRE

    Awata,Shiro; Nakayama,Kazuko; SUZUKI, Isao; Kodama, Hiroyuki

    1989-01-01

    In vivo inactivation of cystathionine gamma-lyase by D,L-propargylglycine, a suicide inhibitor, was found to be less profound in rat kidney than in the liver. We investigated the cause of this difference using rat tissues. We fractionated kidney extract to characterize the substance which protected enzyme, and found that cysteine exhibits protecting action. Addition of 0.3 mM L-cysteine to the incubation mixture containing dialyzed kidney supernatant and 0.5 mM D,L-propargylglycine resulted i...

  3. Crystallization and preliminary X-ray analysis of pectin lyase A from Aspergillus niger.

    Science.gov (United States)

    Jenkins, J; Scott, M; Mayans, O; Pickersgill, R; Harris, G; Connerton, I; Gravesen, T

    1996-03-01

    The major secreted pectin lyase (E.C. 4.2.2.10) from Aspergillus niger, strain 4M-147, has been purified and crystallized by the hanging-drop method using polyethylene glycol as precipitant. The crystals belong to the space group P2(1)2(1)2(1) with cell dimensions a = 45.2, b = 83.2, c = 93.1 A (1 A = 0.1 nm) and a single molecule in the asymmetric unit. The crystals diffract to at least 2.0 A resolution and are suitable for structure determination.

  4. Bacterial Anabaena variabilis phenylalanine ammonia lyase: a biocatalyst with broad substrate specificity.

    Science.gov (United States)

    Lovelock, Sarah L; Turner, Nicholas J

    2014-10-15

    Phenylalanine ammonia lyases (PALs) catalyse the regio- and stereoselective hydroamination of cinnamic acid analogues to yield optically enriched α-amino acids. Herein, we demonstrate that a bacterial PAL from Anabaena variabilis (AvPAL) displays significantly higher activity towards a series of non-natural substrates than previously described eukaryotic PALs. Biotransformations performed on a preparative scale led to the synthesis of the 2-chloro- and 4-trifluoromethyl-phenylalanine derivatives in excellent ee, highlighting the enormous potential of bacterial PALs as biocatalysts for the synthesis of high value, non-natural amino acids.

  5. Enhancing Production of Alkaline Polygalacturonate Lyase from Bacillus subtilis by Fed-Batch Fermentation

    OpenAIRE

    Mouyong Zou; Fenfen Guo; Xuezhi Li; Jian Zhao; Yinbo Qu

    2014-01-01

    Alkaline polygalacturonate lyase (PGL, EC 4.2.2.2) is an enzyme used in many industries. We developed a fed-batch fermentation process that combines the enzymatic pretreatment of the carbon source with controlling the pH of the fermentative broth to enhance the PGL production from Bacillus subtilis 7-3-3 to decrease the production cost. Maintaining the fermentation broth at pH 6.5 prior to feeding with ammonia and at pH 6.0 after feeding significantly improved PGL activity (743.5 U mL-1) comp...

  6. One-step purification and characterization of alginate lyase from a clinical Pseudomonas aeruginosa with destructive activity on bacterial biofilm

    Directory of Open Access Journals (Sweden)

    Parinaz Ghadam

    2017-05-01

    Full Text Available Objective(s: Pseudomonas aeruginosais a Gram-negative and aerobic rod bacterium that displays mucoid and non-mucoid phenotype. Mucoid strains secrete alginate, which is the main agent of biofilms in chronic P. aeruginosa infections, show high resistance to antibiotics; consequently, the biological disruption of mucoid P. aeruginosa biofilms is an attractive area of study for researchers. Alginate lyase gene (algl is a member of alginate producing operon which by glycosidase activity produces primer for other enzymes in this cluster. Also this activity can destroy the extracellular alginate; therefore this enzyme participates in alginate production and destruction pathway. Alginate lyase causes detachment of a biofilm by reducing its adhesion to the surfaces, and increases phagocytosis and antibiotic susceptibility. In this study, alginate lyase was purified in just one step and its properties were investigated. Materials and Methods: The purification was done by affinity chromatography, analysed by SDS-PAGE, and its effect on P. aeruginosa biofilms was surveyed by micro titer plate assay and SEM. The substrate specificity of the enzyme was determined by PCR. Results: Alginate lyase from isolate 48 was purified in one step. It is more thermally resistant than alginate lyase from Pseudomonas aeruginosa PAO1 and poly M, poly G and poly MG alginate were the substrate of this enzyme. Moreover, it has an eradication effect on biofilms from P. aeruginosa 48 and PAO1. Conclusion: In this study an alginate lyase with many characteristics suitable in medicine such as thermal stability, effective on poly M alginate, and bacterial biofilm destructive was introduced and purified.

  7. TALE-induced bHLH transcription factors that activate a pectate lyase contribute to water soaking in bacterial spot of tomato

    Science.gov (United States)

    Schwartz, Allison R.; Morbitzer, Robert; Lahaye, Thomas; Staskawicz, Brian J.

    2017-01-01

    AvrHah1 [avirulence (avr) gene homologous to avrBs3 and hax2, no. 1] is a transcription activator-like (TAL) effector (TALE) in Xanthomonas gardneri that induces water-soaked disease lesions on fruits and leaves during bacterial spot of tomato. We observe that water from outside the leaf is drawn into the apoplast in X. gardneri-infected, but not X. gardneriΔavrHah1 (XgΔavrHah1)-infected, plants, conferring a dark, water-soaked appearance. The pull of water can facilitate entry of additional bacterial cells into the apoplast. Comparing the transcriptomes of tomato infected with X. gardneri vs. XgΔavrHah1 revealed the differential up-regulation of two basic helix–loop–helix (bHLH) transcription factors with predicted effector binding elements (EBEs) for AvrHah1. We mined our RNA-sequencing data for differentially up-regulated genes that could be direct targets of the bHLH transcription factors and therefore indirect targets of AvrHah1. We show that two pectin modification genes, a pectate lyase and pectinesterase, are targets of both bHLH transcription factors. Designer TALEs (dTALEs) for the bHLH transcription factors and the pectate lyase, but not for the pectinesterase, complement water soaking when delivered by XgΔavrHah1. By perturbing transcriptional networks and/or modifying the plant cell wall, AvrHah1 may promote water uptake to enhance tissue damage and eventual bacterial egression from the apoplast to the leaf surface. Understanding how disease symptoms develop may be a useful tool for improving the tolerance of crops from damaging disease lesions. PMID:28100489

  8. A Case of Dilated Cardiomyopathy Associated with 3-Hydroxy-3-Methylglutaryl-Coenzyme A (HMG CoA Lyase Deficiency

    Directory of Open Access Journals (Sweden)

    Alexander A. C. Leung

    2009-01-01

    Full Text Available 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA lyase deficiency is an inborn error of metabolism characterized by impairment of ketogenesis and leucine catabolism resulting in an organic acidopathy. In 1994, a case of dilated cardiomyopathy and fatal arrhythmia was reported in a 7-month-old infant. We report a case of dilated cardiomyopathy in association with HMG CoA lyase deficiency in a 23-year-old man with the acute presentation of heart failure. To our knowledge, this is the first case reported in an adult.

  9. Dysregulation of cystathionine γ-lyase (CSE)/hydrogen sulfide pathway contributes to ox-LDL-induced inflammation in macrophage.

    Science.gov (United States)

    Wang, Xian-Hui; Wang, Fen; You, Shou-Jiang; Cao, Yong-Jun; Cao, Li-Dan; Han, Qiao; Liu, Chun-Feng; Hu, Li-Fang

    2013-11-01

    Hydrogen sulfide (H2S), mainly produced by cystathionine γ-lyase (CSE) in vascular system, emerges as a novel gasotransmitter exerting anti-inflammatory and anti-atherosclerotic effects. Alterations of CSE/H2S pathway may thus be involved in atherosclerosis pathogenesis. However, the underlying mechanisms are poorly understood. The present study showed that the levels of CSE mRNA and protein expression, as well as H2S production were decreased in ox-LDL-treated macrophage. CSE overexpression reduced the ox-LDL-stimulated tumor necrosis factor-α (TNF-α) generation in Raw264.7 and primary macrophage while CSE knockdown enhanced it. Exogenous supplementation of H2S with NaHS and Na2S also decreased the production of TNF-α and intercellular adhesion molecule-1 (ICAM-1) in ox-LDL-stimulated macrophage, and alleviated the adhesion of macrophage to endothelial monolayer. Cysteine, a CSE preferential substrate for H2S biosynthesis, produced similar effects on the pro-inflammatory cytokine generation, which were reversed by CSE inhibitors PAG and BCA, respectively. Moreover, NaHS and Na2S attenuated the phosphorylation and degradation of IκBα and p65 nuclear translocation, as well as JNK activation caused by ox-LDL. The JNK inhibitor suppressed the NF-κB transcription activity in ox-LDL-treated cells. Furthermore, inhibitors of NF-κB (PDTC), ERK (U0126 and PD98059) and JNK (SP600125) partially blocked the suppression by ox-LDL on the CSE mRNA levels. Taken together, the findings demonstrate that ox-LDL may down-regulate the CSE/H2S pathway, which plays an anti-inflammatory role in ox-LDL-stimulated macrophage by suppressing JNK/NF-κB signaling. The study reveals new therapeutic strategies for atherosclerosis, based on modulating CSE/H2S pathway.

  10. Entropic origin of cobalt-carbon bond cleavage catalysis in adenosylcobalamin-dependent ethanolamine ammonia-lyase.

    Science.gov (United States)

    Wang, Miao; Warncke, Kurt

    2013-10-09

    Adenosylcobalamin-dependent enzymes accelerate the cleavage of the cobalt-carbon (Co-C) bond of the bound coenzyme by >10(10)-fold. The cleavage-generated 5'-deoxyadenosyl radical initiates the catalytic cycle by abstracting a hydrogen atom from substrate. Kinetic coupling of the Co-C bond cleavage and hydrogen-atom-transfer steps at ambient temperatures has interfered with past experimental attempts to directly address the factors that govern Co-C bond cleavage catalysis. Here, we use time-resolved, full-spectrum electron paramagnetic resonance spectroscopy, with temperature-step reaction initiation, starting from the enzyme-coenzyme-substrate ternary complex and (2)H-labeled substrate, to study radical pair generation in ethanolamine ammonia-lyase from Salmonella typhimurium at 234-248 K in a dimethylsulfoxide/water cryosolvent system. The monoexponential kinetics of formation of the (2)H- and (1)H-substituted substrate radicals are the same, indicating that Co-C bond cleavage rate-limits radical pair formation. Analysis of the kinetics by using a linear, three-state model allows extraction of the microscopic rate constant for Co-C bond cleavage. Eyring analysis reveals that the activation enthalpy for Co-C bond cleavage is 32 ± 1 kcal/mol, which is the same as for the cleavage reaction in solution. The origin of Co-C bond cleavage catalysis in the enzyme is, therefore, the large, favorable activation entropy of 61 ± 6 cal/(mol·K) (relative to 7 ± 1 cal/(mol·K) in solution). This represents a paradigm shift from traditional, enthalpy-based mechanisms that have been proposed for Co-C bond-breaking in B12 enzymes. The catalysis is proposed to arise from an increase in protein configurational entropy along the reaction coordinate.

  11. 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.

  12. Utilization of Glyphosate as Phosphate Source: Biochemistry and Genetics of Bacterial Carbon-Phosphorus Lyase

    Science.gov (United States)

    Zechel, David L.; Jochimsen, Bjarne

    2014-01-01

    SUMMARY After several decades of use of glyphosate, the active ingredient in weed killers such as Roundup, in fields, forests, and gardens, the biochemical pathway of transformation of glyphosate phosphorus to a useful phosphorus source for microorganisms has been disclosed. Glyphosate is a member 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 of the carbon-phosphorus lyase pathway, which involves reactions for activation of phosphonate, carbon-phosphorus bond cleavage, and further chemical transformation before a useful phosphate ion is generated in a series of seven or eight enzyme-catalyzed reactions. The phn genes, encoding the enzymes for this pathway, are widespread among bacterial species. The processes are described with emphasis on glyphosate as a substrate. Additionally, the catabolism of glyphosate is intimately connected with that of aminomethylphosphonate, which is also treated in this review. Results of physiological and genetic analyses are combined with those of bioinformatics analyses. PMID:24600043

  13. Characterization of pectin lyase produced by an endophytic strain isolated from coffee cherries.

    Science.gov (United States)

    Sakiyama, C C; Paula, E M; Pereira, P C; Borges, A C; Silva, D O

    2001-08-01

    The effect of endophytic bacterial activity on the quality of coffee beverage was studied. A survey of the micro-organisms in coffee cherries was performed before harvesting, and their growth on the main nutrients available in coffee cherries was determined in vitro. Many endophytic bacteria were isolated from surface-sterilized coffee cherries. One of the pectinolytic strains was physiologically and phenotypically characterized, and was tentatively identified by partial 16S rDNA sequencing as Paenibacillus amylolyticus. This endophytic strain produced an extracellular pectinase with maximal activity at 40 degrees C and pH 7.9, and was thermostable up to 45 degrees C. EDTA and metal ions had little effect on pectin lyase activity. Km and Vmax values were 4.6 mg ml(-1) and 94.0 10(-8) mol min(-1) ml(-1), respectively. Pectin lyases have been found in fungi but rarely in bacteria, and this isolate is a promising tool for regulation studies of these enzymes.

  14. Modulation of Central Carbon Metabolism by Acetylation of Isocitrate Lyase in Mycobacterium tuberculosis

    Science.gov (United States)

    Bi, Jing; Wang, Yihong; Yu, Heguo; Qian, Xiaoyan; Wang, Honghai; Liu, Jun; Zhang, Xuelian

    2017-01-01

    Several enzymes involved in central carbon metabolism such as isocitrate lyase and phosphoenolpyruvate carboxykinase are key determinants of pathogenesis of Mycobacterium tuberculosis (M. tb). In this study, we found that lysine acetylation plays an important role in the modulation of central carbon metabolism in M. tb. Mutant of M. tb defective in sirtuin deacetylase exhibited improved growth in fatty acid-containing media. Global analysis of lysine acetylome of M. tb identified three acetylated lysine residues (K322, K331, and K392) of isocitrate lyase (ICL1). Using a genetically encoding system, we demonstrated that acetylation of K392 increased the enzyme activity of ICL1, whereas acetylation of K322 decreased its activity. Antibodies that specifically recognized acetyllysine at 392 and 322 of ICL1 were used to monitor the levels of ICL1 acetylation in M. tb cultures. The physiological significance of ICL1 acetylation was demonstrated by the observation that M. tb altered the levels of acetylated K392 in response to changes of carbon sources, and that acetylation of K392 affected the abundance of ICL1 protein. Our study has uncovered another regulatory mechanism of ICL1. PMID:28322251

  15. Isolation and characterization of an Antarctic Flavobacterium strain with agarase and alginate lyase activities

    Directory of Open Access Journals (Sweden)

    Lavín Paris

    2016-09-01

    Full Text Available Several bacteria that are associated with macroalgae can use phycocolloids as a carbon source. Strain INACH002, isolated from decomposing Porphyra (Rhodophyta, in King George Island, Antarctica, was screened and characterized for the ability to produce agarase and alginate-lyase enzymatic activities. Our strain INACH002 was identified as a member of the genus Flavobacterium, closely related to Flavobacterium faecale, using 16S rRNA gene analysis. The INACH002 strain was characterized as psychrotrophic due to its optimal temperature (17ºC and maximum temperature (20°C of growth. Agarase and alginate-lyase displayed enzymatic activities within a range of 10°C to 50°C, with differences in the optimal temperature to hydrolyze agar (50°C, agarose (50°C and alginate (30°C during the first 30 min of activity. Strain Flavobacterium INACH002 is a promising Antarctic biotechnological resource; however, further research is required to illustrate the structural and functional bases of the enzymatic performance observed during the degradation of different substrates at different temperatures.

  16. [Characterization and properties of two dehydroquinate hydro-lyases in higher plants].

    Science.gov (United States)

    Boudet, A M; Lécussan, R; Boudet, A

    1975-01-01

    Two dehydroquinate hydro-lyases (E.C. 4.2.1.10) have been routinely separated from different organs of Zea mays L. by chromatography on Cellex-D Bio-Rad or hydroxypatite using linear salt gradients. Dehydroquinate hydro-lyase 1 is associated with shikimate: NADP(+) oxidoreductase (E.C. 1.1.1.25). DHQase 2 is a free constitutive enzyme; in this respect it differs from the inducible enzyme of microorganisms which appears only when dehydroquinate or quinate is the principal carbon source. DHQase 1 and DHQase 2 have a similar apparent Michaelis constant and pH optimum, but they differ in their molecular weight, thermal stability and sensitivity to metabolic effectors. DHQase 2 is specifically activated by shikimic acid. This strong activation and the channeling properties of the complex involved in the shikimate pathway can provide an effective means of control in the utilization of dehydroquinate between two different pathways. The significance of such a system involving both a specific regulation of isoenzymes and a molecular compartmentation by means of an enzymatic complex is discussed.

  17. 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.

  18. Characterization of C-S Lyase from C. diphtheriae: A Possible Target for New Antimicrobial Drugs

    Directory of Open Access Journals (Sweden)

    Alessandra Astegno

    2013-01-01

    Full Text Available The emergence of antibiotic resistance in microbial pathogens requires the identification of new antibacterial drugs. The biosynthesis of methionine is an attractive target because of its central importance in cellular metabolism. Moreover, most of the steps in methionine biosynthesis pathway are absent in mammals, lowering the probability of unwanted side effects. Herein, detailed biochemical characterization of one enzyme required for methionine biosynthesis, a pyridoxal-5′-phosphate (PLP- dependent C-S lyase from Corynebacterium diphtheriae, a pathogenic bacterium that causes diphtheria, has been performed. We overexpressed the protein in E. coli and analyzed substrate specificity, pH dependence of steady state kinetic parameters, and ligand-induced spectral transitions of the protein. Structural comparison of the enzyme with cystalysin from Treponema denticola indicates a similarity in overall folding. We used site-directed mutagenesis to highlight the importance of active site residues Tyr55, Tyr114, and Arg351, analyzing the effects of amino acid replacement on catalytic properties of enzyme. Better understanding of the active site of C. diphtheriae C-S lyase and the determinants of substrate and reaction specificity from this work will facilitate the design of novel inhibitors as antibacterial therapeutics.

  19. Purification and Characterization of a New Alginate Lyase from Marine Bacterium Vibrio sp. SY08.

    Science.gov (United States)

    Li, Shangyong; Wang, Linna; Hao, Jianhua; Xing, Mengxin; Sun, Jingjing; Sun, Mi

    2016-12-23

    Unsaturated alginate disaccharides (UADs), enzymatically derived from the degradation of alginate polymers, are considered powerful antioxidants. In this study, a new high UAD-producing alginate lyase, AlySY08, has been purified from the marine bacterium Vibrio sp. SY08. AlySY08, with a molecular weight of about 33 kDa and a specific activity of 1070.2 U/mg, showed the highest activity at 40 °C in phosphate buffer at pH 7.6. The enzyme was stable over a broad pH range (6.0-9.0) and retained about 75% activity after incubation at 40 °C for 2 h. Moreover, the enzyme was active in the absence of salt ions and its activity was enhanced by the addition of NaCl and KCl. AlySY08 resulted in an endo-type alginate lyase that degrades both polyM and polyG blocks, yielding UADs as the main product (81.4% of total products). All these features made AlySY08 a promising candidate for industrial applications in the production of antioxidants from alginate polysaccharides.

  20. The Saccharomyces cerevisiae ICL2 Gene Encodes a Mitochondrial 2-Methylisocitrate Lyase Involved in Propionyl-Coenzyme A Metabolism

    NARCIS (Netherlands)

    Luttik, Marijke A.H.; Kötter, Peter; Salomons, Florian A.; Klei, Ida J. van der; Dijken, Johannes P. van; Pronk, Jack T.

    2000-01-01

    The Saccharomyces cerevisiae ICL1 gene encodes isocitrate lyase, an essential enzyme for growth on ethanol and acetate. Previous studies have demonstrated that the highly homologous ICL2 gene (YPR006c) is transcribed during the growth of wild-type cells on ethanol. However, even when multiple copies

  1. 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

  2. Characterization of an extracellular biofunctional alginate lyase from marine Microbulbifer sp. ALW1 and antioxidant activity of enzymatic hydrolysates.

    Science.gov (United States)

    Zhu, Yanbing; Wu, Liyun; Chen, Yanhong; Ni, Hui; Xiao, Anfeng; Cai, Huinong

    2016-01-01

    A novel alginate-degrading marine bacterium Microbulbifer sp. ALW1 was isolated from rotten brown alga. An extracellular alginate lyase was purified to electrophoretic homogeneity and had a molecular mass of about 26.0 kDa determined by SDS-PAGE and size exclusion chromatography. This enzyme showed activities towards both polyguluronate and polymannuronate indicating its bifunctionality while with preference for the former substrate. Using sodium alginate as a substrate, strain ALW1 alginate lyase was optimally active at 45 °C and pH 7.0. It was stable at 25 °C, 30 °C, 35 °C and 40 °C, but not stable at 50 °C. This alginate lyase showed good stability over a broad pH range (5.0-9.0). The enzyme activity was increased to 5.1 times by adding NaCl to a final concentration of 0.5M. Strain ALW1 alginate lyase produced disaccharide (majority) and trisaccharide from alginate indicating that this enzyme could be a good tool for preparation of alginate oligosaccharides with low degree of polymerization (DP). The alginate oligosaccharides displayed the scavenging abilities towards radicals (DPPH, ABTS(+) and hydroxyl) and the reducing power. Therefore, the hydrolysates exhibited the antioxidant activity and had potential as a natural antioxidant.

  3. 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

  4. Crystallization and preliminary X-ray crystallographic studies of the ArsI C–As lyase from Thermomonospora curvata

    Energy Technology Data Exchange (ETDEWEB)

    Nadar, S. Venkadesh; Yoshinaga, Masafumi; Kandavelu, Palani; Sankaran, Banumathi; Rosen, Barry P., E-mail: brosen@fiu.edu

    2014-05-10

    The ArsI C-As lyase from Thermomonospora curvata was expressed, purified and crystallized. The crystals diffracted to 1.46 Å and belong to space group P4{sub 3}2{sub 1}2 or its enantiomer P4{sub 1}2{sub 1}2.

  5. 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 ...

  6. 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

  7. Purification and characterization of a novel alginate lyase from the marine bacterium Cobetia sp. NAP1 isolated from brown algae.

    Science.gov (United States)

    Yagi, Hisashi; Fujise, Asako; Itabashi, Narumi; Ohshiro, Takashi

    2016-12-01

    The application of marine resources, instead of fossil fuels, for biomass production is important for building a sustainable society. Seaweed is valuable as a source of marine biomass for producing biofuels such as ethanol, and can be used in various fields. Alginate is an anionic polysaccharide that forms the main component of brown algae. Various alginate lyases (e.g. exo- and endo-types and oligoalginate lyase) are generally used to degrade alginate. We herein describe a novel alginate lyase, AlgC-PL7, which belongs to the polysaccharide lyase 7 family. AlgC-PL7 was isolated from the halophilic Gram-negative bacterium Cobetia sp. NAP1 collected from the brown algae Padina arborescens Holmes. The optimal temperature and pH for AlgC-PL7 activity were 45 °C and 8, respectively. Additionally, AlgC-PL7 was thermostable and salt-tolerant, exhibited broad substrate specificity, and degraded alginate into monosaccharides. Therefore, AlgC-PL7 is a promising enzyme for the production of biofuels.

  8. 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.

  9. 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

  10. 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.

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

    Directory of Open Access Journals (Sweden)

    Danielle Biscaro Pedrolli

    2014-01-01

    Full Text Available 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 dependent on Ca2+. 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.

  12. Mutant form C115H of Clostridium sporogenes methionine γ-lyase efficiently cleaves S-Alk(en)yl-l-cysteine sulfoxides to antibacterial thiosulfinates.

    Science.gov (United States)

    Kulikova, Vitalia V; Anufrieva, Natalya V; Revtovich, Svetlana V; Chernov, Alexander S; Telegin, Georgii B; Morozova, Elena A; Demidkina, Tatyana V

    2016-10-01

    Pyridoxal 5'-phosphate-dependent methionine γ-lyase (MGL) catalyzes the β-elimination reaction of S-alk(en)yl-l-cysteine sulfoxides to thiosulfinates, which possess antimicrobial activity. Partial inactivation of the enzyme in the course of the reaction occurs due to oxidation of active site cysteine 115 conserved in bacterial MGLs. In this work, the C115H mutant form of Clostridium sporogenes MGL was prepared and the steady-state kinetic parameters of the enzyme were determined. The substitution results in an increase in the catalytic efficiency of the mutant form towards S-substituted l-cysteine sulfoxides compared to the wild type enzyme. We used a sulfoxide/enzyme system to generate antibacterial activity in situ. Two-component systems composed of the mutant enzyme and three S-substituted l-cysteine sulfoxides were demonstrated to be effective against Gram-positive and Gram-negative bacteria and three clinical isolates from mice. © 2016 IUBMB Life, 68(10):830-835, 2016.

  13. Cysteine sulfinate desulfinase, a NIFS-like protein of Escherichia coli with selenocysteine lyase and cysteine desulfurase activities. Gene cloning, purification, and characterization of a novel pyridoxal enzyme.

    Science.gov (United States)

    Mihara, H; Kurihara, T; Yoshimura, T; Soda, K; Esaki, N

    1997-09-05

    Selenocysteine lyase (EC 4.4.1.16) exclusively decomposes selenocysteine to alanine and elemental selenium, whereas cysteine desulfurase (NIFS protein) of Azotobacter vinelandii acts indiscriminately on both cysteine and selenocysteine to produce elemental sulfur and selenium respectively, and alanine. These proteins exhibit some sequence homology. The Escherichia coli genome contains three genes with sequence homology to nifS. We have cloned the gene mapped at 63.4 min in the chromosome and have expressed, purified to homogeneity, and characterized the gene product. The enzyme comprises two identical subunits with 401 amino acid residues (Mr 43,238) and contains pyridoxal 5'-phosphate as a coenzyme. The enzyme catalyzes the removal of elemental sulfur and selenium atoms from L-cysteine, L-cystine, L-selenocysteine, and L-selenocystine to produce L-alanine. Because L-cysteine sulfinic acid was desulfinated to form L-alanine as the preferred substrate, we have named this new enzyme cysteine sulfinate desulfinase. Mutant enzymes having alanine substituted for each of the four cysteinyl residues (Cys-100, Cys-176, Cys-323, and Cys-358) were all active. Cys-358 corresponds to Cys-325 of A. vinelandii NIFS, which is conserved among all NIFS-like proteins and catalytically essential (Zheng, L., White, R. H., Cash, V. L., and Dean, D. R. (1994) Biochemistry 33, 4714-4720), is not required for cysteine sulfinate desulfinase. Thus, the enzyme is distinct from A. vinelandii NIFS in this respect.

  14. Shewanella haliotis BP-1海藻酸裂解酶基因的克隆表达%Gene Cloning and Expression of Alginate Lyase from Shewanella haliotis BP-1

    Institute of Scientific and Technical Information of China (English)

    黄桂媛; 温顺华; 李锋; 卢明倩; 王巧贞; 廖威; 黄庶识

    2016-01-01

    Objective]Alginate lyase in Shewanella haliotis BP-1 strains was studied illustrate its biological activity of degrading alginate.[Methods]The gene cloning technology and the Escherichia coli heterologous expression technology were applied to overexpress the alginate lyase;And the enzyme activity was analyzed after the crude enzyme was separated and purified by DEAE Sepharose FF chromatogra-phy.[Results]The alginate lyase gene Alg 1 7S , with a size of 2 1 5 7 bp,was cloned from S. haliotis BP-1 strain genomic DNA and encoded an alginate lyase Alg17S,which belonged to pol-ysaccharide lyase(PL)1 7 family and had a size of 79 726 Da protein(including an N-terminal signal peptide of 26 amino acid signal peptide).Alg17S showed high sequence identity of 5 2% with PL-17 protein sequence Alg17C from Saccharophagus degradans 2-40.Both the purified recombi-nase Alg17S and the △snAlg17S(without the N-terminal signal peptide of 26 amino acids)can degrade alginate,but the enzymatic activity of △snAlg17S revealed a specific activity of 9 635 U/mg,which was more efficient than Alg17S.[Conclusion]The recombinant alginate lyase △s-nAlg17S that has both high-level expression and high enzymatic activity could be a potential en-zyme for further researching on the alginate saccharification and the biofuels production.%【目的】了解海洋细菌Shewanella haliotis B P-1中海藻酸裂解酶降解海藻酸钠的生物活性。【方法】应用基因克隆和大肠杆菌异源表达技术,过量表达海藻酸裂解酶,将粗酶液通过 DEAE Sepharose FF柱分离纯化后检测其酶活性。【结果】从S.haliotis BP-1菌株的基因组DNA中克隆得到一个大小为2157 bp的海藻酸裂解酶基因Alg17S ,该基因编码的海藻酸裂解酶 Alg17S属于PL17家族的蛋白,大小为79726 Da,其中包括N端26个氨基酸的信号肽,与Saccharophagus degradans 2-40菌株产生的海藻酸裂解酶 Alg17C 具有高度同源性,相似性为52%。

  15. 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.

  16. 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.

  17. Rapid induction of the synthesis of phenylalanine ammonia-lyase and of chalcone synthase in elicitor-treated plant cells.

    Science.gov (United States)

    Lawton, M A; Dixon, R A; Hahlbrock, K; Lamb, C

    1983-01-01

    Changes in the rate of synthesis of phenylalanine ammonia-lyase and chalcone synthase, two characteristic enzymes of phenylpropanoid biosynthesis, have been investigated by direct immunoprecipitation of in vivo [35S]methionine-labelled enzyme subunits in elicitor-treated cells of dwarf French bean (Phaseolus vulgaris). Elicitor, heat-released from cell walls of Colletotrichum lindemuthianum, the causal agent of anthracnose disease of bean, causes marked but transient increases in the rates of synthesis of both enzymes concomitant with the phase of rapid increase in enzyme activity at the onset of phaseollin accumulation during the phytoalexin defence response. Increased rates of synthesis of both enzymes can be observed 20 min after elicitor treatment and the pattern of induction of synthesis of phenylalanine ammonia-lyase and chalcone synthase are broadly similar with respect to elicitor concentration and time, maximum rates of synthesis being attained between 2.5 h and 3.0 h after elicitor treatment. Within this overall co-ordination small but distinct differences between the enzymes were observed in: (a) the elicitor concentrations giving maximum enzyme synthesis, and (b) the precise timing of maximum enzyme synthesis, with that for chalcone synthase occurring 20-30 min earlier than that for phenylalanine ammonia-lyase. However, for a given rate of enzyme synthesis, induction of the activities of phenylalanine ammonia-lyase and chalcone synthase is more efficient at high elicitor concentrations. This may reflect the operation under certain circumstances of post-translational control of the activity levels of these enzymes as implicated for phenylalanine ammonia-lyase by previous density-labelling experiments [Lawton et al. (1980) Biochim. Biophys. Acta, 633, 162-175]. The same pattern of induction of enzyme synthesis is observed with elicitor preparations from a variety of sources.

  18. 草鱼腺苷酸基琥珀酸裂解酶克隆及序列分析%MOLECULAR CLONING AND CHARACTERIZATION OF ADENYLOSUCCINATE LYASE cDNA AND GENOMIC DNA IN GRASS CARP

    Institute of Scientific and Technical Information of China (English)

    屈刚; 顾继锐; 辜文博; 朱文漓; 吴江; 刘汉元; 徐恒

    2009-01-01

    ) comprising 482 codons preceded by a 5'-untranslated region of 63 nucleotides and followed by 3'-untranslated region of 75 nucleotides. The typical polyadenylation signal AATA-AA is found 13bp upstream of the polyA tail. The predicted mass of encoded protein is 54,552 Da with a calculated iso-electric point of 6.72 and-4.11 charges at pH 7. The ADSL araino acid sequence of grass carp has classical "signature" sequence of enzymes that catalyzing β-elimination reactions and two conservative histidine residues as general acid-base catalysis active site. The deduced amino acid sequence shares high homology with other five vertebrates ADSL and has 94.6% similarity with Danio rerio, 78.5% with Xenopus laevis, 70.8% with Gallus gallus, 76.2% with Mus musculus and 76.0% with Homo sapiens. To obtain the grass carp ADSL genomic DNA, we design four pairs primers based on the full length cDNA of ADSL and result 8557bp ADSL genomic DNA of grass carp which encompass 13 exons and 12 in-trons. The splice sites are well conserved through evolution, and observe the regulation of GT-AG except for the ninth in-tron whose 5' site was GC. The alignment of five vertebrates ADSL genomic DNA sequence indicate that they have the same numbers of intron and exon, and the number of base pairs is identical in 2-12 exons. This reveals that the structure of ADSL gene is well conserved through evolution in exon.

  19. Mini-review: recent developments in hydroxynitrile lyases for industrial biotechnology.

    Science.gov (United States)

    Lanfranchi, Elisa; Steiner, Kerstin; Glieder, Anton; Hajnal, Ivan; Sheldon, Roger A; van Pelt, Sander; Winkler, Margit

    2013-12-01

    Hydroxynitrile lyases (HNLs) catalyze the cleavage as well as the formation of cyanohydrins. The latter reaction is valuable for the stereoselective C-C bond formation by condensation of HCN with carbonyl compounds. The resulting cyanohydrins serve as versatile building blocks for a broad range of chemical and enzymatic follow-up reactions. A significant number of (R)- and (S)-selective HNLs are known today and the number is still increasing. HNLs not only exhibit varying substrate scope but also differ in sequence and structure. Tailor-made enzymes for large-scale manufacturing of cyanohydrins with improved yield and enantiomeric excess are very interesting targets, which is reflected in a solid number of patents. This review will complement and extend our recent review with a strong focus on applications of HNLs for the synthesis of highly functionalized, chiral compounds with newest literature, recent and current patent literature.

  20. Watermelon (Citrullus lanatus) hydroperoxide lyase greatly increases C6 aldehyde formation in transgenic leaves.

    Science.gov (United States)

    Fukushige, Hirotada; Hildebrand, David F

    2005-03-23

    Fatty acid hydroperoxide lyase (HL) is the key enzyme for the production of the "green note"compounds, leaf aldehyde [(2E)-hexenal] and leaf alcohol [(3Z)-hexenol], in plant tissues. A cDNA encoding HL was cloned from leaves of watermelon (Citrullus lanatus) and expressed in Nicotiana tabacum. The enzyme is 3 times more active with 13-hydroperoxylinolenic acid than with 13-hydroperoxylinoleic acid. The activity against 9-hydroperoxides of polyunsaturated fatty acids is minimal. Enzyme activity of the watermelon HL in the transgenic leaves was approximately 50 times higher than endogenous HL activity in the wild-type N. tabacum plants. When compared with Arabidopsis HL also expressed in N. tabacum, the highest HL activity is 10 times higher in watermelon HL overexpressing leaves than in Arabidopsis HL overexpressers.

  1. 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

    With the availability of the genome sequence of the filamentous fungus Aspergillus niger, the use of targeted genetic modifications has become feasible. This, together with the fact that A. niger is well established industrially, makes this fungus an attractive micro-organism for creating a cell...... 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...... the acl gene. Additionally, the total amount of organic acids produced in the deletion strain was significantly increased. Genome-scale stoichiometric metabolic model predictions can be used for identifying gene targets. Deletion of the acl led to increased succinic acid production by A. niger....

  2. Probing reversible chemistry in coenzyme B12 -dependent ethanolamine ammonia lyase with kinetic isotope effects.

    Science.gov (United States)

    Jones, Alex R; Rentergent, Julius; Scrutton, Nigel S; Hay, Sam

    2015-06-08

    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 during single-turnover stopped-flow measurements. These data are interpreted within the context of a kinetic model where the 5'-deoxyadenosyl radical intermediate may be quasi-stable and rearrangement of the substrate radical is essentially irreversible. Global fitting of these data allows estimation of the intrinsic rate constants associated with CoC homolysis and initial H-abstraction steps. In contrast to previous stopped-flow studies, the apparent kinetic isotope effects are found to be relatively small.

  3. Expression in E. coli of the gene encoding phenylalanine ammonia-lyase from Rhodosporidium toruloides.

    Science.gov (United States)

    Orum, H; Rasmussen, O F

    1992-03-01

    The active sites of the enzyme phenylalanine ammonia-lyase (Pal) from Rhodosporidium toruloides contains a dehydroalanine residue that is believed to be essential for catalytic activity. Furthermore, the dehydroalanine is believed to be added post-translationally as part of a prosthetic group covalently attached to the enzyme. Perhaps for this reason no attempts to produce Pal in foreign host cells have been reported. We have inserted the entire uninterupted pal gene from R. toruloides into the Escherichia coli expression vector pKK 223-3. E. coli cells containing this vector synthesize a protein of the expected size, and extracts prepared from these cells contain a Pal-like activity. The potential implications of this finding are discussed.

  4. Paraffin as oxygen vector modulates tyrosine phenol lyase production by Citrobacter freundii MTCC 2424.

    Science.gov (United States)

    Azmi, Wamik; Kumar, Ajay; Dev, Varun

    2013-06-01

    The efficiency of three oxygen-vectors liquid paraffin, silicone oil and n-dodecane in the production of tyrosine phenol lyase (TPL) by Citrobacter freundii MTCC 2424 was evaluated at 4% (v/v) concentration. The liquid paraffin as oxygenvectors was found to exhibit a stimulatory effect on TPL synthesis. The liquid paraffin at 6% (v/v) resulted in 34% increase in the TPL synthesis accompanied by a 13% increase in the production of cell mass at a 10 L scale. This improvement in TPL and cell mass production in the presence of liquid paraffin can be related to the fact that liquid paraffin was capable of maintaining dissolved O2 concentration above 28% throughout the course of the fermentation. Maintenance of the dissolved O2 concentration above 28% could be viewed in terms of an adequate oxygen supply to the rapidly dividing cells of the bacterium, which in turn resulted in enhanced synthesis of TPL and cell mass.

  5. Protein packing interactions and polymorphy of chorismate lyase from E. Coli

    Science.gov (United States)

    Gallagher, Travis

    2001-11-01

    The enzyme chorismate lyase from E. coli crystallizes into three well characterized polymorphs in identical conditions. The Wild-type enzyme tends to aggregate, even in the presence of a reducing agent, and yields monoclinic crystals that grow in intricate clusters. Protein aggregation was largely eliminated by mutating the protein's two cysteines to serines. The double mutant retains full enzymatic activity and grows singly in two new forms: triclinic and orthorhombic. The triclinic crystals diffract to 0.9 Å resolution. A single-cysteine mutant that crystallizes in the orthorhombic form was used to determine the structure, enabling examination of the packing interactions at 2.0 Å resolution or better in all three forms. A novel system for labeling contacts is proposed, and relations between packing patterns and crystal properties are discussed. Diffraction resolution is found to correlate with coordination number and with the root-mean-square deviation from mean extent of the contacts. Implications for contact energies are considered.

  6. 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.

  7. Enhancing Production of Alkaline Polygalacturonate Lyase from Bacillus subtilis by Fed-Batch Fermentation

    Science.gov (United States)

    Zou, Mouyong; Guo, Fenfen; Li, Xuezhi; Zhao, Jian; Qu, Yinbo

    2014-01-01

    Alkaline polygalacturonate lyase (PGL, EC 4.2.2.2) is an enzyme used in many industries. We developed a fed-batch fermentation process that combines the enzymatic pretreatment of the carbon source with controlling the pH of the fermentative broth to enhance the PGL production from Bacillus subtilis 7-3-3 to decrease the production cost. Maintaining the fermentation broth at pH 6.5 prior to feeding with ammonia and at pH 6.0 after feeding significantly improved PGL activity (743.5 U mL−1) compared with the control (202.5 U mL−1). The average PGL productivity reached 19.6 U mL−1 h−1 after 38 h of fermentation. The crude PGL was suitable for environmentally friendly ramie enzymatic degumming. PMID:24603713

  8. Enzyme discovery beyond homology: a unique hydroxynitrile lyase in the Bet v1 superfamily

    Science.gov (United States)

    Lanfranchi, Elisa; Pavkov-Keller, Tea; Koehler, Eva-Maria; Diepold, Matthias; Steiner, Kerstin; Darnhofer, Barbara; Hartler, Jürgen; van den Bergh, Tom; Joosten, Henk-Jan; Gruber-Khadjawi, Mandana; Thallinger, Gerhard G.; Birner-Gruenberger, Ruth; Gruber, Karl; Winkler, Margit; Glieder, Anton

    2017-05-01

    Homology and similarity based approaches are most widely used for the identification of new enzymes for biocatalysis. However, they are not suitable to find truly novel scaffolds with a desired function and this averts options and diversity. Hydroxynitrile lyases (HNLs) are an example of non-homologous isofunctional enzymes for the synthesis of chiral cyanohydrins. Due to their convergent evolution, finding new representatives is challenging. Here we show the discovery of unique HNL enzymes from the fern Davallia tyermannii by coalescence of transcriptomics, proteomics and enzymatic screening. It is the first protein with a Bet v1-like protein fold exhibiting HNL activity, and has a new catalytic center, as shown by protein crystallography. Biochemical properties of D. tyermannii HNLs open perspectives for the development of a complementary class of biocatalysts for the stereoselective synthesis of cyanohydrins. This work shows that systematic integration of -omics data facilitates discovery of enzymes with unpredictable sequences and helps to extend our knowledge about enzyme diversity.

  9. Phenylalanine ammonia lyase catalyzed synthesis of amino acids by an MIO-cofactor independent pathway.

    Science.gov (United States)

    Lovelock, Sarah L; Lloyd, Richard C; Turner, Nicholas J

    2014-04-25

    Phenylalanine ammonia lyases (PALs) belong to a family of 4-methylideneimidazole-5-one (MIO) cofactor dependent enzymes which are responsible for the conversion of L-phenylalanine into trans-cinnamic acid in eukaryotic and prokaryotic organisms. Under conditions of high ammonia concentration, this deamination reaction is reversible and hence there is considerable interest in the development of PALs as biocatalysts for the enantioselective synthesis of non-natural amino acids. Herein the discovery of a previously unobserved competing MIO-independent reaction pathway, which proceeds in a non-stereoselective manner and results in the generation of both L- and D-phenylalanine derivatives, is described. The mechanism of the MIO-independent pathway is explored through isotopic-labeling studies and mutagenesis of key active-site residues. The results obtained are consistent with amino acid deamination occurring by a stepwise E1 cB elimination mechanism.

  10. Synthesis of d- and l-Phenylalanine Derivatives by Phenylalanine Ammonia Lyases: A Multienzymatic Cascade Process**

    Science.gov (United States)

    Parmeggiani, Fabio; Lovelock, Sarah L; Weise, Nicholas J; Ahmed, Syed T; Turner, Nicholas J

    2015-01-01

    The synthesis of substituted d-phenylalanines in high yield and excellent optical purity, starting from inexpensive cinnamic acids, has been achieved with a novel one-pot approach by coupling phenylalanine ammonia lyase (PAL) amination with a chemoenzymatic deracemization (based on stereoselective oxidation and nonselective reduction). A simple high-throughput solid-phase screening method has also been developed to identify PALs with higher rates of formation of non-natural d-phenylalanines. The best variants were exploited in the chemoenzymatic cascade, thus increasing the yield and ee value of the d-configured product. Furthermore, the system was extended to the preparation of those l-phenylalanines which are obtained with a low ee value using PAL amination. PMID:25728350

  11. Synthesis of d‐ and l‐Phenylalanine Derivatives by Phenylalanine Ammonia Lyases: A Multienzymatic Cascade Process†

    Science.gov (United States)

    Parmeggiani, Fabio; Lovelock, Sarah L.; Weise, Nicholas J.; Ahmed, Syed T.

    2015-01-01

    Abstract The synthesis of substituted d‐phenylalanines in high yield and excellent optical purity, starting from inexpensive cinnamic acids, has been achieved with a novel one‐pot approach by coupling phenylalanine ammonia lyase (PAL) amination with a chemoenzymatic deracemization (based on stereoselective oxidation and nonselective reduction). A simple high‐throughput solid‐phase screening method has also been developed to identify PALs with higher rates of formation of non‐natural d‐phenylalanines. The best variants were exploited in the chemoenzymatic cascade, thus increasing the yield and ee value of the d‐configured product. Furthermore, the system was extended to the preparation of those l‐phenylalanines which are obtained with a low ee value using PAL amination. PMID:27478261

  12. Synthesis of d- and l-Phenylalanine Derivatives by Phenylalanine Ammonia Lyases: A Multienzymatic Cascade Process.

    Science.gov (United States)

    Parmeggiani, Fabio; Lovelock, Sarah L; Weise, Nicholas J; Ahmed, Syed T; Turner, Nicholas J

    2015-04-07

    The synthesis of substituted d-phenylalanines in high yield and excellent optical purity, starting from inexpensive cinnamic acids, has been achieved with a novel one-pot approach by coupling phenylalanine ammonia lyase (PAL) amination with a chemoenzymatic deracemization (based on stereoselective oxidation and nonselective reduction). A simple high-throughput solid-phase screening method has also been developed to identify PALs with higher rates of formation of non-natural d-phenylalanines. The best variants were exploited in the chemoenzymatic cascade, thus increasing the yield and ee value of the d-configured product. Furthermore, the system was extended to the preparation of those l-phenylalanines which are obtained with a low ee value using PAL amination.

  13. Suvanine Sesterterpenes from a Tropical Sponge Coscinoderma sp. Inhibit Isocitrate Lyase in the Glyoxylate Cycle

    Directory of Open Access Journals (Sweden)

    So-Hyoung Lee

    2014-10-01

    Full Text Available The glyoxylate cycle is a sequence of anaplerotic reactions catalyzed by the key enzymes isocitrate lyase (ICL and malate synthase (MLS. Mutants of Candida albicans lacking ICL are markedly less virulent in mice than the wild-type. Suvanine sesterterpenes (1−9 isolated from a tropical sponge Coscinoderma sp. were evaluated for their inhibitory activities toward recombinant ICL from C. albicans. These studies led to the identification of a potent ICL inhibitor, suvanine salt (2, which possesses a sodium counterion and displays an inhibitory concentration value (IC50 of 6.35 μM. The growth phenotype of ICL deletion mutants and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR analyses indicated that compound 2 inhibits the ICL mRNA expression in C. albicans under C2-carbon-utilizing conditions. The present data highlight the potential for suvanine sesterterpenes treatment of C. albicans infections via inhibition of ICL activity.

  14. Synthesis of D- and L-phenylalanine derivatives by phenylalanine ammonia lyases: a multienzymatic cascade process.

    Science.gov (United States)

    Parmeggiani, Fabio; Lovelock, Sarah L; Weise, Nicholas J; Ahmed, Syed T; Turner, Nicholas J

    2015-04-01

    The synthesis of substituted D-phenylalanines in high yield and excellent optical purity, starting from inexpensive cinnamic acids, has been achieved with a novel one-pot approach by coupling phenylalanine ammonia lyase (PAL) amination with a chemoenzymatic deracemization (based on stereoselective oxidation and nonselective reduction). A simple high-throughput solid-phase screening method has also been developed to identify PALs with higher rates of formation of non-natural D-phenylalanines. The best variants were exploited in the chemoenzymatic cascade, thus increasing the yield and ee value of the D-configured product. Furthermore, the system was extended to the preparation of those L-phenylalanines which are obtained with a low ee value using PAL amination.

  15. Preparation of bioimprinting cross-linked enzyme aggregates of phenylalanine ammonia lyase and it's partial properties

    Directory of Open Access Journals (Sweden)

    Jiandong CUI

    2015-12-01

    Full Text Available Phenylalanine ammonia lyase (PAL is a key enzyme for production of L-phenylalanine. Currently, PAL is mainly obtained from Rhodotorula PAL However, Rhodotorula PAL exhibits poor stability, which limits its industrial application. In this study, bioimprinting cross-linked enzyme aggregates of PAL (PAL-iCLEAs is developed by combining cross-linked enzyme aggregates technology and imprinted enzyme method. The most optimal imprinting molecule substrate is screened. Moreover, some characteristics of the PAL-iCLEAs are examined. The results show that the most suitable substrates for preparing PAL-iCLEAs is tran-cinnamic acid. The optimal temperature and pH was 50 ℃ and 10.5, respectively. In addition, PAL-iCLEAs shows good reusability, the recovery of PAL activity still remained 32% after reusing 9 times.

  16. Relationship between cystathionine γ-lyase gene polymorphism and essential hypertension in Northern Chinese Han population

    Institute of Scientific and Technical Information of China (English)

    LI Yun; ZHAO Qi; LIU Xiao-li; WANG Lai-yuan; LU Xiang-feng; LI Hong-fang; CHEN Shu-feng; HUANG Jian-feng; GU Dong-feng

    2008-01-01

    Background Hydrogen sulfide(H2S)plays an important role in the smooth muscle cell relaxation and thereby participates in the development of hypertension. Cystathionine γ-lyase is the key enzyme in the endogenous production of H2S. Up to now, the reports on the relationship between the polymorphisms of cystathionine γ-lyase gene (CTH) and essential hypertension(EH)are limited. This study was designed to assess their underlying relationship. Methods A total of 503 hypertensive patients and 490 age-, gender-and area-matched normotensive controls were enrolled in this study. Based on the FASTSNP, a web server to identify putative functional single nucleotide polymorphisms (SNPs) of genes, we selected two SNPs, rs482843 and rs1021737, in the CTH gene for genotyping. Genotyping was performed by the polymerase chain reaction and restriction fragment length polymorphism method (PCR-RFLP). The frequencies of the alleles and genotypes between cases and controls were compared by the chi-square test. The program Haplo. stats was used to investigate the relationship between the haplotypes and EH. Results These two SNPs were in Hardy-Weinberg Equilibrium in both cases and controls. The genotype distribution and allele frequencies of them did not significantly differ between cases and controls(all P>0.05). In the stepwise logistic regression analysis we failed to observe their association with hypertension. In addition, none of the four estimated haplotypes or diplotypes significantly increased or decreased the risk of hypertension before or after adjustment for several known risk factors. Conclusions The present study suggests that the SNPs rs482843 and rs1021737 of the CTH gene were not associated with essential hypertension in the Northern Chinese Han population. However, replications in other populations and further functional studies are still necessary to clarify the role of the CTH gene in the pathogenesis of EH.

  17. A C⋅As lyase for degradation of environmental organoarsenical herbicides and animal husbandry growth promoters

    Science.gov (United States)

    Yoshinaga, Masafumi; Rosen, Barry P.

    2014-01-01

    Arsenic is the most widespread environmental toxin. Substantial amounts of pentavalent organoarsenicals have been used as herbicides, such as monosodium methylarsonic acid (MSMA), and as growth enhancers for animal husbandry, such as roxarsone (4-hydroxy-3-nitrophenylarsonic acid) [Rox(V)]. These undergo environmental degradation to more toxic inorganic arsenite [As(III)]. We previously demonstrated a two-step pathway of degradation of MSMA to As(III) by microbial communities involving sequential reduction to methylarsonous acid [MAs(III)] by one bacterial species and demethylation from MAs(III) to As(III) by another. In this study, the gene responsible for MAs(III) demethylation was identified from an environmental MAs(III)-demethylating isolate, Bacillus sp. MD1. This gene, termed arsenic inducible gene (arsI), is in an arsenic resistance (ars) operon and encodes a nonheme iron-dependent dioxygenase with C⋅As lyase activity. Heterologous expression of ArsI conferred MAs(III)-demethylating activity and MAs(III) resistance to an arsenic-hypersensitive strain of Escherichia coli, demonstrating that MAs(III) demethylation is a detoxification process. Purified ArsI catalyzes Fe2+-dependent MAs(III) demethylation. In addition, ArsI cleaves the C⋅As bond in trivalent roxarsone and other aromatic arsenicals. ArsI homologs are widely distributed in prokaryotes, and we propose that ArsI-catalyzed organoarsenical degradation has a significant impact on the arsenic biogeocycle. To our knowledge, this is the first report of a molecular mechanism for organoarsenic degradation by a C⋅As lyase. PMID:24821808

  18. A C⋅As lyase for degradation of environmental organoarsenical herbicides and animal husbandry growth promoters.

    Science.gov (United States)

    Yoshinaga, Masafumi; Rosen, Barry P

    2014-05-27

    Arsenic is the most widespread environmental toxin. Substantial amounts of pentavalent organoarsenicals have been used as herbicides, such as monosodium methylarsonic acid (MSMA), and as growth enhancers for animal husbandry, such as roxarsone (4-hydroxy-3-nitrophenylarsonic acid) [Rox(V)]. These undergo environmental degradation to more toxic inorganic arsenite [As(III)]. We previously demonstrated a two-step pathway of degradation of MSMA to As(III) by microbial communities involving sequential reduction to methylarsonous acid [MAs(III)] by one bacterial species and demethylation from MAs(III) to As(III) by another. In this study, the gene responsible for MAs(III) demethylation was identified from an environmental MAs(III)-demethylating isolate, Bacillus sp. MD1. This gene, termed arsenic inducible gene (arsI), is in an arsenic resistance (ars) operon and encodes a nonheme iron-dependent dioxygenase with C ⋅ As lyase activity. Heterologous expression of ArsI conferred MAs(III)-demethylating activity and MAs(III) resistance to an arsenic-hypersensitive strain of Escherichia coli, demonstrating that MAs(III) demethylation is a detoxification process. Purified ArsI catalyzes Fe(2+)-dependent MAs(III) demethylation. In addition, ArsI cleaves the C ⋅ As bond in trivalent roxarsone and other aromatic arsenicals. ArsI homologs are widely distributed in prokaryotes, and we propose that ArsI-catalyzed organoarsenical degradation has a significant impact on the arsenic biogeocycle. To our knowledge, this is the first report of a molecular mechanism for organoarsenic degradation by a C ⋅ As lyase.

  19. Crystal structures of a yeast 14-3-3 protein from Lachancea thermotolerans in the unliganded form and bound to a human lipid kinase PI4KB-derived peptide reveal high evolutionary conservation.

    Science.gov (United States)

    Eisenreichova, Andrea; Klima, Martin; Boura, Evzen

    2016-11-01

    14-3-3 proteins bind phosphorylated binding partners to regulate several of their properties, including enzymatic activity, stability and subcellular localization. Here, two crystal structures are presented: the crystal structures of the 14-3-3 protein (also known as Bmh1) from the yeast Lachancea thermotolerans in the unliganded form and bound to a phosphopeptide derived from human PI4KB (phosphatidylinositol 4-kinase B). The structures demonstrate the high evolutionary conservation of ligand recognition by 14-3-3 proteins. The structural analysis suggests that ligand recognition by 14-3-3 proteins evolved very early in the evolution of eukaryotes and remained conserved, underlying the importance of 14-3-3 proteins in physiology.

  20. Molecular cloning and promoter analysis of the specific salicylic acid biosynthetic pathway gene phenylalanine ammonia-lyase (AaPAL1) from Artemisia annua.

    Science.gov (United States)

    Zhang, Ying; Fu, Xueqing; Hao, Xiaolong; Zhang, Lida; Wang, Luyao; Qian, Hongmei; Zhao, Jingya

    2016-07-01

    Phenylalanine ammonia-lyase (PAL) is the key enzyme in the biosynthetic pathway of salicylic acid (SA). In this study, a full-length cDNA of PAL gene (named as AaPAL1) was cloned from Artemisia annua. The gene contains an open reading frame of 2,151 bps encoding 716 amino acids. Comparative and bioinformatics analysis revealed that the polypeptide protein of AaPAL1 was highly homologous to PALs from other plant species. Southern blot analysis revealed that it belonged to a gene family with three members. Quantitative RT-PCR analysis of various tissues of A. annua showed that AaPAL1 transcript levels were highest in the young leaves. A 1160-bp promoter region was also isolated resulting in identification of distinct cis-regulatory elements including W-box, TGACG-motif, and TC-rich repeats. Quantitative RT-PCR indicated that AaPAL1 was upregulated by salinity, drought, wounding, and SA stresses, which were corroborated positively with the identified cis-elements within the promoter region. AaPAL1 was successfully expressed in Escherichia. coli and the enzyme activity of the purified AaPAL1 was approximately 287.2 U/mg. These results substantiated the involvement of AaPAL1 in the phenylalanine pathway.

  1. INFLUENCE OF COBALT IONS ON ENZYME ACTIVTY OF ISOCITRIATE LYASE AND ITS REGULATION IN CONDITION OF SEED GERMINATION OF GLYCINE MAX L

    National Research Council Canada - National Science Library

    Chechui O. F

    2012-01-01

    We investigated the activity of isocitrate lyase in seeds of Glycine max L. after 24, 72, and 120 hours of germination and effect of cobalt ions on the activity of the enzyme in time limit of the experiment...

  2. Structure/Function Analysis of Recurrent Mutations in SETD2 Protein Reveals a Critical and Conserved Role for a SET Domain Residue in Maintaining Protein Stability and Histone H3 Lys-36 Trimethylation.

    Science.gov (United States)

    Hacker, Kathryn E; Fahey, Catherine C; Shinsky, Stephen A; Chiang, Yun-Chen J; DiFiore, Julia V; Jha, Deepak Kumar; Vo, Andy H; Shavit, Jordan A; Davis, Ian J; Strahl, Brian D; Rathmell, W Kimryn

    2016-09-30

    The yeast Set2 histone methyltransferase is a critical enzyme that plays a number of key roles in gene transcription and DNA repair. Recently, the human homologue, SETD2, was found to be recurrently mutated in a significant percentage of renal cell carcinomas, raising the possibility that the activity of SETD2 is tumor-suppressive. Using budding yeast and human cell line model systems, we examined the functional significance of two evolutionarily conserved residues in SETD2 that are recurrently mutated in human cancers. Whereas one of these mutations (R2510H), located in the Set2 Rpb1 interaction domain, did not result in an observable defect in SETD2 enzymatic function, a second mutation in the catalytic domain of this enzyme (R1625C) resulted in a complete loss of histone H3 Lys-36 trimethylation (H3K36me3). This mutant showed unchanged thermal stability as compared with the wild type protein but diminished binding to the histone H3 tail. Surprisingly, mutation of the conserved residue in Set2 (R195C) similarly resulted in a complete loss of H3K36me3 but did not affect dimethylated histone H3 Lys-36 (H3K36me2) or functions associated with H3K36me2 in yeast. Collectively, these data imply a critical role for Arg-1625 in maintaining the protein interaction with H3 and specific H3K36me3 function of this enzyme, which is conserved from yeast to humans. They also may provide a refined biochemical explanation for how H3K36me3 loss leads to genomic instability and cancer.

  3. Family 13 carbohydrate-binding module of alginate lyase from Agarivorans sp. L11 enhances its catalytic efficiency and thermostability, and alters its substrate preference and product distribution.

    Science.gov (United States)

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

    2015-05-01

    The carbohydrate-binding module (CBM) in polysaccharide hydrolases plays a key role in the hydrolysis of cellulose, xylan and chitin. However, the function of CBM in alginate lyases has not been elucidated. A new alginate lyase gene, alyL2, was cloned from the marine bacterium Agarivorans sp. L11 by using degenerate and site-finding PCR. The alginate lyase, AlyL2, contained an N-terminal CBM13 and a C-terminal catalytic family 7 polysaccharide lyase (PL7) module. To better understand the function of CBM13 in alginate lyase AlyL2, the full-length enzyme (AlyL2-FL) and its catalytic module (AlyL2-CM) were expressed in Escherichia coli and characterized. The specific activity and catalytic efficiency of AlyL2-FL were approximately twice those of AlyL2-CM. The half-lives of AlyL2-FL were 4.7-6.6 times those of AlyL2-CM at 30-50°C. In addition, the presence of CBM13 in AlyL2 changed its substrate preference and increased the percentage of disaccharides from 50.5% to 64.6% in the total products. This first report of the function of CBM13 in alginate lyase provides new insights into the degradation of alginate by marine microorganisms.

  4. Local Responses to Participatory Conservation in Annapurna Conservation Area, Nepal

    Science.gov (United States)

    Khadka, Damodar; Nepal, Sanjay K.

    2010-02-01

    Biodiversity conservation has undergone a profound change in philosophy, policies and management approaches over the last forty years. The traditional top-down approach to nature protection has been widely criticized for failing to include critical social elements in management practices, and is being gradually replaced by a slew of participatory strategies under the rubric of bottom-up conservation. The new approach recognizes local communities as key partners in wildlife management and seeks their participation in social development and biodiversity conservation. However, every social context is different in its structure and functions, and in the way social groups respond to calls for participation. In order to gain a better understanding of the approach and the barriers encountered in its implementation, a questionnaire survey of 188 households was employed in the communities of the Upper Mustang extension of Annapurna Conservation Area (ACA) in Nepal. The study provides a comparative analysis of community participation and its barriers between Non-Tourist (NT) and Tourist (TV) villages. The results revealed important differences between the two groups in terms of their participation in community programs, barriers to participation, and perception of benefits from participation. Owing to their distinct spatial, demographic and attitudinal differences, the two village groups have their own sets of needs, values and motivation factors which cannot be generalized and treated as such. The research clearly identifies the need for the conservation agency to be creative in devising strategies and initiatives appropriate to specific social groups so as to optimize their input in participatory conservation.

  5. 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

    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/mg; KM 1.2mg....../ml). The addition of both Ca2+ and Mn2+ was essential for enzyme activity. The enzyme retained its catalytic activity at higher temperatures and the enzyme has a half life at 61°C of 15min. The work thus demonstrated the workability of in silico based screening coupled with a synthetic biology approach for gene...

  6. Karyotypic evolution in squamate reptiles: comparative gene mapping revealed highly conserved linkage homology between the butterfly lizard (Leiolepis reevesii rubritaeniata, Agamidae, Lacertilia) and the Japanese four-striped rat snake (Elaphe quadrivirgata, Colubridae, Serpentes).

    Science.gov (United States)

    Srikulnath, Kornsorn; Nishida, Chizuko; Matsubara, Kazumi; Uno, Yoshinobu; Thongpan, Amara; Suputtitada, Saowanee; Apisitwanich, Somsak; Matsuda, Yoichi

    2009-01-01

    The butterfly lizard (Leiolepis reevesii rubritaeniata) has the diploid chromosome number of 2n = 36, comprising two distinctive components, macrochromosomes and microchromosomes. To clarify the conserved linkage homology between lizard and snake chromosomes and to delineate the process of karyotypic evolution in Squamata, we constructed a cytogenetic map of L. reevesii rubritaeniata with 54 functional genes and compared it with that of the Japanese four-striped rat snake (E. quadrivirgata, 2n = 36). Six pairs of the lizard macrochromosomes were homologous to eight pairs of the snake macrochromosomes. The lizard chromosomes 1, 2, 4, and 6 corresponded to the snake chromosomes 1, 2, 3, and Z, respectively. LRE3p and LRE3q showed the homology with EQU5 and EQU4, respectively, and LRE5p and LRE5q corresponded to EQU7 and EQU6, respectively. These results suggest that the genetic linkages have been highly conserved between the two species and that their karyotypic difference might be caused by the telomere-to-telomere fusion events followed by inactivation of one of two centromeres on the derived dicentric chromosomes in the lineage of L. reevesii rubritaeniata or the centric fission events of the bi-armed macrochromosomes and subsequent centromere repositioning in the lineage of E. quadrivirgata. The homology with L. reevesii rubritaeniata microchromosomes were also identified in the distal regions of EQU1p and 1q, indicating the occurrence of telomere-to-telomere fusions of microchromosomes to the p and q arms of EQU1.

  7. 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...

  8. Inactivation, complementation, and heterologous expression of encP, a novel bacterial phenylalanine ammonia-lyase gene.

    Science.gov (United States)

    Xiang, Longkuan; Moore, Bradley S

    2002-09-06

    The enzyme phenylalanine ammonia-lyase, which catalyzes the nonoxidative deamination of l-phenylalanine to trans-cinnamic acid, is ubiquitously distributed in plants. We now report its characterization for the first time in a bacterium. The phenylalanine ammonia-lyase homologous gene encP from the "Streptomyces maritimus" enterocin biosynthetic gene cluster was functionally characterized and shown to encode the first enzyme in the pathway to the enterocin polyketide synthase starter unit benzoyl-coenzyme A. The disruption of the encP gene completely inhibited the production of cinnamate and enterocin, whereas complementation of the mutant with benzoyl-coenzyme A pathway intermediates or with the wild-type gene encP restored the formation of the benzoate-primed polyketide antibiotic enterocin. Heterologous expression of the encP gene under the control of the ermE* promoter in Streptomyces coelicolor furthermore led to the production of cinnamic acid in the fermented cultures, confirming that the encP gene indeed encodes a novel bacterial phenylalanine ammonia-lyase.

  9. Mechanistic studies of a novel C-S lyase in ergothioneine biosynthesis: the involvement of a sulfenic acid intermediate.

    Science.gov (United States)

    Song, Heng; Hu, Wen; Naowarojna, Nathchar; Her, Ampon Sae; Wang, Shu; Desai, Rushil; Qin, Li; Chen, Xiaoping; Liu, Pinghua

    2015-01-01

    Ergothioneine is a histidine thio-derivative isolated in 1909. In ergothioneine biosynthesis, the combination of a mononuclear non-heme iron enzyme catalyzed oxidative C-S bond formation reaction and a PLP-mediated C-S lyase (EgtE) reaction results in a net sulfur transfer from cysteine to histidine side-chain. This demonstrates a new sulfur transfer strategy in the biosynthesis of sulfur-containing natural products. Due to difficulties associated with the overexpression of Mycobacterium smegmatis EgtE protein, the proposed EgtE functionality remained to be verified biochemically. In this study, we have successfully overexpressed and purified M. smegmatis EgtE enzyme and evaluated its activities under different in vitro conditions: C-S lyase reaction using either thioether or sulfoxide as a substrate in the presence or absence of reductants. Results from our biochemical characterizations support the assignment of sulfoxide 4 as the native EgtE substrate and the involvement of a sulfenic acid intermediate in the ergothioneine C-S lyase reaction.

  10. Structural and catalytic properties of the four phenylalanine ammonia-lyase isoenzymes from parsley (Petroselinum crispum Nym.).

    Science.gov (United States)

    Appert, C; Logemann, E; Hahlbrock, K; Schmid, J; Amrhein, N

    1994-10-01

    Near-full-length cDNAs for the four phenylalanine ammonia-lyase (PAL) isoenzymes in parsley (Petroselium crispum Nym.) were cloned and the complete amino acid sequences deduced. Fusion proteins with glutathione S-transferase were expressed in Escherichia coli, purified and cleaved. All of the resulting phenylalanine ammonia-lyase proteins, as well as the fusion proteins, were catalytically active. The turnover number of one selected isoenzyme, PAL-1, was estimated to be around 22 s-1 for each active site. In contrast to a certain degree of differential expression in various parts of parsley plants, the four phenylalanine ammonia-lyase isoenzymes exhibited very similar apparent Km values for L-phenylalanine (15-24.5 microM) as well as identical temperature (58 degrees C) and pH (8.5) optima. All of them were competitively inhibited by (E)-cinnamate with similar efficiency (Ki values: 9.1-21.5 microM), lacked cooperative behaviour, and accepted L-tyrosine as a substrate with low affinity (Km values: 2.6-7.8 mM). These results suggest that the occurrence of multiple gene copies has a function other than encoding isoenzymes with different enzyme kinetic properties.

  11. Expression and enzymatic activity of phenylalanine ammonia-lyase and p-coumarate 3-hydroxylase in mango (Mangifera indica 'Ataulfo') during ripening.

    Science.gov (United States)

    Palafox-Carlos, H; Contreras-Vergara, C A; Muhlia-Almazán, A; Islas-Osuna, M A; González-Aguilar, G A

    2014-05-16

    Phenylalanine ammonia lyase (PAL) and p-coumarate 3-hydroxylase (C3H) are key enzymes in the phenylpropanoid pathway. The relative expression of PAL and C3H was evaluated in mango fruit cultivar 'Ataulfo' in four ripening stages (RS1, RS2, RS3, and RS4) by quantitative polymerase chain reaction. In addition, enzyme activity of PAL and C3H was determined in mango fruits during ripening. The PAL levels were downregulated at the RS2 and RS3 stages, while C3H levels were upregulated in fruits only at RS3. The enzyme activity of PAL followed a pattern that was different from that of the PAL expression, thus suggesting regulation at several levels. For C3H, a regulation at the transcriptional level is suggested because a similar pattern was revealed by its activity and transcript level. In this study, the complexity of secondary metabolite biosynthesis regulation is emphasized because PAL and C3H enzymes are involved in the biosynthesis of several secondary metabolites that are active during all mango ripening stages.

  12. Expression Analysis of Phenylalanine Ammonia Lyase Gene and Rosmarinic Acid Production in Salvia officinalis and Salvia virgata Shoots Under Salicylic Acid Elicitation.

    Science.gov (United States)

    Ejtahed, Roghayeh Sadat; Radjabian, Tayebeh; Hoseini Tafreshi, Sayed Ali

    2015-08-01

    Partial fragments of phenylalanine ammonia lyase (PAL) genes were cloned and characterized from Salvia officinalis (SoPAL) and Salvia virgata (SvPAL). Different concentrations (250 and 500 μM) of exogenous salicylic acid (SA) were used when correlation between PAL expression and rosmarinic acid (RA) accumulation was compared. The results showed that the deduced cDNA sequences of the partial genes had high similarities with those of known PAL gene from other plant species. Semi-quantitative reverse transcription PCR (RT-PCR) analysis revealed that exogenous application of SA led to up-regulating of the PAL expression. Further analysis showed that in S. virgata, at higher concentration of SA, higher accumulation of RA was achieved, while in S. officinalis, the higher RA accumulation was observed at lower concentration of SA. It was concluded that there was no positive correlation between the intensity of PAL transcription and the RA accumulation in the studied species. Therefore, despite of the increase in transcription rate of the PAL at the higher concentration of SA, the lower amounts of RA were accumulated in the case of S. officinalis. Consequently, the hypothesis that PAL is the rate-determining step in RA biosynthesis is not always valid and probably some other unknown factors participate in the synthesis of phenolics.

  13. Changes in levels of argininosuccinate lyase mRNA during induction by glucagon and cyclic AMP in cultured foetal-rat hepatocytes.

    Science.gov (United States)

    Renouf, S; Buquet, C; Fairand, A; Benamar, M; Husson, A

    1993-01-01

    During the perinatal period, the activity of the urea-cycle enzyme argininosuccinate lyase (ASL) is regulated by glucocorticoids, glucagon and insulin. In this study, the effects of glucagon and cyclic AMP (cAMP) analogues were examined on the synthesis of ASL and on the level of its corresponding mRNA in cultured foetal hepatocytes. Northern-blot analysis revealed that these agents only gave a transient induction of ASL mRNA amount, which reached a peak at 6 h and declined thereafter. This induction preceded the increase in enzyme activity and amount which could be observed for 2 or 3 days of culture. Stimulation of ASL mRNA accumulation by a combination of cAMP analogues and dexamethasone was additive, indicating that glucocorticoids and cAMP are both necessary to promote hepatocyte differentiation and that inductions could occur via independent pathways. Induction by cAMP analogues could be abolished by actinomycin D, suggesting a control mechanism at the transcriptional level. Puromycin was without effect on ASL mRNA induction by cAMP, indicating that no ongoing protein synthesis was required in the stimulation process. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8387274

  14. High resolution crystal structure of Clostridium propionicum β-alanyl-CoA:ammonia lyase, a new member of the "hot dog fold" protein superfamily.

    Science.gov (United States)

    Heine, Andreas; Herrmann, Gloria; Selmer, Thorsten; Terwesten, Felix; Buckel, Wolfgang; Reuter, Klaus

    2014-09-01

    Clostridium propionicum is the only organism known to ferment β-alanine, a constituent of coenzyme A (CoA) and the phosphopantetheinyl prosthetic group of holo-acyl carrier protein. The first step in the fermentation is a CoA-transfer to β-alanine. Subsequently, the resulting β-alanyl-CoA is deaminated by the enzyme β-alanyl-CoA:ammonia lyase (Acl) to reversibly form ammonia and acrylyl-CoA. We have determined the crystal structure of Acl in its apo-form at a resolution of 0.97 Å as well as in complex with CoA at a resolution of 1.59 Å. The structures reveal that the enyzme belongs to a superfamily of proteins exhibiting a so called "hot dog fold" which is characterized by a five-stranded antiparallel β-sheet with a long α-helix packed against it. The functional unit of all "hot dog fold" proteins is a homodimer containing two equivalent substrate binding sites which are established by the dimer interface. In the case of Acl, three functional dimers combine to a homohexamer strongly resembling the homohexamer formed by YciA-like acyl-CoA thioesterases. Here, we propose an enzymatic mechanism based on the crystal structure of the Acl·CoA complex and molecular docking.

  15. ATP-Citrate Lyase Controls a Glucose-to-Acetate Metabolic Switch

    Directory of Open Access Journals (Sweden)

    Steven Zhao

    2016-10-01

    Full Text Available Mechanisms of metabolic flexibility enable cells to survive under stressful conditions and can thwart therapeutic responses. Acetyl-coenzyme A (CoA plays central roles in energy production, lipid metabolism, and epigenomic modifications. Here, we show that, upon genetic deletion of Acly, the gene coding for ATP-citrate lyase (ACLY, cells remain viable and proliferate, although at an impaired rate. In the absence of ACLY, cells upregulate ACSS2 and utilize exogenous acetate to provide acetyl-CoA for de novo lipogenesis (DNL and histone acetylation. A physiological level of acetate is sufficient for cell viability and abundant acetyl-CoA production, although histone acetylation levels remain low in ACLY-deficient cells unless supplemented with high levels of acetate. ACLY-deficient adipocytes accumulate lipid in vivo, exhibit increased acetyl-CoA and malonyl-CoA production from acetate, and display some differences in fatty acid content and synthesis. Together, these data indicate that engagement of acetate metabolism is a crucial, although partial, mechanism of compensation for ACLY deficiency.

  16. 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.

  17. Changes in rice allelopathy and rhizosphere microflora by inhibiting rice phenylalanine ammonia-lyase gene expression.

    Science.gov (United States)

    Fang, Changxun; Zhuang, Yuee; Xu, Tiecheng; Li, Yingzhe; Li, Yue; Lin, Wenxiong

    2013-02-01

    Gene expression of phenylalanine ammonia-lyase (PAL) in allelopathic rice PI312777 was inhibited by RNA interference (RNAi). Transgenic rice showed lower levels of PAL gene expression and PAL activity than wild type rice (WT). The concentrations of phenolic compounds were lower in the root tissues and root exudates of transgenic rice than in those of wild type plants. When barndyardgrass (BYG) was used as the receiver plant, the allelopathic potential of transgenic rice was reduced. The sizes of the bacterial and fungal populations in rice rhizospheric soil at the 3-, 5-, and 7-leaf stages were estimated by using quantitative PCR (qPCR), which showed a decrease in both populations at all stages of leaf development analyzed. However, PI312777 had a larger microbial population than transgenic rice. In addition, in T-RFLP studies, 14 different groups of bacteria were detected in WT and only 6 were detected in transgenic rice. This indicates that there was less rhizospheric bacterial diversity associated with transgenic rice than with WT. These findings collectively suggest that PAL functions as a positive regulator of rice allelopathic potential.

  18. 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.

  19. Role of the cystathionine γ lyase/hydrogen sulfide pathway in human melanoma progression.

    Science.gov (United States)

    Panza, Elisabetta; De Cicco, Paola; Armogida, Chiara; Scognamiglio, Giosuè; Gigantino, Vincenzo; Botti, Gerardo; Germano, Domenico; Napolitano, Maria; Papapetropoulos, Andreas; Bucci, Mariarosaria; Cirino, Giuseppe; Ianaro, Angela

    2015-01-01

    In humans, two main metabolic enzymes synthesize hydrogen sulfide (H2 S): cystathionine γ lyase (CSE) and cystathionine β synthase (CBS). A third enzyme, 3-mercaptopyruvate sulfurtransferase (3-MST), synthesizes H2 S in the presence of the substrate 3-mercaptopyruvate (3-MP). The immunohistochemistry analysis performed on human melanoma samples demonstrated that CSE expression was highest in primary tumors, decreased in the metastatic lesions and was almost silent in non-lymph node metastases. The primary role played by CSE was confirmed by the finding that the overexpression of CSE induced spontaneous apoptosis of human melanoma cells. The same effect was achieved using different H2 S donors, the most active of which was diallyl trisulfide (DATS). The main pro-apoptotic mechanisms involved were suppression of nuclear factor-κB activity and inhibition of AKT and extracellular signal-regulated kinase pathways. A proof of concept was obtained in vivo using a murine melanoma model. In fact, either l-cysteine, the CSE substrate, or DATS inhibited tumor growth in mice. In conclusion, we have determined that the l-cysteine/CSE/H2 S pathway is involved in melanoma progression.

  20. Characterization of a bifunctional glyoxylate cycle enzyme, malate synthase/isocitrate lyase, of Euglena gracilis.

    Science.gov (United States)

    Nakazawa, Masami; Nishimura, Masaaki; Inoue, Kengo; Ueda, Mitsuhiro; Inui, Hiroshi; Nakano, Yoshihisa; Miyatake, Kazutaka

    2011-01-01

    The glyoxylate cycle is a modified form of the tricarboxylic acid cycle, which enables organisms to synthesize carbohydrates from C2 compounds. In the protozoan Euglena gracilis, the key enzyme activities of the glyoxylate cycle, isocitrate lyase (ICL) and malate synthase (MS), are conferred by a single bifunctional protein named glyoxylate cycle enzyme (Euglena gracilis glyoxylate cycle enzyme [EgGCE]). We analyzed the enzymatic properties of recombinant EgGCE to determine the functions of its different domains. The 62-kDa N-terminal domain of EgGCE was sufficient to provide the MS activity as expected from an analysis of the deduced amino acid sequence. In contrast, expression of the 67-kDa C-terminal domain of EgGCE failed to yield ICL activity even though this domain was structurally similar to ICL family enzymes. Analyses of truncation mutants suggested that the N-terminal residues of EgGCE are critical for both the ICL and MS activities. The ICL activity of EgGCE increased in the presence of micro-molar concentrations of acetyl-coenzyme A (CoA). Acetyl-CoA also increased the activity in a mutant type EgGCE with a mutation at the acetyl-CoA binding site in the MS domain of EgGCE. This suggests that acetyl-CoA regulates the ICL reaction by binding to a site other than the catalytic center of the MS reaction.

  1. 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.

  2. 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

    2016-03-02

    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.

  3. 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. Furtherm