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Sample records for lyase gene ogl

  1. 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.

  2. 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.

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

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    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.

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

    Science.gov (United States)

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

    2015-11-01

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

  5. 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

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

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    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.

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

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    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.

  8. 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....

  9. 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

  10. Relationship between cystathionine γ-lyase gene polymorphism and essential hypertension in Northern Chinese Han population

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    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.

  11. Expression in E. coli of the gene encoding phenylalanine ammonia-lyase from Rhodosporidium toruloides.

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    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.

  12. 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

  13. 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

  14. 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

  15. Inactivation, complementation, and heterologous expression of encP, a novel bacterial phenylalanine ammonia-lyase gene.

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    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.

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

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    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.

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

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    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.

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

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    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.

  19. Phenylalanine ammonia-lyase gene families incucurbit species:Structure, evolution, and expression

    Institute of Scientific and Technical Information of China (English)

    DONG Chun-juan; CAO Ning; ZHANG Zhi-gang; SHANG Qing-mao

    2016-01-01

    Phenylalanine ammonia-lyase (PAL), the ifrst enzyme of phenylpropanoid pathway, is always encoded by multigene families in plants. In this study, using genome-wide searches, 13PAL genes in cucumber (CsPAL1–13) and 13PALsin melon (Cm-PAL1–13) were identiifed. In the corresponding genomes, ten of thesePAL genes were located in tandem in two clusters, while the others were widely dispersed in different chromosomes as a single copy. The protein sequences of CsPALs and CmPALs shared an overal high identity to each other. In our previous report, 12PAL genes were identiifed in watermelon (ClPAL1–12). Thereby, a total of 38 cucurbitPAL members were included. Here, a comprehensive comparison ofPAL gene families was performed among three cucurbit plants. The phylogenetic and syntenic analyses placed the cucurbit PALs as 11 CsPAL-CmPAL-ClPAL triples, of which ten triples were clustered into the dicot group, and the remaining one, CsPAL1-CmPAL8-ClPAL2, was grouped with gymnosperm PALs and might serve as an ancestor of cucurbit PALs. By comparing the syntenic relationships and gene structure of these PAL genes, the expansion of cucurbit PALfamilies might arise from a series of segmental and tandem duplications and intron insertion events. Furthermore, the expression proifling in different tissues suggested that different cucurbit PALs displayed divergent but overlapping expression proifles, and the CsPAL-CmPAL-ClPAL orthologs showed correlative expression patterns among three cucurbit plants. Taken together, this study provided an extensive description on the evolution and expression of cucurbit PAL gene families and might facilitate the further studies for elucidating the functions of PALs in cucurbit plants.

  20. 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

  1. 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.

  2. Changes in rice allelopathy and rhizosphere microflora by inhibiting rice phenylalanine ammonia-lyase gene expression.

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    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.

  3. 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.

  4. Silencing of grapevine pectate lyase-like genes VvPLL2 and VvPLL3 confers resistance against Erysiphe necator and differentially modulates gene expression

    Science.gov (United States)

    Broad-spectrum resistance against powdery mildew (PM) has been reported by silencing susceptibility genes in the model plant Arabidopsis. Here we used artificial microRNA constructs in PM-susceptible Vitis vinifera cv. Chardonnay to stably silence two pectate lyase-like orthologs (VvPLL2 and VvPLL3)...

  5. Molecular cloning and characterization of an Erwinia carotovora subsp. carotovora pectin lyase gene that responds to DNA-damaging agents.

    OpenAIRE

    McEvoy, J L; Murata, H.; Chatterjee, A. K.

    1990-01-01

    recA-mediated production of pectin lyase (PNL) and the bacteriocin carotovoricin occurs in Erwinia carotovora subsp. carotovora 71 when this organism is subjected to agents that damage or inhibit the synthesis of DNA. The structural gene pnlA was isolated from a strain 71 cosmid gene library following mobilization of the cosmids into a moderate PNL producer, strain 193. The cosmid complemented pnl::Tn5 but not ctv::Tn5 mutations. A constitutive level of PNL activity was detected in RecA+ and ...

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

    Science.gov (United States)

    Sun, Lingxia; van Nocker, Steven

    2010-07-22

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

  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. The pectate lyase encoded by the pecCl1 gene is an important determinant for the aggressiveness of Colletotrichum lindemuthianum.

    Science.gov (United States)

    Cnossen-Fassoni, Andréia; Bazzolli, Denise Mara Soares; Brommonschenkel, Sérgio Hermínio; Fernandes de Araújo, Elza; de Queiroz, Marisa Vieira

    2013-08-01

    Colletotrichum lindemuthianum is the causal agent of anthracnose in the common bean, and the genes that encode its cell-wall-degrading enzymes are crucial for the development of the disease. Pectinases are the most important group of cell wall-degrading enzymes produced by phytopathogenic fungi. The pecC1l gene, which encodes a pectate lyase in C. lindemuthianum, was isolated and characterized. Possible cis-regulatory elements and transcription factor binding sites that may be involved in the regulation of genetic expression were detected in the promoter region of the gene. pecCl1 is represented by a single copy in the genome of C. lindemuthianum, though in silico analyses of the genomes of Colletotrichum graminicola and Colletotrichum higginsianum suggest that the genome of C. lindemuthianum includes other genes that encode pectate lyases. Phylogenetic analysis detected two groups that clustered based on different members of the pectate lyase family. Analysis of the differential expression of pecCl1 during different stages of infection showed a significant increase in pecCl1 expression five days after infection, at the onset of the necrotrophic phase. The split-maker technique proved to be an efficient method for inactivation of the pecCl1 gene, which allowed functional study of a mutant with a site-specific integration. Though gene inactivation did not result in complete loss of pectate lyase activity, the symptoms of anthracnose were reduced. Analysis of pectate lyases might not only contribute to the understanding of anthracnose in the common bean but might also lead to the discovery of an additional target for controlling anthracnose.

  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. 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.

  13. Mutation R96W in cytochrome P450c17 gene causes combined 17{alpha}-hydroxylase/17-20-lyase deficiency in two french canadian patients

    Energy Technology Data Exchange (ETDEWEB)

    LaFlamme, N.; Leblanc, J.F.; Mailloux, J. [Laval Univ., Quebec (Canada)

    1996-01-01

    Congenital adrenal hyperplasia (CAH) is the most frequent cause of adrenal insufficiency and ambiguous genitalia in newborn children. In contrast to CAH caused by 21{alpha}-hydroxylase and 11{beta}-hydroxylase deficiencies, which impairs steroid formation in the adrenal exclusively, 17{alpha}-hydroxylase/17,20-lyase deficiency impairs steroid biosynthesis in the adrenals and gonads. The sequence of CYP17 gene was determined by direct sequencing of asymmetric PCR products in two French-Canadian 46,XY pseudohermaphrodite siblings suffering from combined 17{alpha}-hydroxylase/17,20-lyase deficiency. The two patients are homozygous for the novel missense mutation R96W caused by a C to T transition converting codon Arg{sup 96} (CGG) into a Trp (TGG) in exon 1. Both parents are heterozygous for this missense mutation. We assessed the effect of the R96W mutation on 17{alpha}-hydroxylase/17,20-lyase activity by analysis of mutant enzyme, generated by site-directed mutagenesis, expressed in COS-1 cells. The presence of R96W substitution almost completely abolished the activity of the mutant protein. The present findings provide a molecular explanation for the signs and symptoms of combined 17 {alpha}-hydroxylase/17,20-lyase deficiency in these two patients and provide useful information on the structure-activity relationships of the P450c17 enzyme. 31 refs., 4 figs., 1 tab.

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  16. 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.

  17. cDNA cloning, Phylogenic Analysis and Gene Expression Pattern of Phenylalanine ammonia-lyase in Sugarcane (Saccharum officinarum L.

    Directory of Open Access Journals (Sweden)

    Mahmoud Hashemitabar

    2014-08-01

    Full Text Available The aim of the present study was to clone and characterize a full length cDNA of sugarcane (Saccharum officinarum phenylalanine ammonia-lyase (SoPAL. Differential tissue expression pattern of the SoPAL transcript and its enzyme activity was also analyzed during the tillering stage of growth. The full-length of SoPAL cDNA was 2118 bp long and contained a protein with 706 amino acids, determined by encoding technique. The amino acid sequence and phylogenic analysis of the cloned SoPAL showed high similarity to PAL from other monocotyledonous such as sorghum (96%, maize (93% and Bamboos (87.12%. The highest levels of SoPAL transcript were observed in the root and stem, while its minimal gene expression levels were in the leaves and sheath, respectively. The highest level of SoPAL enzyme activity was in the leaves. These results helped to understanding the characteristics of PAL biosynthesis and its regulation at the molecular level in sugarcane. This information could be critical for the manipulation of phenylpropanoid biosynthesis in the plant using biotechnological processes.

  18. 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

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

    Directory of Open Access Journals (Sweden)

    Sau Ha Cheung

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

  20. 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

  1. 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 ...

  2. 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

  3. 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.

  4. 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.

  5. Some of the out genes involved in the secretion of pectate lyases in Erwinia chrysanthemi are regulated by kdgR.

    Science.gov (United States)

    Condemine, G; Dorel, C; Hugouvieux-Cotte-Pattat, N; Robert-Baudouy, J

    1992-11-01

    The out genes of Erwinia chrysanthemi are required for the translocation across the outer membrane of pectate lyases and cellulases. We present the characterization and the nucleotide sequence of five genes of the out cluster. The products of outS, B, C, D and E have significant homology with the PulS, B, C, D and E proteins necessary to the secretion of pullulanase in Klebsiella pneumoniae. An open reading frame, outT, located between outB and outC has no homology with the pul cluster but is involved in secretion. outC, outD and outE form an operon while outS, outB and outT constitute independent transcription units. outT and the outCDE operon are regulated by kdgR, the negative regulatory gene controlling pectinase production. outB and outS seem to be expressed constitutively.

  6. A nifS-like gene, csdB, encodes an Escherichia coli counterpart of mammalian selenocysteine lyase. Gene cloning, purification, characterization and preliminary x-ray crystallographic studies.

    Science.gov (United States)

    Mihara, H; Maeda, M; Fujii, T; Kurihara, T; Hata, Y; Esaki, N

    1999-05-21

    Selenocysteine lyase is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the exclusive decomposition of L-selenocysteine to L-alanine and elemental selenium. An open reading frame, named csdB, from Escherichia coli encodes a putative protein that is similar to selenocysteine lyase of pig liver and cysteine desulfurase (NifS) of Azotobacter vinelandii. In this study, the csdB gene was cloned and expressed in E. coli cells. The gene product was a homodimer with the subunit Mr of 44,439, contained 1 mol of PLP as a cofactor per mol of subunit, and catalyzed the release of Se, SO2, and S from L-selenocysteine, L-cysteine sulfinic acid, and L-cysteine, respectively, to yield L-alanine; the reactivity of the substrates decreased in this order. Although the enzyme was not specific for L-selenocysteine, the high specific activity for L-selenocysteine (5.5 units/mg compared with 0.019 units/mg for L-cysteine) supports the view that the enzyme can be regarded as an E. coli counterpart of mammalian selenocysteine lyase. We crystallized CsdB, the csdB gene product, by the hanging drop vapor diffusion method. The crystals were of suitable quality for x-ray crystallography and belonged to the tetragonal space group P43212 with unit cell dimensions of a = b = 128.1 A and c = 137.0 A. Consideration of the Matthews parameter Vm (3.19 A3/Da) accounts for the presence of a single dimer in the crystallographic asymmetric unit. A native diffraction dataset up to 2.8 A resolution was collected. This is the first crystallographic analysis of a protein of NifS/selenocysteine lyase family.

  7. 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.

  8. Overexpression of Rice Sphingosine-1-Phoshpate Lyase Gene OsSPL1 in Transgenic Tobacco Reduces Salt and Oxidative Stress Tolerance

    Institute of Scientific and Technical Information of China (English)

    Huijuan Zhang; Jing Zhai; Jibo Mo; Dayong Li; Fengming Song

    2012-01-01

    Sphingolipids,including sphingosine-1-phosphate (S1P),have been shown to function as signaling mediators to regulate diverse aspects of plant growth,development,and stress response.In this study,we performed functional analysis of a rice (Oryza sativa) S1P lyase gene OsSPL1 in transgenic tobacco plants and explored its possible involvement in abiotic stress response.Overexpression of OsSPL1 in transgenic tobacco resulted in enhanced sensitivity to exogenous abscisic acid (ABA),and decreased tolerance to salt and oxidative stress,when compared with the wild type.Furthermore,the expression levels of some selected stress-related genes in OsSPL1-overexpressing plants were reduced after application of salt or oxidative stress,indicating that the altered responsiveness of stress-related genes may be responsible for the reduced tolerance in OsSPL1-overexpressing tobacco plants under salt and oxidative stress.Our results suggest that rice OsSPL1 plays an important role in abiotic stress responses.

  9. 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.

  10. 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.

  11. Cloning and Expression Analysis of Phenylalanine Ammonia-Lyase Gene in the Mycelium and Fruit Body of the Edible Mushroom Flammulina velutipes.

    Science.gov (United States)

    Yun, Yeo Hong; Koo, Ja Sun; Kim, Seong Hwan; Kong, Won Sik

    2015-09-01

    Phenylalanine ammonia-lyase (PAL) gene is known to be expressed in plants, and is involved in the differentiation, growth and synthesis of secondary metabolites. However, its expression in fungi remains to be explored. To understand its expression in mushroom fungi, the PAL gene of the edible mushroom Flammulina velutipes (Fvpal) was cloned and characterized. The cloned Fvpal consists of 2,175 bp, coding for a polypeptide containing 724 amino acids and having 11 introns. The translated amino acid sequence of Fvpal shares a high identity (66%) with that of ectomycorrhizal fungus Tricholoma matsutake. Distinctively, the Fvpal expression in the mycelium was higher in minimal medium supplemented with L-tyrosine than with other aromatic amino acids. During cultivation of the mushroom on sawdust medium, Fvpal expression in the fruit body correspondingly increased as the mushroom grew. In the fruiting body, Fvpal was expressed more in the stipe than in the pileus. These results suggest that F. velutipes PAL activity differs in the different organs of the mushroom. Overall, this is first report to show that the PAL gene expression is associated with mushroom growth in fungi.

  12. 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.

  13. Identification, cloning and characterization of cysK, the gene encoding O-acetylserine (thiol)-lyase from Azospirillum brasilense, which is involved in tellurite resistance.

    Science.gov (United States)

    Ramírez, Alberto; Castañeda, Miguel; Xiqui, María L; Sosa, Araceli; Baca, Beatriz E

    2006-08-01

    O-Acetylserine (thiol)-lyase (cysteine synthase) was purified from Azospirillum brasilense Sp7. After hydrolysis of the purified protein, amino acid sequences of five peptides were obtained, which permitted the cloning and sequencing of the cysK gene. The deduced amino acid sequence of cysteine synthase exhibited homology with several putative proteins from Alpha- and Gammaproteobacteria. Azospirillum brasilense Sp7 cysK exhibited 58% identity (72% similarity) with Escherichia coli K12 and Salmonella enterica serovar Typhimurium cysteine synthase proteins. An E. coli auxotroph lacking cysteine synthase loci could be complemented with A. brasilense Sp7 cysK. The 3.0-kb HindIII-EcoRI fragment bearing cysK contained two additional ORFs encoding a putative transcriptional regulator and dUTPase. Insertional disruption of the cysK gene did not produce a cysteine auxotroph, indicating that gene redundancy in the cysteine biosynthetic or other biosynthetic pathways exists in Azospirillum, as already described in other bacteria. Nitrogen fixation was not altered in the mutant strain as determined by acetylene reduction. However, this strain showed an eight-fold reduction in tellurite resistance as compared to the wild-type strain, which was only observed during growth in minimal medium. These data confirm earlier observations regarding the importance of cysteine metabolism in tellurite resistance.

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

    Indian Academy of Sciences (India)

    A Badiei; ST Chambers; RR Gaddam; M Bhatia

    2016-03-01

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

  15. Developmental role of phenylalanine-ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) genes during adventitious rooting of Juglans regia L. microshoots.

    Science.gov (United States)

    Cheniany, Monireh; Ganjeali, Ali

    2016-12-01

    Phenylalanine-ammonia-lyase and cinnamate-4-hydroxylase play important role in the phenylpropanoid pathway, which produces many biologically important secondary metabolites participating in normal plant development. Flavonol quercetin is the main representant of these compounds that has been identified in numerous Juglans spp. In this survey, the developmental expression patterns of PAL and C4H genes during in vitro rooting of two walnut cultivars 'Sunland' and 'Howard' was examined by RT-PCR. To understand the potential role in rooting, the changing pattern of endogenous content of quercetin was also analyzed by HPLC. The 'Sunland' with better capacity to root had more quercetin content during the "inductive phase" of rooting than 'Howard'. In each cultivar, the level of PAL transcripts showed the same behavior with the changing patterns of quercetin during root formation of microshoots. The positive correlation between the changes of quercetin and PAL-mRNA indicated that PAL gene may have an immediate effect on flavonoid pathway metabolites including quercetin. Although the behavioral change of C4H expression was similar in both cultivars during root formation (with significantly more level for 'Howard'), it was not coincide with the changes of quercerin concentrations. Our results showed that C4H function is important for the normal development, but its transcriptional regulation does not correlate with quercetin as an efficient phenolic compound for walnut rhizogenesis.

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

    Science.gov (United States)

    Hu, Xiao-Mei; Shi, Cai-Yun; Liu, Xiao; Jin, Long-Fei; Liu, Yong-Zhong; Peng, Shu-Ang

    2015-02-01

    ATP-citrate lyase (ACL, EC4.1.3.8) catalyzes citrate to oxaloacetate and acetyl-CoA in the cell cytosol, and has important roles in normal plant growth and in the biosynthesis of some secondary metabolites. We identified three ACL genes, CitACLα1, CitACLα2, and CitACLβ1, in the citrus genome database. Both CitACLα1 and CitACLα2 encode putative ACL α subunits with 82.5 % amino acid identity, whereas CitACLβ1 encodes a putative ACL β subunit. Gene structure analysis showed that CitACLα1 and CitACLα2 had 12 exons and 11 introns, and CitACLβ1 had 16 exons and 15 introns. CitACLα1 and CitACLβ1 were predominantly expressed in flower, and CitACLα2 was predominantly expressed in stem and fibrous roots. As fruits ripen, the transcript levels of CitACLα1, CitACLβ1, and/or CitACLα2 in cultivars 'Niuher' and 'Owari' increased, accompanied by significant decreases in citrate content, while their transcript levels decreased significantly in 'Egan No. 1' and 'Iyokan', although citrate content also decreased. In 'HB pummelo', in which acid content increased as fruit ripened, and in acid-free pummelo, transcript levels of CitACLα2, CitACLβ1, and/or CitACLα1 increased. Moreover, mild drought stress and ABA treatment significantly increased citrate contents in fruits. Transcript levels of the three genes were significantly reduced by mild drought stress, and the transcript level of only CitACLβ1 was significantly reduced by ABA treatment. Taken together, these data indicate that the effects of ACL on citrate use during fruit ripening depends on the cultivar, and the reduction in ACL gene expression may be attributed to citrate increases under mild drought stress or ABA treatment.

  17. 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

  18. 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

  19. Cloning, bioinformatics and the enzyme activity analyses of a phenylalanine ammonia-lyase gene involved in dragon's blood biosynthesis in Dracaena cambodiana.

    Science.gov (United States)

    Wang, Xing-Hong; Gong, Min; Tang, Liang; Zheng, Shui; Lou, Ji-Dong; Ou, Lingcheng; Gomes-Laranjo, José; Zhang, Changhe

    2013-01-01

    Phenylalanine ammonia-lyase (PAL) is the key enzyme of the phenylpropanoid pathway, playing an important role in plant development and defence. We cloned a partial cDNA of PAL gene, DcPAL1, from Dracaena cambodiana seedlings using RT-PCR with degenerate primers that were designed based on a multiple sequence alignment of known PAL genes from other plant species. DcPAL1 shows highly homologous to other known PAL genes registered in GenBank, being closest to that of Musa acuminata. DcPAL1 has a relatively high GC content and most of the GC is in the third codon position. It has 768 bp in size with a maximum open reading frame (ORF) of 765 bp, encoding a 255 amino acid-polypeptide. The deduced PAL protein is a stable protein, having classical PAL domains and consisting of three major hydrophobic domains. Analysis of effective number of codons (ENC) shows that DcPAL1 codons are used at equal frequency. Relatively higher usage frequency appears randomly in codons ended with any of the four bases; six codons have no usage bias. There are 45 codons showing distinct usage preference between DcPAL1 and E. coli, 20 between DcPAL1 and yeast. Therefore, the yeast system may be more suitable for the expression of DcPAL1. Upon the elicitation of Fusarium proliferatum, a potent elicitor of dragon's blood, the PAL enzyme activity in the leaves and stems of D. cambodiana and other two Dracaena spp. significantly increased, accompanying with the formation of dragon's blood, indicating the involvement of PAL in the biosynthesis of dragon's blood, a precious traditional medicine.

  20. Adaptive Evolutionary Analysis of Chicken Adenylosuccinate Lyase Gene%鸡腺苷琥珀酸裂解酶基因适用性进化分析

    Institute of Scientific and Technical Information of China (English)

    朱云芬; 张学余; 韩威; 李慧芳; 李新

    2011-01-01

    采用反转录-聚合酶链式反应(RT-PCR)方法,以肝脏总RNA为模板,对泰和乌骨鸡、隐性白羽肉鸡腺苷琥珀酸裂解酶基因(ADSL)编码区序列进行克隆和测序;并结合Gen-Bank中已提交的家鸡、鱼类、哺乳动物和人类等ADSL基因序列,采用基于最大似然法的密码子置换模型分析ADSL基因编码区序列在进化过程中承受的选择压力.结果RT-PCR方法准确获得了泰和乌骨鸡和隐性白羽肉鸡ADSL基因编码区序列,全长为1 380 bp.“分支特异模型”检验结果表明,ADSL基因在不同物种进化过程中较为保守,未受到正选择作用.鸡ADSL基因序列的“位点特异模型”检验未发现正选择位点.研究结果有助于了解鸡ADSL基因的结构及进化历程.%The coding region of ADSL gene (Adenylosuccinate Lyase)was amplified from the extracted liver total RNA in Taihe Silkies and Recessive White Boilers , and were subcloned and sequenced in both directions. Some ADSL gene sequences that had been submitted in GenBank, including sequences of domestic chicken, fish, mammals and human were also used in this study. Maximum-likelihood models of codon substitution were used to analyze diversifying selection pressures of ADSL gene coding region. The results showed RT-PCR methods acquired the aimed fragments, in a total of 1380bp. "Branch-special model" test showed ADSL genes of different species were rather conservative, not having suffered positive selection pressure. "Branch-special model" also did not find positive sites in chicken ADSL genes. The results could help to trace the route of chicken ADSL gene evolution and knowledge of its structure.

  1. HMG-CoA lyase (HL) gene: Cloning and characterization of the 5{prime} end of the mouse gene, gene targeting in ES cells, and demonstration of large deletions in three HL-deficient patients

    Energy Technology Data Exchange (ETDEWEB)

    Wang, S.; Robert, M.F.; Mitchell, G.A. [Hopital Sainte-Justine, Quebec (Canada)] [and others

    1994-09-01

    3-hydroxy-3-methylglutaryl CoA lyase (HL) is a mitochondrial matrix enzyme which catalyzes the last step of leucine catabolism and of ketogenesis. Autosomal recessive HL deficiency in humans results in episodes of hypoglycemia and coma. We are interested in the pathophysiology of HL deficiency as a model for both amino acid and fatty acid inborn errors. We have cloned the human and mouse HL genes. In order to analyze the 5{prime} nontranslated region of mouse HL gene, we cloned and sequenced a 1.8 kb fragment containing the 5{prime} extremity including exon 1 and about 1.6 kb of 5{prime} nontranslated sequence. The region surrounding exon 1 is CpG-rich (66.4%). Using the criteria of West, the Observed/Expected ratio for CpG dinucleotides is 0.7 ({ge}0.6 is consistent with a CpG island). We are carrying out primer extension and RNase protection experiments to determine the transcription initiation site. We constructed a gene targeting vector by introducing the neomycin resistance gene into exon 2 of a 7.5 kb genomic subclone of the mouse HL gene. Targeting was performed by electroporating 10 mg linearized vector into 10{sup 7} ES cells and selecting for 12 days with G418. 5/228 colonies (2.2%) had homologous recombination as shown by PCR screening and Southern analysis. We are microinjecting the 5 targeted clones into blastocysts to create an HL-deficient mouse. To date we have obtained two chimeras with contributions of 95% and 55% from 129, by coat color estimates. Three of 27 (11%) of the HL-deficient patients studied were suggested by genomic Southern analysis to be homozygous for large intragenic deletions. We confirmed this and defined the boundaries using exonic PCR.

  2. 3-Hydroxy-3-methylglutaryl CoA lyase (HL): Mouse and human HL gene (HMGCL) cloning and detection of large gene deletions in two unrelated HL-deficient patients

    Energy Technology Data Exchange (ETDEWEB)

    Wang, S.P.; Robert, M.F.; Mitchell, G.A. [Hopital Sainte-Justine, Quebec (Canada)] [and others

    1996-04-01

    3-hydroxy-3-methylglutaryl CoA lyase (HL, EC 4.1.3.4) catalyzes the cleavage of 3-hydroxy-3-methylglutaryl CoA to acetoacetic acid and acetyl CoA, the final reaction of both ketogenesis and leucine catabolism. Autosomal-recessive HL deficiency in humans results in episodes of hypoketotic hypoglycemia and coma. Using a mouse HL cDNA as a probe, we isolated a clone containing the full-length mouse HL gene that spans about 15 kb of mouse chromosome 4 and contains nine exons. The promoter region of the mouse HL gene contains elements characteristic of a housekeeping gene: a CpG island containing multiple Sp1 binding sites surrounds exon 1, and neither a TATA nor a CAAT box are present. We identified multiple transcription start sites in the mouse HL gene, 35 to 9 bases upstream of the translation start codon. We also isolated two human HL genomic clones that include HL exons 2 to 9 within 18 kb. The mouse and human HL genes (HGMW-approved symbol HMGCL) are highly homologous, with identical locations of intron-exon junctions. By genomic Southern blot analysis and exonic PCR, was found 2 of 33 HL-deficient probands to be homozygous for large deletions in the HL gene. 26 refs., 4 figs., 2 tabs.

  3. Isolation and Functional Characterization of a Phenylalanine Ammonia-Lyase Gene (SsPAL1 from Coleus (Solenostemon scutellarioides (L. Codd

    Directory of Open Access Journals (Sweden)

    Qinlong Zhu

    2015-09-01

    Full Text Available Phenylalanine ammonia-lyase (PAL is the first enzyme involved in the phenylpropanoid pathway and plays important roles in the secondary metabolisms, development and defense of plants. To study the molecular function of PAL in anthocyanin synthesis of Coleus (Solenostemon scutellarioides (L. Codd, a Coleus PAL gene designated as SsPAL1 was cloned and characterized using a degenerate oligonucleotide primer PCR and RACE method. The full-length SsPAL1 was 2450 bp in size and consisted of one intron and two exons encoding a polypeptide of 711 amino acids. The deduced SsPAL1 protein showed high identities and structural similarities with other functional plant PAL proteins. A series of putative cis-acting elements involved in transcriptional regulation, light and stress responsiveness were found in the upstream regulatory sequence of SsPAL1. Transcription pattern analysis indicated that SsPAL1 was constitutively expressed in all tissues examined and was enhanced by light and different abiotic factors. The recombinant SsPAL1 protein exhibited high PAL activity, at optimal conditions of 60 °C and pH 8.2. Although the levels of total PAL activity and total anthocyanin concentration have a similar variation trend in different Coleus cultivars, there was no significant correlation between them (r = 0.7529, p > 0.1, suggesting that PAL was not the rate-limiting enzyme for the downstream anthocyanin biosynthetic branch in Coleus. This study enables us to further understand the role of SsPAL1 in the phenylpropanoid (flavonoids, anthocyanins biosynthesis in Coleus at the molecular level.

  4. Cloning and knockout of formate hydrogen lyase and H{sub 2}-uptake hydrogenase genes in Enterobacter aerogenes for enhanced hydrogen production

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Hongxin; Ma, Kun; Lu, Yuan; Zhang, Chong; Wang, Liyan; Xing, Xin-Hui [Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Tsinghua Yuan, Beijing 100084 (China)

    2009-01-15

    A 5431-bp DNA fragment partially encoding the formate hydrogen lyase (FHL) gene cluster hycABCDE was isolated and identified from Enterobacter aerogenes IAM1183 chromosomal DNA. All the five putative gene products showed a high degree of homology to the reported bacterial FHL proteins. The gene hycA, encoding the FHL repressor protein, and hybO, encoding the small subunit of the uptake hydrogenase, were targeted for genetic knockout for improving the hydrogen production. The pYM-Red recombination system was adopted to form insertional mutations in the E. aerogenes genome, thereby creating mutant strains of IAM1183-A ({delta} hycA), IAM1183-O ({delta} hybO), and IAM1183-AO ({delta} hycA/ {delta} hybO double knockout). The hydrogen production experiments with these mutants showed that the maximum specific hydrogen productivities of IAM1183-A, IAM1183-O, and IAM1183-AO were 2879.466 {+-} 38.59, 2747.203 {+-} 13.25 and 3372.019 {+-} 4.39 (ml h{sup -1} g{sup -1}dry cell weight), respectively, higher than that of the wild strain (2321.861 {+-} 15.34 ml h{sup -1} g{sup -1}dry cell weight). The total H{sub 2} yields by the three mutants IAM1183-A, IAM1183-O and IAM1183-AO were 0.73, 0.78, and 0.83 mol-H{sub 2}/mol glucose, respectively, while the wild-type IAM1183 was only 0.65 mol-H{sub 2}/mol glucose. The metabolites of the mutants including acetate, ethanol, 2,3-butanediol and succinate were all increased compared with that of the wild type, implying the changed metabolic flux by the mutation. In the fermentor cultivation with IAM1183 {delta} hycA/ {delta} hybO, the total hydrogen volume after 16 h cultivation reached 4.4 L, while that for the wild type was only 2.9 L. (author)

  5. Millimagnitude Photometry for Transiting Extrasolar Planetary Candidates. V. Follow-up of 30 OGLE Transits. New Candidates

    CERN Document Server

    Pietrukowicz, P; Diaz, R F; Fernández, J M; Zoccali, M; Gieren, W; Pietrzynski, G; Ruiz, M T; Udalski, A; Szeifert, T; Hempel, M

    2009-01-01

    We used VLT/VIMOS images in the V band to obtain light curves of extrasolar planetary transits OGLE-TR-111 and OGLE-TR-113, and candidate planetary transits: OGLE-TR-82, OGLE-TR-86, OGLE-TR-91, OGLE-TR-106, OGLE-TR-109, OGLE-TR-110, OGLE-TR-159, OGLE-TR-167, OGLE-TR-170, OGLE-TR-171. Using difference imaging photometry, we were able to achieve millimagnitude errors in the individual data points. We present the analysis of the data and the light curves, by measuring transit amplitudes and ephemerides, and by calculating geometrical parameters for some of the systems. We observed 9 OGLE objects at the predicted transit moments. Two other transits were shifted in time by a few hours. For another seven objects we expected to observe transits during the VIMOS run, but they were not detected. The stars OGLE-TR-111 and OGLE-TR-113 are probably the only OGLE objects in the observed sample to host planets, with the other objects being very likely eclipsing binaries or multiple systems. In this paper we also report on ...

  6. OGLE-ing the Magellanic System: Photometric Metallicity from Fundamental Mode RR Lyrae Stars

    CERN Document Server

    Skowron, D M; Udalski, A; Szymański, M K; Pietrukowicz, P; Poleski, R; Wyrzykowski, Ł; Ulaczyk, K; Kozłowski, S; Skowron, J; Mróz, P; Pawlak, M

    2016-01-01

    In an era of extensive photometric observations, the catalogs of RR Lyrae type variable stars number tens of thousands of objects. The relation between the iron abundance [Fe/H] and the Fourier parameters of the stars light curve allows us to investigate mean metallicities and metallicity gradients in various stellar environments, independently of time-consuming spectroscopic observations. In this paper we use almost 15000 V- and I-band light curves of fundamental mode RR Lyrae stars from the OGLE-IV survey to provide a precise relation between the V- and I-band phase parameter phi_31 used to estimate [Fe/H]. We apply this relation to metallicity formulae developed for the Johnson V- and the Kepler Kp-band to obtain the relation between [Fe/H] and phi_31 for the I-band photometry. Last, we apply the new relation of Nemec et al. (2013) to the OGLE-IV fundamental mode RR Lyrae stars data and construct a metallicity map of the Magellanic Clouds. Median [Fe/H] is -1.39 +- 0.44 dex for the LMC and -1.77 +- 0.48 de...

  7. 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

    by expression in E. coli and purification of Phn-polypeptides. PhnG, PhnH, PhnI, PhnJ, and PhnK copurify as a protein complex by ion-exchange, size-exclusion, and affinity chromatography. The five polypeptides also comigrate in native-PAGE. Cross-linking of the purified protein complex reveals a close proximity...... is suggested to be PhnG4H2I2J2K. Deletion of individual phn genes reveals that a strain harboring plasmid-borne phnGHIJ produces a protein complex consisting of PhnG, PhnH, PhnI, and PhnJ, whereas a strain harboring plasmid-borne phnGIJK produces a protein complex consisting of PhnG and PhnI. We conclude...

  8. Developmental gonadal expression of the transcription factor SET and its target gene, P450c17 (17alpha-hydroxylase/c17,20 lyase).

    Science.gov (United States)

    Zhang, P; Compagnone, N A; Fiore, C; Vigne, J L; Culp, P; Musci, T J; Mellon, S H

    2001-10-01

    Cytochrome P450c17 catalyzes the 17alpha-hydroxylase/17,20 lyase activity needed for sex steroid synthesis. We recently characterized the nuclear phosphoprotein SET as a novel transcriptional regulator that binds to the -447/-399 region of the rat P450c17 gene, along with the transcription factors COUP-TF II, NGF-IB, and SF-1. Gel shift studies localized SET binding to nucleotides -410/-402. We have shown that SET activates transcription of the rat P450c17 gene in neuronal precursor cells and now show that it also activates transcription from the -418/-399 region of the rat P450c17 gene in mouse Leydig MA-10 cells. Studying the ontogenic expression of SET and P450c17 in the rodent gonad, we found that SET expression preceded P450c17 expression in the embryonic genital ridge, suggesting that SET may be important for initiating P450c17 expression in this region. Expression of SET also preceded P450c17 expression in the testis and ovary, and its expression was much greater during embryogenesis than in the adult gonad. In the adult rat testis, P450c17 was expressed only in Leydig cells, while SET was expressed in Leydig cells and in spermatocytes. In the adult rat ovary, P450c17 was expressed only in theca cells, while SET was expressed in theca cells and also in oocytes. Because SET is expressed early in development in the genital ridge and in the testis and ovary, and because SET has many functions in addition to its activity as a transcription factor, we determined whether SET acts a transcription factor in oocytes. The SET protein was detected by Western blots in Xenopus oocytes from stages II through VI and in mature oocytes. Using extracts of Xenopus oocytes in gel shift assays, we detected a protein that bound to the -418/-399 region of the rat P450c17 gene, to which SET binds. Nuclear injection of either a -418/-399TK32LUC wildtype reporter construct or a construct containing a mutant SET site into Xenopus oocytes from stages III through VI resulted in

  9. 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%。

  10. OGLE-ING the Magellanic System: Stellar Populations in the Magellanic Bridge

    Science.gov (United States)

    Skowron, D. M.; Jacyszyn, A. M.; Udalski, A.; Szymański, M. K.; Skowron, J.; Poleski, R.; Kozłowski, S.; Kubiak, M.; Pietrzyński, G.; Soszyński, I.; Mróz, P.; Pietrukowicz, P.; Ulaczyk, K.; Wyrzykowski, Ł.

    2014-11-01

    We report the discovery of a young stellar bridge that forms a continuous connection between the Magellanic Clouds. This finding is based on number density maps for stellar populations found in data gathered by OGLE-IV that fully cover over 270 deg2 of the sky in the Magellanic Bridge area. This is the most extensive optical survey of this region to date. We find that the young population is present mainly in the western half of the MBR, which, together with the newly discovered young population in the eastern Bridge, form a continuous stream of stars connecting both galaxies along δ ~ -73.5 deg. The young population distribution is clumped, with one of the major densities close to the SMC and the other fairly isolated and located approximately mid-way between the Clouds, which we call the OGLE island. These overdensities are well matched by H I surface density contours, although the newly found young population in the eastern Bridge is offset by ~2 deg north from the highest H I density contour. We observe a continuity of red clump stars between the Magellanic Clouds which represent an intermediate-age population. Red clump stars are present mainly in the southern and central parts of the Magellanic Bridge, below its gaseous part, and their presence is reflected by a strong deviation from the radial density profiles of the two galaxies. This may indicate either a tidal stream of stars, or that the stellar halos of the two galaxies overlap. On the other hand, we do not observe such an overlap within an intermediate-age population represented by the top of the red giant branch and the asymptotic giant branch stars. We also see only minor mixing of the old populations of the Clouds in the southern part of the Bridge, represented by the lowest part of the red giant branch.

  11. OGLE-ing the Magellanic System: Photometric Metallicity from Fundamental Mode RR Lyrae Stars

    Science.gov (United States)

    Skowron, D. M.; Soszyński, I.; Udalski, A.; Szymański, M. K.; Pietrukowicz, P.; Skowron, J.; Poleski, R.; Wyrzykowski, Ł.; Ulaczyk, K.; Kozłowski, S.; Mróz, P.; Pawlak, M.

    2016-09-01

    In an era of extensive photometric observations, the catalogs of RR Lyr type variable stars number tens of thousands of objects. The relation between the iron abundance [Fe/H] and the Fourier parameters of the stars light curve allows us to investigate mean metallicities and metallicity gradients in various stellar environments, independently of time-consuming spectroscopic observations. In this paper we use almost 6500 V- and I-band light curves of fundamental mode RR Lyr stars from the OGLE-IV survey to provide a relation between the V- and I-band phase parameter ϕ31 used to estimate [Fe/H]. The relation depends on metallicity, which limits its applicability. We apply this relation to metallicity formulae developed for the Johnson V- and the Kepler Kp-band to obtain the relation between [Fe/H] and ϕ31 for the I-band photometry. Last, we apply the new relation of Nemec to the OGLE-IV fundamental mode RR Lyr stars data and construct a metallicity map of the Magellanic Clouds. Median [Fe/H] is -1.39±0.44 dex for the LMC and -1.77±0.48 dex for the SMC, on the Jurcsik metallicity scale. We also find a metallicity gradient within the LMC with a slope of -0.029±0.002 dex/kpc in the inner 5 kpc and -0.030±0.003 dex/kpc beyond 8 kpc, and no gradient in-between (-0.019±0.002 dex/kpc integrally). We do not observe a metallicity gradient in the SMC, although we show that the metal-rich RRab stars are more concentrated toward the SMC center than the metal-poor.

  12. REANALYSES OF ANOMALOUS GRAVITATIONAL MICROLENSING EVENTS IN THE OGLE-III EARLY WARNING SYSTEM DATABASE WITH COMBINED DATA

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, J.; Park, H.; Han, C. [Department of Physics, Institute for Astrophysics, Chungbuk National University, Cheongju 361-763 (Korea, Republic of); Gould, A.; Poleski, R. [Department of Astronomy, Ohio State University, 140 W. 18th Ave., Columbus, OH 43210 (United States); Udalski, A.; Szymański, M. K.; Pietrzyński, G.; Soszyński, I.; Ulaczyk, K.; Wyrzykowski, Ł. [Warsaw University Observatory, Al. Ujazdowskie 4, 00-478 Warszawa (Poland); Abe, F.; Fukunaga, D.; Itow, Y. [Solar-Terrestrial Environment Laboratory, Nagoya University, Nagoya, 464-8601 (Japan); Bennett, D. P. [Department of Physics, University of Notre Dame, 225 Nieuwland Science Hall, Notre Dame, IN 46556-5670 (United States); Bond, I. A. [Institute of Information and Mathematical Sciences, Massey University, Private Bag 102-904, North Shore Mail Centre, Auckland (New Zealand); Botzler, C. S.; Freeman, M. [Department of Physics, University of Auckland, Private Bag 92-019, Auckland 1001 (New Zealand); Fukui, A. [Okayama Astrophysical Observatory, National Astronomical Observatory of Japan, Asakuchi, Okayama 719-0232 (Japan); Koshimoto, N. [Department of Earth and Space Science, Osaka University, Osaka 560-0043 (Japan); Collaboration: (The OGLE Collaboration); (The MOA Collaboration); (The PLANET Collaboration); (The μFUN Collaboration); (The RoboNet Collaboration); and others

    2015-05-01

    We reanalyze microlensing events in the published list of anomalous events that were observed from the Optical Gravitational Lensing Experiment (OGLE) lensing survey conducted during the 2004–2008 period. In order to check the existence of possible degenerate solutions and extract extra information, we conduct analyses based on combined data from other survey and follow-up observation and consider higher-order effects. Among the analyzed events, we present analyses of eight events for which either new solutions are identified or additional information is obtained. We find that the previous binary-source interpretations of five events are better interpreted by binary-lens models. These events include OGLE-2006-BLG-238, OGLE-2007-BLG-159, OGLE-2007-BLG-491, OGLE-2008-BLG-143, and OGLE-2008-BLG-210. With additional data covering caustic crossings, we detect finite-source effects for six events including OGLE-2006-BLG-215, OGLE-2006-BLG-238, OGLE-2006-BLG-450, OGLE-2008-BLG-143, OGLE-2008-BLG-210, and OGLE-2008-BLG-513. Among them, we are able to measure the Einstein radii of three events for which multi-band data are available. These events are OGLE-2006-BLG-238, OGLE-2008-BLG-210, and OGLE-2008-BLG-513. For OGLE-2008-BLG-143, we detect higher-order effects induced by the changes of the observer’s position caused by the orbital motion of the Earth around the Sun. In addition, we present degenerate solutions resulting from the known close/wide or ecliptic degeneracy. Finally, we note that the masses of the binary companions of the lenses of OGLE-2006-BLG-450 and OGLE-2008-BLG-210 are in the brown-dwarf regime.

  13. Two pectin lyase genes, pnl-1 and pnl-2, from Colletotrichum gloeosporioides f. sp. malvae differ in a cellulose-binding domain and in their expression during infection of Malva pusilla.

    Science.gov (United States)

    Wei, Yangdou; Shih, Jenny; Li, Jieran; Goodwin, Paul H

    2002-07-01

    Two pectin lyase genes, designated pnl-1 and pnl-2, were cloned from Colletotrichum gloeosporioides f. sp. malvae, a pathogen of round-leaved mallow (Malva pusilla). pnl-1 was isolated using cDNA from infected plant material; pnl-2 was isolated using cDNA from 3-day-old mycelia grown in mallow-cell-wall extract (MCWE) broth. pnl-1 is the first pectinase gene described thus far to encode a cellulose-binding domain (CBD), which is common in cellulases and xylanases, whereas pnl-2 encodes a pectin lyase that lacks a CBD. In pure culture, pnl-1 expression could be detected when purified pectin or glucose was the sole carbon source, but not when MCWE was the sole carbon source. The lack of pnl-1 expression appeared to be due to gene repression by some unknown factor(s) in the cell-wall extract. In contrast, expression of pnl-2 was detected in cultures when MCWE, but not when purified pectin or glucose, was the sole carbon source. In infected tissue, detection of pnl-1 expression by Northern-blot hybridization and by RT-PCR began with the onset of the necrotrophic phase of infection. Expression ofpnl-2 was not detectable by Northern-blot hybridization, but was observed byRT-PCR in both the biotrophic and necrotrophic phases of infection. The differences between pnl-1 and pnl-2 (i.e. pnl-1 encoding a CBD and differences in the expression patterns of both genes) may be related to the requirements of C. gloeosporioides f. sp. malvae to be able to grow in host tissue under the different conditions present during the biotrophic and necrotrophic phases of infection.

  14. 植物苯丙氨酸解氨酶基因的研究进展%Research Advances on Plant Phenylalanine Ammonia-Lyase Gene

    Institute of Scientific and Technical Information of China (English)

    董艳珍

    2006-01-01

    苯丙氨酸解氨酶(phenylalanine ammonia-lyase, PAL)是连接植物初级代谢和苯丙烷类代谢、催化苯丙烷类代谢第一步反应的酶.综述植物PAL基因的研究进展,主要包括PAL基因的结构特点、表达特点和PAL基因表达的调控机制,并指出今后对PAL基因的研究方向.

  15. Stress enhances the gene expression and enzyme activity of phenylalanine ammonia-lyase and the endogenous content of salicylic acid to induce flowering in pharbitis.

    Science.gov (United States)

    Wada, Kaede C; Mizuuchi, Kaori; Koshio, Aya; Kaneko, Kentaro; Mitsui, Toshiaki; Takeno, Kiyotoshi

    2014-07-01

    The involvement of salicylic acid (SA) in the regulation of stress-induced flowering in the short-day plant pharbitis (also called Japanese morning glory) Ipomoea nil (formerly Pharbitis nil) was studied. Pharbitis cv. Violet was induced to flower when grown in 1/100-strength mineral nutrient solution under non-inductive long-day conditions. All fully expanded true leaves were removed from seedlings, leaving only the cotyledons, and flowering was induced under poor-nutrition stress conditions. This indicates that cotyledons can play a role in the regulation of poor-nutrition stress-induced flowering. The expression of the pharbitis homolog of PHENYLALANINE AMMONIA-LYASE, the enzyme activity of phenylalanine ammonia-lyase (PAL; E.C. 4.3.1.5) and the content of SA in the cotyledons were all up-regulated by the stress treatment. The Violet was also induced to flower by low-temperature stress, DNA demethylation and short-day treatment. Low-temperature stress enhanced PAL activity, whereas non-stress factors such as DNA demethylation and short-day treatment decreased the activity. The PAL enzyme activity was also examined in another cultivar, Tendan, obtaining similar results to Violet. The exogenously applied SA did not induce flowering under non-stress conditions but did promote flowering under weak stress conditions in both cultivars. These results suggest that stress-induced flowering in pharbitis is induced, at least partly, by SA, and the synthesis of SA is promoted by PAL.

  16. Isolation of the patC gene encoding the cystathionine beta-lyase of Lactobacillus delbrueckii subsp. bulgaricus and molecular analysis of inter-strain variability in enzyme biosynthesis.

    Science.gov (United States)

    Aubel, Dominique; Germond, Jacques Edouard; Gilbert, Christophe; Atlan, Danièle

    2002-07-01

    The patC gene encoding the cystathionine beta-lyase (CBL) of Lactobacillus delbrueckii subsp. bulgaricus NCDO 1489 was cloned and expressed in Escherichia coli. Overexpression of CBL complemented the methionine auxotrophy of an E. coli metC mutant, demonstrating in vivo that this enzyme functions as a CBL. However, PatC is distinguishable from the MetC CBLs by a low identity in amino acid sequence, a sensitivity to iodoacetic acid, greater thermostability and a lower substrate affinity. Homologues of patC were detected in the 13 Lb. delbrueckii strains studied, but only seven of them showed CBL activity. In constrast to CBL(+) strains, all CBL-deficient strains analysed were auxotrophic for methionine. This supports the hypothesis that CBLs from lactobacilli are probably involved in methionine biosynthesis. Moreover, the results of this study suggest that post-transcriptional mechanisms account for the differences in CBL activities observed between strains of Lb. delbrueckii.

  17. Reanalyses of Anomalous Gravitational Microlensing Events in the OGLE-III Early Warning System Database with Combined Data

    CERN Document Server

    Jeong, J; Han, C; Gould, A; Udalski, A; Szymański, M K; Pietrzyński, G; Soszyński, I; Poleski, R; Ulaczyk, K; Wyrzykowski, Ł; Abe, F; Bennett, D P; Bond, I A; Botzler, C S; Freeman, M; Fukui, A; Fukunaga, D; Itow, Y; Koshimoto, N; Masuda, K; Matsubara, Y; Muraki, Y; Namba, S; Ohnishi, K; Rattenbury, N J; Saito, To; Sullivan, D J; Sweatman, W L; Sumi, T; Suzuki, D; Tristram, P J; Tsurumi, N; Wada, K; Yamai, N; Yock, P C M; Yonehara, A; Albrow, M D; Batista, V; Beaulieu, J -P; Caldwell, J A R; Cassan, A; Cole, A; Coutures, C; Dieters, S; Dominik, M; Prester, D Dominis; Donatowicz, J; Fouqué, P; Greenhill, J; Hoffman, M; Huber, M; Jørgensen, U G; Kane, S R; Kubas, D; Martin, R; Marquette, J -B; Menzies, J; Pitrou, C; Pollard, K; Sahu, K C; Vinter, C; Wambsganss, J; Williams, A; Allen, W; Bolt, G; Choi, J -Y; Christie, G W; DePoy, D L; Drummond, J; Gaudi, B S; Hwang, K -H; Jung, Y K; Lee, C -U; Mallia, F; Maoz, D; Maury, A; McCormick, J; Monard, L A G; Moorhouse, D; Natusch, T; Ofek, E O; Park, B -G; Pogge, R W; Santallo, R; Shin, I -G; Thornley, G; Yee, J C; Bramich, D M; Horne, K; Hundertmark, M; Kains, N; Snodgrass, C; Steele, I; Street, R; Tsapras, Y

    2015-01-01

    We reanalyze microlensing events in the published list of anomalous events that were observed from the OGLE lensing survey conducted during 2004-2008 period. In order to check the existence of possible degenerate solutions and extract extra information, we conduct analyses based on combined data from other survey and follow-up observation and consider higher-order effects. Among the analyzed events, we present analyses of 8 events for which either new solutions are identified or additional information is obtained. We find that the previous binary-source interpretations of 5 events are better interpreted by binary-lens models. These events include OGLE-2006-BLG-238, OGLE-2007-BLG-159, OGLE-2007-BLG-491, OGLE-2008-BLG-143, and OGLE-2008-BLG-210. With additional data covering caustic crossings, we detect finite-source effects for 6 events including OGLE-2006-BLG-215, OGLE-2006-BLG-238, OGLE-2006-BLG-450, OGLE-2008-BLG-143, OGLE-2008-BLG-210, and OGLE-2008-BLG-513. Among them, we are able to measure the Einstein ...

  18. 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.

  19. 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.

  20. OGLE-BLG182.1.162852: An Eclipsing Binary with a Circumstellar Disk

    CERN Document Server

    Rattenbury, N J; Kostrzewa-Rutkowska, Z; Udalski, A; Kozłowski, S; Szymański, M K; Pietrzyński, G; Soszyński, I; Poleski, R; Ulaczyk, K; Skowron, J; Pietrukowicz, P; Mróz, P; Skowron, D

    2014-01-01

    We present the discovery of a plausible disk-eclipse system OGLE-BLG182.1.162852. The OGLE light curve for OGLE-BLG182.1.162852 shows three episodes of dimming by $I \\simeq 2 - 3$ magnitudes, separated by 1277 days. The shape of the light curve during dimming events is very similar to that of known disk eclipse system OGLE-LMC-ECL-11893 (Dong et al. 2014). The event is presently undergoing a dimming event, predicted to end on December 30th, 2014. We encourage spectroscopic and multi-band photometric observations now. The next dimming episode for OGLE-BLG182.1.162852 is expected to occur in March 2018.

  1. Multiple Genes in a Single Host: Cost-Effective Production of Bacterial Laccase (cotA), Pectate Lyase (pel), and Endoxylanase (xyl) by Simultaneous Expression and Cloning in Single Vector in E. coli

    Science.gov (United States)

    Kumar, Sandeep; Jain, Kavish Kumar; Bhardwaj, Kailash N.; Chakraborty, Subhojit; Kuhad, Ramesh Chander

    2015-01-01

    This study attempted to reduce the enzyme production cost for exploiting lignocellulosic materials by expression of multiple genes in a single host. Genes for bacterial laccase (CotA), pectate lyase (Pel) and endoxylanase (Xyl), which hold significance in lignocellulose degradation, were cloned in pETDuet-1 vector containing two independent cloning sites (MCS). CotA and xyl genes were cloned in MCS1 and MCS 2, respectively. Pel gene was cloned by inserting complete cassette (T7 promoter, ribosome binding site, pel gene, His tag and complete gene ORF) preceded by cotA open reading frame in the MCS1. IPTG induction of CPXpDuet-1 construct in E. coli BL21(DE3) resulted in expression of all three heterologous proteins of ~65 kDa (CotA), ~45 kDa (Pel) and ~25 kDa (Xyl), confirmed by SDS-PAGE and western blotting. Significant portions of the enzymes were also found in culture supernatant (~16, ~720 and ~370 IU/ml activities of CotA, Pel and Xyl, respectively). Culture media optimization resulted in 2, 3 and 7 fold increased secretion of recombinant CotA, Pel and Xyl, respectively. Bioreactor level optimization of the recombinant cocktail expression resulted in production of 19 g/L dry cell biomass at OD600nm 74 from 1 L induced culture after 15 h of cultivation, from which 9, 627 and 1090 IU/ml secretory enzyme activities of CotA, Xyl and Pel were obtained, respectively. The cocktail was also found to increase the saccharification of orange peel in comparison to the xylanase alone. Thus, simultaneous expression as well as extra cellular secretion of these enzymes as cocktail can reduce the enzyme production cost which increases their applicability specially for exploiting lignocellulosic materials for their conversion to value added products like alcohol and animal feed. PMID:26642207

  2. 紫苏苯丙氨酸解氨酶基因片段克隆及序列分析%Molecular Cloning and Sequence Analysis of Phenylalanine Ammonia-lyase Gene Fragment in Perilla frutescens

    Institute of Scientific and Technical Information of China (English)

    吕晓玲; 孙雪梅; 王芳; 郝磊; 孙晶磊

    2011-01-01

    Phenylalanine ammonia-lyase (PAL), responsible for catalyzing the conversion of phenylalanine to cinnamic acid to finish the first step of phenylalanine pathway, was the key enzyme during the biosynthesis of rosmarinic acid. The cDNA fragment of PAL gene was successfully cloned by homology cloning method (Accession No. HQ388347.1), 399 bp and encoded 133 amino acids. It was designated as PerPAL-1. The results of amino acid sequence analysis showed that the identity of the sequence of PerPAL-1 amino acid with that of Salvia miltiorrhiza and Agastache rugosa was 96f and 95%, respectively. Phylogenetic tree analysis revealed that PerPAL-1 had closer relationship with PALs from Lamiaceac plants than those of other plants.The expression of PerPAL-1 gene was the strongest in young leaves and the weakest in stems.%苯丙氨酸解氨酶(Phenylalanine ammonia-lyase,PAL)是迷迭香酸合成途径中苯丙氨酸支路的关键酶,它催化苯丙氨酸生成肉桂酸,完成该支路第一步反应.本实验利用同源克隆方法成功克隆了紫苏PAL基因cDNA片段,命名为PerPAL-1(GenBank登录号:HQ388347.1),该片段长399bp,编码133个氨基酸.通过氨基酸序列比对分析,发现其氨基酸序列与丹参和藿香PAL该片段的同源性分别高达96%和95%.PAL系统进化树表明PerPAL-1与唇形科植物的PAL亲缘关系最近.PerPAL-1基因在叶中表达最强,根中次之,而在茎中表达最弱.

  3. Cystathionine γ-lyase, a H2S-generating enzyme, is a GPBAR1-regulated gene and contributes to vasodilation caused by secondary bile acids.

    Science.gov (United States)

    Renga, Barbara; Bucci, Mariarosaria; Cipriani, Sabrina; Carino, Adriana; Monti, Maria Chiara; Zampella, Angela; Gargiulo, Antonella; d'Emmanuele di Villa Bianca, Roberta; Distrutti, Eleonora; Fiorucci, Stefano

    2015-07-01

    GPBAR1 is a bile acid-activated receptor (BAR) for secondary bile acids, lithocholic (LCA) and deoxycholic acid (DCA), expressed in the enterohepatic tissues and in the vasculature by endothelial and smooth muscle cells. Despite that bile acids cause vasodilation, it is unclear why these effects involve GPBAR1, and the vascular phenotype of GPBAR1 deficient mice remains poorly defined. Previous studies have suggested a role for nitric oxide (NO) in regulatory activity exerted by GPBAR1 in liver endothelial cells. Hydrogen sulfide (H2S) is a vasodilatory agent generated in endothelial cells by cystathionine-γ-lyase (CSE). Here we demonstrate that GPBAR1 null mice had increased levels of primary and secondary bile acids and impaired vasoconstriction to phenylephrine. In aortic ring preparations, vasodilation caused by chenodeoxycholic acid (CDCA), a weak GPBAR1 ligand and farnesoid-x-receptor agonist (FXR), was iberiotoxin-dependent and GPBAR1-independent. In contrast, vasodilation caused by LCA was GPBAR1 dependent and abrogated by propargyl-glycine, a CSE inhibitor, and by 5β-cholanic acid, a GPBAR1 antagonist, but not by N(5)-(1-iminoethyl)-l-ornithine (l-NIO), an endothelial NO synthase inhibitor, or iberiotoxin, a large-conductance calcium-activated potassium (BKCa) channels antagonist. In venular and aortic endothelial (HUVEC and HAEC) cells GPBAR1 activation increases CSE expression/activity and H2S production. Two cAMP response element binding protein (CREB) sites (CREs) were identified in the CSE promoter. In addition, TLCA stimulates CSE phosphorylation on serine residues. In conclusion we demonstrate that GPBAR1 mediates the vasodilatory activity of LCA and regulates the expression/activity of CSE. Vasodilation caused by CDCA involves BKCa channels. The GPBAR1/CSE pathway might contribute to endothelial dysfunction and hyperdynamic circulation in liver cirrhosis.

  4. 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.

  5. OGLE-IV Real-Time Transient Search

    CERN Document Server

    Wyrzykowski, L; Kozlowski, S; Udalski, A; Poleski, R; Skowron, J; Blagorodnova, N; Kubiak, M; Szymanski, M K; Pietrzynski, G; Soszynski, I; Ulaczyk, K; Pietrukowicz, P; Mroz, P

    2014-01-01

    We present the design and first results of a real-time search for transients within the 650 sq. deg. area around the Magellanic Clouds, conducted as part of the OGLE-IV project and aimed at detecting supernovae, novae and other events. The average sampling of about 4 days from September to May, yielded a detection of 238 transients in 2012/2013 and 2013/2014 seasons. The superb photometric and astrometric quality of the OGLE data allows for numerous applications of the discovered transients. We use this sample to prepare and train a Machine Learning-based automated classifier for early light curves, which distinguishes major classes of transients with more than 80% of correct answers. Spectroscopically classified 49 supernovae Type Ia are used to construct a Hubble Diagram with statistical scatter of about 0.3 mag and fill the least populated region of the redshifts range in the Union sample. We investigate the influence of host galaxy environments on supernovae statistics and find the mean host extinction of...

  6. OGLE Atlas of Classical Novae II. Magellanic Clouds

    CERN Document Server

    Mroz, P; Poleski, R; Soszynski, I; Szymanski, M K; Pietrzynski, G; Wyrzykowski, L; Ulaczyk, K; Kozlowski, S; Pietrukowicz, P; Skowron, J

    2016-01-01

    The population of classical novae in the Magellanic Clouds was poorly known because of a lack of systematic studies. There were some suggestions that nova rates per unit mass in the Magellanic Clouds were higher than in any other galaxy. Here, we present an analysis of data collected over sixteen years by the OGLE survey with the aim of characterizing nova population in the Clouds. We found twenty eruptions of novae, half of them are new discoveries. We robustly measure the nova rates of $2.4 \\pm 0.8$ yr$^{-1}$ (LMC) and $0.9 \\pm 0.4$ yr$^{-1}$ (SMC) and confirm that K-band luminosity-specific nova rates in both Clouds are 2-3 times higher than in other galaxies. This can be explained by the star formation history in the Magellanic Clouds, specifically a re-ignition of the star formation rate a few Gyr ago. We also present the discovery of an intriguing system OGLE-MBR133.25.1160 which mimics recurrent nova eruptions.

  7. OGLE Atlas of Classical Novae. II. Magellanic Clouds

    Science.gov (United States)

    Mróz, P.; Udalski, A.; Poleski, R.; Soszyński, I.; Szymański, M. K.; Pietrzyński, G.; Wyrzykowski, Ł.; Ulaczyk, K.; Kozłowski, S.; Pietrukowicz, P.; Skowron, J.

    2016-01-01

    The population of classical novae in the Magellanic Clouds was poorly known because of a lack of systematic studies. There were some suggestions that nova rates per unit mass in the Magellanic Clouds were higher than in any other galaxy. Here, we present an analysis of data collected over 16 years by the OGLE survey with the aim of characterizing the nova population in the Clouds. We found 20 eruptions of novae, half of which are new discoveries. We robustly measure nova rates of 2.4 ± 0.8 yr-1 (LMC) and 0.9 ± 0.4 yr-1 (SMC) and confirm that the K-band luminosity-specific nova rates in both Clouds are 2-3 times higher than in other galaxies. This can be explained by the star formation history in the Magellanic Clouds, specifically the re-ignition of the star formation rate a few Gyr ago. We also present the discovery of the intriguing system OGLE-MBR133.25.1160, which mimics recurrent nova eruptions.

  8. 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 ...

  9. [Cystathionine γ-lyase].

    Science.gov (United States)

    Jurkowska, Halina; Kaczor-Kamińska, Marta; Bronowicka-Adamska, Patrycja; Wróbel, Maria

    2014-01-01

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

  10. Cystathionine γ-lyase

    Directory of Open Access Journals (Sweden)

    Halina Jurkowska

    2014-01-01

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

  11. OGLE-TR-211 - a new transiting inflated hot Jupiter from the OGLE survey and ESO LP666 spectroscopic follow-up program

    CERN Document Server

    Udalski, A; Naef, D; Melo, C; Bouchy, F; Santos, N C; Moutou, C; Diaz, R F; Gieren, W; Gillon, M; Hoyer, S; Mayor, M; Mazeh, T; Minniti, D; Pietrzynski, G; Queloz, D; Ramírez, S; Ruiz, M T; Tamuz, O; Udry, S; Zoccali, M; Kubiak, M; Szymanski, M K; Soszynski, I; Szewczyk, O; Ulaczyk, K; Wyrzykowski, L

    2007-01-01

    We present results of the photometric campaign for planetary and low-luminosity object transits conducted by the OGLE survey in 2005 season (Campaign #5). About twenty most promising candidates discovered in these data were subsequently verified spectroscopically with the VLT/FLAMES spectrograph. One of the candidates, OGLE-TR-211, reveals clear changes of radial velocity with small amplitude of 82 m/sec, varying in phase with photometric transit ephemeris. Thus, we confirm the planetary nature of the OGLE-TR-211 system. Follow-up precise photometry of OGLE-TR-211 with VLT/FORS together with radial velocity spectroscopy supplemented with high resolution, high S/N VLT/UVES spectra allowed us to derive parameters of the planet and host star. OGLE-TR-211b is a hot Jupiter orbiting a F7-8 spectral type dwarf star with the period of 3.68 days. The mass of the planet is equal to 1.03+/-0.20 M_Jup while its radius 1.36+0.18-0.09 R_Jup. The radius is about 20% larger than the typical radius of hot Jupiters of similar...

  12. VizieR Online Data Catalog: OGLE LC classification of MC Cepheids (Garcia-Varela+, 2016)

    Science.gov (United States)

    Garcia-Varela, A.; Munoz, J. R.; Sabogal, B. E.; Vargas Dominguez, S.; Martinez, J.

    2016-08-01

    OGLE-II and OGLE-IV observations of Cepheid variables in the LMC and SMC galaxies were collected with the 1.3m Warsaw telescope, at Las Campanas Observatory, Chile (Udalski et al. 1999, J/AcA/49/223; 1999, J/AcA/49/437; 2015AcA....65....1U). While Cepheid catalogs for the OGLE-II fundamental mode contain 771 and 1319 stars for the LMC and SMC, respectively, OGLE-IV has a nearly complete collection (2429 and 2739 for the LMC and SMC, respectively), covering practically the whole Magellanic System with a time baseline of a little more than five years (Soszynski et al. 2015AcA....65..329S). (1 data file).

  13. 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...

  14. Over 38000 RR Lyrae Stars in the OGLE Galactic Bulge Fields

    CERN Document Server

    Soszynski, I; Szymanski, M K; Pietrukowicz, P; Mroz, P; Skowron, J; Kozlowski, S; Poleski, R; Skowron, D; Pietrzynski, G; Wyrzykowski, L; Ulaczyk, K; Kubiak, M

    2014-01-01

    We present the most comprehensive picture ever obtained of the central parts of the Milky Way probed with RR Lyrae variable stars. This is a collection of 38257 RR Lyr stars detected over 182 square degrees monitored photometrically by the Optical Gravitational Lensing Experiment (OGLE) in the most central regions of the Galactic bulge. The sample consists of 16804 variables found and published by the OGLE collaboration in 2011 and 21453 RR Lyr stars newly detected in the photometric databases of the fourth phase of the OGLE survey (OGLE-IV). 93% of the OGLE-IV variables were previously unknown. The total sample consists of 27258 RRab, 10825 RRc, and 174 RRd stars. We provide OGLE-IV I- and V-band light curves of the variables along with their basic parameters. About 300 RR Lyr stars in our collection are plausible members of 15 globular clusters. Among others, we found the first pulsating variables that may belong to the globular cluster Terzan 1 and the first RRd star in the globular cluster M54. Our survey...

  15. Molecular evolution and functional characterisation of an ancient phenylalanine ammonia-lyase gene (NnPAL1) from Nelumbo nucifera: novel insight into the evolution of the PAL family in angiosperms.

    Science.gov (United States)

    Wu, Zhihua; Gui, Songtao; Wang, Shuzhen; Ding, Yi

    2014-05-09

    Phenylalanine ammonia-lyase (PAL; E.C.4.3.1.5) is a key enzyme of the phenylpropanoid pathway in plant development, and it catalyses the deamination of phenylalanine to trans-cinnamic acid, leading to the production of secondary metabolites. This enzyme has been identified in many organisms, ranging from prokaryotes to higher plants. Because Nelumbo nucifera is a basal dicot rich in many secondary metabolites, it is a suitable candidate for research on the phenylpropanoid pathway. Three PAL members, NnPAL1, NnPAL2 and NnPAL3, have been identified in N. nucifera using genome-wide analysis. NnPAL1 contains two introns; however, both NnPAL2 and NnPAL3 have only one intron. Molecular and evolutionary analysis of NnPAL1 confirms that it is an ancient PAL member of the angiosperms and may have a different origin. However, PAL clusters, except NnPAL1, are monophyletic after the split between dicots and monocots. These observations suggest that duplication events remain an important occurrence in the evolution of the PAL gene family. Molecular assays demonstrate that the mRNA of the NnPAL1 gene is 2343 bp in size and encodes a 717 amino acid polypeptide. The optimal pH and temperature of the recombinant NnPAL1 protein are 9.0 and 55°C, respectively. The NnPAL1 protein retains both PAL and weak TAL catalytic activities with Km values of 1.07 mM for L-phenylalanine and 3.43 mM for L-tyrosine, respectively. Cis-elements response to environmental stress are identified and confirmed using real-time PCR for treatments with abscisic acid (ABA), indoleacetic acid (IAA), ultraviolet light, Neurospora crassa (fungi) and drought. We conclude that the angiosperm PAL genes are not derived from a single gene in an ancestral angiosperm genome; therefore, there may be another ancestral duplication and vertical inheritance from the gymnosperms. The different evolutionary histories for PAL genes in angiosperms suggest different mechanisms of functional regulation. The expression

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. Binding sequences for RdgB, a DNA damage-responsive transcriptional activator, and temperature-dependent expression of bacteriocin and pectin lyase genes in Pectobacterium carotovorum subsp. carotovorum.

    Science.gov (United States)

    Yamada, Kazuteru; Kaneko, Jun; Kamio, Yoshiyuki; Itoh, Yoshifumi

    2008-10-01

    Pectobacterium carotovorum subsp. carotovorum strain Er simultaneously produces the phage tail-like bacteriocin carotovoricin (Ctv) and pectin lyase (Pnl) in response to DNA-damaging agents. The regulatory protein RdgB of the Mor/C family of proteins activates transcription of pnl through binding to the promoter. However, the optimal temperature for the synthesis of Ctv (23 degrees C) differs from that for synthesis of Pnl (30 degrees C), raising the question of whether RdgB directly activates ctv transcription. Here we report that RdgB directly regulates Ctv synthesis. Gel mobility shift assays demonstrated RdgB binding to the P(0), P(1), and P(2) promoters of the ctv operons, and DNase I footprinting determined RdgB-binding sequences (RdgB boxes) on these and on the pnl promoters. The RdgB box of the pnl promoter included a perfect 7-bp inverted repeat with high binding affinity to the regulator (K(d) [dissociation constant] = 150 nM). In contrast, RdgB boxes of the ctv promoters contained an imperfect inverted repeat with two or three mismatches that consequently reduced binding affinity (K(d) = 250 to 350 nM). Transcription of the rdgB and ctv genes was about doubled at 23 degrees C compared with that at 30 degrees C. In contrast, the amount of pnl transcription tripled at 30 degrees C. Thus, the inverse synthesis of Ctv and Pnl as a function of temperature is apparently controlled at the transcriptional level, and reduced rdgB expression at 30 degrees C obviously affected transcription from the ctv promoters with low-affinity RdgB boxes. Pathogenicity toward potato tubers was reduced in an rdgB knockout mutant, suggesting that the RdgAB system contributes to the pathogenicity of this bacterium, probably by activating pnl expression.

  1. 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.

  2. 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.)

  3. Production of AI-2 is mediated by the S-ribosylhomocystein lyase gene luxS in Bacteroides fragilis and Bacteroides vulgatus.

    Science.gov (United States)

    Peixoto, Rafael José Marques; Miranda, Karla Rodrigues; Ferreira, Eliane Oliveira; de Paula, Geraldo Renato; Rocha, Edson Ribeiro; Lobo, Leandro Araujo; Domingues, Regina Maria Cavalcanti Pilotto

    2014-07-01

    Quorum sensing is a cell-cell signaling mechanism based on cell density and that involves the production of hormone-like molecules called autoinducers (AI). One of the most studied AIs has been termed AI-2, and its biosynthesis requires the enzyme encoded by luxS. We have previously described for the first time that Bacteroides species can produce molecules with AI-2 activity. In this study, we focus on the detection of luxS and its activity as the AI-2 synthase in Bacteroides species. The strains Bacteroides fragilis B3b and Bacteroides vulgatus ATCC 8482 were selected based on a positive phenotype for AI-2 production and the presence of a putative luxS in the genome, respectively. In order to identify the luxS gene, cloning and heterologous expression strategies were utilized. We demonstrate that both strains contain functional luxS orthologs that can complement AI-2 production in Escherichia coli. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. In search of RR Lyrae type stars in eclipsing binary systems. OGLE052218.07-692827.4: an optical blend

    CERN Document Server

    Prsa, A; Devinney, E J; Engle, S G

    2008-01-01

    During the OGLE-2 operation, Soszynski et al. (2003) found 3 LMC candidates for an RR Lyr-type component in an eclipsing binary system. Two of those have orbital periods that are too short to be physically plausible and hence have to be optical blends. For the third, OGLE052218.07-692827.4, we developed a model of the binary that could host the observed RR Lyr star. After being granted HST/WFPC2 time, however, we were able to resolve 5 distinct sources within a 1.3" region that is typical of OGLE resolution, proving that OGLE052218.07-692827.4 is also an optical blend. Moreover, the putative eclipsing binary signature found in the OGLE data does not seem to correspond to a physically plausible system; the source is likely another background RR Lyr star. There are still no RR Lyr stars discovered so far in an eclipsing binary system.

  5. Nonradial modes in RR Lyrae stars from the OGLE Collection of Variable Stars

    CERN Document Server

    Netzel, Henryka; Moskalik, Pawel

    2016-01-01

    The Optical Gravitational Lensing Experiment (OGLE) is a great source of top-quality photometry of classical pulsators. Collection of variable stars from the fourth part of the project contains more than 38 000 RR Lyrae stars. These stars pulsate mostly in the radial fundamental mode (RRab), in radial first overtone (RRc) or in both modes simultaneously (RRd). Analysis of the OGLE data allowed to detect additional non-radial modes in RRc and in RRd stars. We have found more than 260 double-mode stars with characteristic period ratio of the additional (shorter) period to first overtone period around 0.61, increasing the number of known stars of this type by factor of 10. Stars from the OGLE sample form three nearly parallel sequences in the Petersen diagram. Some stars show more than one non-radial mode simultaneously. These modes belong to different sequences.

  6. 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.

  7. 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....

  8. OGLE-ing the Magellanic System: Three-dimensional structure of the Clouds and the Bridge using classical Cepheids

    CERN Document Server

    Jacyszyn-Dobrzeniecka, Anna M; Mróz, P; Skowron, J; Soszyński, I; Udalski, A; Pietrukowicz, P; Kozłowski, S; Wyrzykowski, Ł; Poleski, R; Pawlak, M; Szymański, M K; Ulaczyk, K

    2016-01-01

    We analyzed a sample of 9418 fundamental-mode and first-overtone classical Cepheids from the OGLE-IV Collection of Classical Cepheids. The distance to each Cepheid was calculated using the period-luminosity relation for the Wesenheit magnitude, fitted to our data. The classical Cepheids in the LMC are situated mainly in the bar and in the northern arm. The eastern part of the LMC is closer to us and the plane fit to the whole LMC sample yields the inclination i=24.2+-0.6 deg and position angle P.A.=151.4+-1.5 deg. We redefined the LMC bar by extending it in the western direction and found no offset from the plane of the LMC contrary to previous studies. On the other hand, we found that the northern arm is offset from a plane by about -0.5 kpc, which was not observed before. The age distribution of the LMC Cepheids shows one maximum at about 100 Myr. We demonstrate that the SMC has a non-planar structure and can be described as an extended ellipsoid. We identified two large ellipsoidal off-axis structures in t...

  9. OGLE-ing the Magellanic System: Three-Dimensional Structure of the Clouds and the Bridge Using RR Lyrae Stars

    CERN Document Server

    Jacyszyn-Dobrzeniecka, Anna M; Mróz, P; Soszyński, I; Udalski, A; Pietrukowicz, P; Skowron, J; Poleski, R; Kozłowski, S; Wyrzykowski, Ł; Pawlak, M; Szymański, M K; Ulaczyk, K

    2016-01-01

    We present a three-dimensional analysis of a sample of 22 859 type $ab$ RR Lyrae stars in the Magellanic System from the OGLE-IV Collection of RR Lyrae stars. The distance to each object was calculated based on its photometric metallicity and a theoretical relation between color, absolute magnitude and metallicity. The LMC RR Lyrae distribution is very regular and does not show any substructures. We demonstrate that the bar found in previous studies may be an overdensity caused by blending and crowding effects. The halo is asymmetrical with a higher stellar density in its north-eastern area, which is also located closer to us. Triaxial ellipsoids were fitted to surfaces of a constant number density. Ellipsoids farther from the LMC center are less elongated and slightly rotated toward the SMC. The inclination and position angle change significantly with the $a$ axis size. The median axis ratio is $1:1.23:1.45$. The RR Lyrae distribution in the SMC has a very regular, ellipsoidal shape and does not show any sub...

  10. Bright but slow - Type II supernovae from OGLE-IV - Implications for magnitude limited surveys

    OpenAIRE

    Poznanski, Dovi; Kostrzewa-Rutkowska, Zuzanna; Wyrzykowski, Lukasz; Blagorodnova, Nadejda

    2015-01-01

    We study a sample of 11 Type II supernovae (SNe) discovered by the OGLE-IV survey. All objects have well sampled I-band light curves, and at least one spectrum. We find that 2 or 3 of the 11 SNe have a declining light curve, and spectra consistent with other SNe II-L, while the rest have plateaus that can be as short as 70d, unlike the 100d typically found in nearby galaxies. The OGLE SNe are also brighter, and show that magnitude limited surveys find SNe that are different than usually found...

  11. The OGLE Collection of Variable Stars. Eclipsing Binaries in the Magellanic System

    Science.gov (United States)

    Pawlak, M.; Soszyński, I.; Udalski, A.; Szymański, M. K.; Wyrzykowski, Ł.; Ulaczyk, K.; Poleski, R.; Pietrukowicz, P.; Kozłowski, S.; Skowron, D. M.; Skowron, J.; Mróz, P.; Hamanowicz, A.

    2016-12-01

    We present the collection of eclipsing binaries in the Large and Small Magellanic Clouds, based on the OGLE survey. It contains 48 605 systems, 40 204 belonging to the LMC and 8401 to the SMC. Out of the total number of presented here binaries, 16 374 are the new discoveries. We present the time-series photometry obtained for the selected objects during the fourth phase of the OGLE project. The catalog has been created using a two step machine learning procedure based on the Random Forest algorithm.

  12. Detection of period variations in extrasolar transiting planet OGLE-TR-111b

    CERN Document Server

    Díaz, Rodrigo F; Melita, Mario; Hoyer, Sergio; Minniti, Dante; Mauas, Pablo J D; Ruíz, María Teresa

    2008-01-01

    Two consecutive transits of planetary companion OGLE-TR-111b were observed in the I band. Combining these observations with data from the literature, we find that the timing of the transits cannot be explained by a constant period, and that the observed variations cannot be originated by the presence of a satellite. However, a perturbing planet with the mass of the Earth in an exterior orbit could explain the observations if the orbit of OGLE-TR-111b is eccentric. We also show that the eccentricity needed to explain the observations is not ruled out by the radial velocity data found in the literature.

  13. Qualitative and quantitative trait loci conditioning resistance to Puccinia coronata pathotypes NQMG and LGCG in the oat (Avena sativa L.) cultivars Ogle and TAM O-301.

    Science.gov (United States)

    Jackson, E W; Obert, D E; Menz, M; Hu, G; Bonman, J M

    2008-02-01

    Mapping disease resistance loci relies on the type and precision of phenotypic measurements. For crown rust of oat, disease severity is commonly assessed based on visual ratings of infection types (IT) and/or diseased leaf area (DLA) of infected plants in the greenhouse or field. These data can be affected by several variables including; (i) non-uniform disease development in the field; (ii) atypical symptom development in the greenhouse; (iii) the presence of multiple pathogenic races or pathotypes in the field, and (iv) rating bias. To overcome these limitations, we mapped crown rust resistance to single isolates in the Ogle/TAM O-301 (OT) recombinant inbred line (RIL) population using detailed measurements of IT, uredinia length (UL) and relative fungal DNA (FDNA) estimates determined by q-PCR. Measurements were taken on OT parents and recombinant inbred lines (RIL) inoculated with Puccinia coronata pathotypes NQMG and LGCG in separate greenhouse and field tests. Qualitative mapping identified an allele conferred by TAM O-301 on linkage group (LG) OT-11, which produced a bleached fleck phenotype to both NQMG and LGCG. Quantitative mapping identified two major quantitative trait loci (QTL) originating from TAM O-301 on LGs OT-11 and OT-32 which reduced UL and FDNA of both isolates in all experiments. Additionally, minor QTLs that reduced UL and FDNA were detected on LGs OT-15 and OT-8, originating from TAM O-301, and on LG OT-27, originating from Ogle. Detailed assessments of the OT population using two pathotypes in both the greenhouse and field provided comprehensive information to effectively map the genes responsible for crown rust resistance in Ogle and TAM O-301 to NQMG and LGCG.

  14. Molecular Cloning and Expression Analysis of a Phenylalanne Ammonial-lyase Gene from Prunella vulgaris%夏枯草苯丙氨酸解氨酶基因的克隆与表达分析

    Institute of Scientific and Technical Information of China (English)

    许锋; 曹腾; 宁迎晶; 蒋丽阳; 张威威; 程水源

    2012-01-01

    苯丙氨酸解氨酶(PAL)是迷迭香酸合成途径中的关键酶之一,根据其他植物PAL基因的保守区域设计特异引物,利用3′-RACE-PCR技术,本研究首次从夏枯草中克隆得到了PAL基因的cDNA片段序列,命名为PvPAL,GenBank登录号为JN65446.PvPAL基因cDNA片段长1 306 bp,其中编码区域为1 047 bp,编码349个氨基酸.蛋白质序列多重比较结果显示,PvPAL蛋白质序列与丹参、地黄、黄芩、藿香等植物的PAL蛋白质高度同源.PAL系统进化树分析结果表明,PvPAL与唇形科植物的PAL基因亲缘关系最近.组织表达分析结果显示,PvPAL基因在根、茎、叶中均表达,其中根中表达量最高.PvPAL基因的克隆为进一步研究夏枯草迷迭香酸合成的分子机制奠定了基础.%Phenylalanne ammonial-lyase( PAL) is one of the key enzymes involved in rosmarinic acid biosyn-thetic pathway. In this study,a PAL gene,named PvPAL,was cloned from Prunella vulgaris at the first time by 3'-RACE-PCR and using the specific primer,which was designed according to the homologus sequences of PAL genes from other plants. The GenBank accession number of PvPAL is JN65446. The length of PvPAL cDNA fragment is 1 306 bp,including a 1 047 bp-length coding sequence,which encoded a 349-amino-acid protein. Sequence multiple-alignment revealed that PvPAL protein had extensive homology with those of other plants as Salvia miltiorrhiza, Rehmannia glutinosa, Scutellaria baicalensis and Agastache rugosa. Phylogenetic tree analysis showed that PvPAL had closest relationship with PALs from Lamiaceae plants than from other plants. Tissue expression analysis indicated that PvPAL expressed in all tissues examined,but highest in roots. The isolation of PvPAL provided basis for further studying the molecular mechanism of rosmarinic acid biosynthesis in P. vulgaris.

  15. OGLE-ing the Magellanic System: Three-Dimensional Structure of the Clouds and the Bridge Using Classical Cepheids

    Science.gov (United States)

    Jacyszyn-Dobrzeniecka, A. M.; Skowron, D. M.; Mróz, P.; Skowron, J.; Soszyński, I.; Udalski, A.; Pietrukowicz, P.; Kozłowski, S.; Wyrzykowski, Ł.; Poleski, R.; Pawlak, M.; Szymański, M. K.; Ulaczyk, K.

    2016-06-01

    We analyzed a sample of 9418 fundamental-mode and first-overtone classical Cepheids from the OGLE-IV Collection of Classical Cepheids. The distance to each Cepheid was calculated using the period-luminosity relation for the Wesenheit magnitude, fitted to our data. The classical Cepheids in the LMC are situated mainly in the bar and in the northern arm. The eastern part of the LMC is closer to us and the plane fit to the whole LMC sample yields the inclination i=24.°2 ±0.°7 and position angle P.A.=151.°4±1.°7. We redefined the LMC bar by extending it in the western direction and found no offset from the plane of the LMC contrary to previous studies. On the other hand, we found that the northern arm is offset from a plane by about -0.5 kpc, which was not observed before. The age distribution of the LMC Cepheids shows one maximum at about 100 Myr. We demonstrate that the SMC has a non-planar structure and can be described as an extended ellipsoid. We identified two large ellipsoidal off-axis structures in the SMC. The northern one is located closer to us and is younger, while the south-western is farther and older. The age distribution of the SMC Cepheids is bimodal with one maximum at 110 Myr, and another one at 220 Myr. Younger stars are located in the closer part of this galaxy while older ones are more distant. We classified nine Cepheids from our sample as Magellanic Bridge objects. These Cepheids show a large spread in three-dimensions although five of them form a connection between the Clouds. The closest one is closer than any of the LMC Cepheids, while the farthest one - farther than any SMC Cepheid. All but one Cepheids in the Magellanic Bridge are younger than 300 Myr. The oldest one can be associated with the SMC Wing.

  16. 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.

  17. 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

  18. SPITZER OBSERVATIONS OF OGLE-2015-BLG-1212 REVEAL A NEW PATH TOWARD BREAKING STRONG MICROLENS DEGENERACIES

    DEFF Research Database (Denmark)

    Bozza, V.; Shvartzvald, Y.; Udalski, A.

    2016-01-01

    Spitzer microlensing parallax observations of OGLE-2015-BLG-1212 decisively break a degeneracy between planetary and binary solutions that is somewhat ambiguous when only ground-based data are considered. Only eight viable models survive out of an initial set of 32 local minima in the parameter s...

  19. OGLE-IV: Fourth Phase of the Optical Gravitational Lensing Experiment

    CERN Document Server

    Udalski, A; Szymański, G

    2015-01-01

    We present both the technical overview and main science drivers of the fourth phase of the Optical Gravitational Lensing Experiment (hereafter OGLE-IV). OGLE-IV is currently one of the largest sky variability surveys worldwide, targeting the densest stellar regions of the sky. The survey covers over 3000 square degrees in the sky and monitors regularly over a billion sources. The main targets include the inner Galactic Bulge and the Magellanic System. Their photometry spans the range of $12OGLE-IV surveys provide photometry with milli-magnitude accuracy at the bright end. The cadence of observations varies from 19-60 minutes in the inner Galactic bulge to 1-3 days in the remaining Galactic bulge fields, Magellanic System and the Galactic disk. OGLE-IV provides the astronomical com...

  20. Coordinates and 2MASS and OGLE identifications for all stars in Arp's 1965 finding chart for Baade's Window

    CERN Document Server

    Church, Ross P; Feltzing, Sofia

    2011-01-01

    Aims: We seek to provide 2MASS and OGLE identifications and coordinates for all stars in the finding chart published by Arp\\,(1965). This chart covers the low extinction area around NGC 6522, also known as Baade's window, at coordinates (l,b)=(1.02,-3.92). Methods: A cross correlation, using numerical techniques, was performed between a scan of the original finding chart from Arp (1965) and 2MASS and OGLE-II images and stellar coordinates. Results: We provide coordinates for all stars in Arp's finding chart and 2MASS and OGLE identifications wherever possible. Two identifications in quadrant II do not appear in the original finding chart.

  1. Expression of Astragalus membranaceus phenylalanine ammonia-lyase gene in Pichia pastoris%膜荚黄芪苯丙氨酸解氨酶基因在毕赤酵母中的分泌表达

    Institute of Scientific and Technical Information of China (English)

    张健慧; 王首锋

    2014-01-01

    L-phenylalanine , as an essential amino acid for human nutrition , is widely used in pharmaceutical and food industries . Using phenylalanine ammonia-lyase ( PAL ,EC 4 .3 .1 .5) to produce L-phenylalanine is one of the major routes . However , most commercial enzymes are extracted from Rhodotorula glutinis , which is time-consuming and over-priced . Therefore , how to efficiently construct the genetic engineering strain to produce PAL is the hot topic . Pichia pastoris is popular in expressing heterologous proteins due to the advantages of low nutritional demands , excellent genetic stability and high-density fermentation . Inserting the heterologous gene into pPIC 9K vectortoachievesecretedexpressionin P.pastorishasbeenreported.However,unlikeothervectors,pPIC9Khas few desirable restriction enzyme cutting sites , which reduces vector construction efficiency when the classical method of digestion and then ligation is adopted . Under this condition , an efficient cloning strategy , independent of digestion and ligation , is required . Homologous recombination in vitro between pPIC9K and gene can settle this problem .Now ,we intend to employ homologous recombination in vitro cloning method to insert the PAL gene into pPIC9K vector to obtain secreted expression in P . pastoris in order to lay the basis for industrial fermentation . First , total RNA extracted from Astragalus membranaceus was used as template for isolating cDNA . Open reading frame ( ORF) of PA L gene was amplified by PCR from cDNA with a pair of primers designed according to the sequence of PA L gene published in the GenBank . Then , ORF was cloned into vector pUCm-T . The transformant was selected to sequence for further analysis of the PA L gene sequence with the help of bioinformatics tools . After that , pPIC9K-PA L was constructed by homologous recombination in vitro . Similarly , the transformant was selected to sequence to investigate the base mutation caused by PCR . Linearized pPIC 9K-PA L by

  2. 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.

  3. 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.

  4. Self-Organizing Maps. An application to the OGLE data and the Gaia Science Alerts

    CERN Document Server

    Wyrzykowski, Lukasz

    2008-01-01

    Self-Organizing Map (SOM) is a promising tool for exploring large multi-dimensional data sets. It is quick and convenient to train in an unsupervised fashion and, as an outcome, it produces natural clusters of data patterns. An example of application of SOM to the new OGLE-III data set is presented along with some preliminary results. Once tested on OGLE data, the SOM technique will also be implemented within the Gaia mission's photometry and spectrometry analysis, in particular, in so-called classification-based Science Alerts. SOM will be used as a basis of this system as the changes in brightness and spectral behaviour of a star can be easily and quickly traced on a map trained in advance with simulated and/or real data from other surveys.

  5. Spitzer Microlensing Program as a Probe for Globular Cluster Planets. Analysis of OGLE-2015-BLG-0448

    CERN Document Server

    Poleski, Radosław; Christie, Grant W; Udalski, Andrzej; Gould, Andrew; Bachelet, Etienne; Skottfelt, Jesper; Novati, Sebastiano Calchi; Szymański, M K; Soszyński, I; Pietrzyński, G; Wyrzykowski, Ł; Ulaczyk, K; Pietrukowicz, P; Kozłowski, Szymon; Skowron, J; Mróz, P; Pawlak, M; Beichman, C; Bryden, G; Carey, S; Fausnaugh, M; Gaudi, B S; Henderson, C B; Pogge, R W; Shvartzvald, Y; Wibking, B; Yee, J C; Beatty, T G; Eastman, J D; Drummond, J; Friedmann, M; Henderson, M; Johnson, J A; Kaspi, S; Maoz, D; McCormick, J; McCrady, N; Natusch, T; Ngan, H; Porritt, I; Relles, H M; Sliski, D H; Tan, T -G; Wittenmyer, R A; Wright, J T; Street, R A; Tsapras, Y; Bramich, D M; Horne, K; Snodgrass, C; Steele, I A; Menzies, J; Jaimes, R Figuera; Wambsganss, J; Schmidt, R; Cassan, A; Ranc, C; Mao, S; Bozza, V; Dominik, M; Hundertmark, M P G; Jørgensen, U G; Andersen, M I; Burgdorf, M J; Ciceri, S; D'Ago, G; Evans, D F; Gu, S -H; Hinse, T C; Kains, N; Kerins, E; Korhonen, H; Kuffmeier, M; Mancini, L; Popovas, A; Rabus, M; Rahvar, S; Rasmussen, R T; Southworth, G Scarpetta J; Surdej, J; Unda-Sanzana, E; Verma, P; von Essen, C; Wang, Y -B; Wertz, O

    2015-01-01

    The microlensing event OGLE-2015-BLG-0448 was observed by Spitzer and lay within the tidal radius of the globular cluster NGC 6558. The event had moderate magnification and was intensively observed, hence it had the potential to probe the distribution of planets in globular clusters. We measure the proper motion of NGC 6558 ($\\mu_{\\rm cl}$(N,E) = (+0.36+-0.10, +1.42+-0.10) mas/yr) as well as the source and show that the lens is not a cluster member. Even though this particular event does not probe the distribution of planets in globular clusters, other potential cluster lens events can be verified using our methodology. Additionally, we find that microlens parallax measured using OGLE photometry is consistent with the value found based on the light curve displacement between Earth and Spitzer.

  6. Molecular Cloning and Sequence Analysis of Phenylalanine Ammonia-lyase Gene Fragment in Ricinus communis%蓖麻PAL基因片段的克隆与序列分析

    Institute of Scientific and Technical Information of China (English)

    高颖; 李梦竹; 冯紫洲; 佟欢; 李毅; 张继星

    2013-01-01

    利用RACE(rapid-amplification of cDNA end)方法,以蓖麻叶片提取总的RNA为模板克隆了蓖麻苯丙氨酸解氨酶(phenylalanineamononiar-lyase,PAL)基因片段.分析表明:该基因片段编码区为1071bp,推测编码357个氨基酸.与所选取的10种植物同类蛋白氨基酸序列进行对比,一致性在75%-81%之间,并具有PAL蛋白家族典型的结构域,把该基因命名为RcPAL1,为后续试验奠定基础.

  7. 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.

  8. 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.

  9. Automated supervised classification of variable stars II. Application to the OGLE database

    CERN Document Server

    Sarro, L M; López, M; Aerts, C

    2008-01-01

    We aim to extend and test the classifiers presented in a previous work against an independent dataset. We complement the assessment of the validity of the classifiers by applying them to the set of OGLE light curves treated as variable objects of unknown class. The results are compared to published classification results based on the so-called extractor methods.Two complementary analyses are carried out in parallel. In both cases, the original time series of OGLE observations of the Galactic bulge and Magellanic Clouds are processed in order to identify and characterize the frequency components. In the first approach, the classifiers are applied to the data and the results analyzed in terms of systematic errors and differences between the definition samples in the training set and in the extractor rules. In the second approach, the original classifiers are extended with colour information and, again, applied to OGLE light curves. We have constructed a classification system that can process huge amounts of tim...

  10. OGLE16aaa - a signature of a hungry supermassive black hole

    Science.gov (United States)

    Wyrzykowski, Łukasz; Zieliński, M.; Kostrzewa-Rutkowska, Z.; Hamanowicz, A.; Jonker, P. G.; Arcavi, I.; Guillochon, J.; Brown, P. J.; Kozłowski, S.; Udalski, A.; Szymański, M. K.; Soszyński, I.; Poleski, R.; Pietrukowicz, P.; Skowron, J.; Mróz, P.; Ulaczyk, K.; Pawlak, M.; Rybicki, K. A.; Greiner, J.; Krühler, T.; Bolmer, J.; Smartt, S. J.; Maguire, K.; Smith, K.

    2017-02-01

    We present the discovery and first three months of follow-up observations of a currently on-going unusual transient detected by the Optical Gravitational Lensing Experiment (OGLE-IV) survey, located in the centre of a galaxy at redshift z = 0.1655. The long rise to absolute magnitude of -20.5 mag, slow decline, very broad He and H spectral features make OGLE16aaa similar to other optical/UV tidal disruption events (TDEs). Weak narrow emission lines in the spectrum and archival photometric observations suggest the host galaxy is a weak-line active galactic nucleus, which has been accreting at higher rate in the past. OGLE16aaa, along with SDSS J0748, seems to form a sub-class of TDEs by weakly or recently active supermassive black holes (SMBHs). This class might bridge the TDEs by quiescent SMBHs and flares observed as `changing-look quasars', if we interpret the latter as TDEs. If this picture is true, the previously applied requirement for identifying a flare as a TDE that it had to come from an inactive nucleus, could be leading to observational bias in TDE selection, thus affecting TDE-rate estimations.

  11. OGLE16aaa - a Signature of a Hungry Super Massive Black Hole

    CERN Document Server

    Wyrzykowski, Łukasz; Kostrzewa-Rutkowska, Z; Hamanowicz, A; Jonker, P G; Arcavi, I; Guillochon, J; Brown, P J; Kozłowski, S; Udalski, A; Szymański, M K; Soszyński, I; Poleski, R; Pietrukowicz, P; Skowron, J; Mróz, P; Ulaczyk, K; Pawlak, M; Rybicki, K A; Greiner, J; Krühler, T; Bolmer, J

    2016-01-01

    We present the discovery and first three months of follow-up observations of a currently on-going unusual transient detected by the OGLE-IV survey, located in the centre of a galaxy at redshift z=0.1655. The long rise to absolute magnitude of -20.5 mag, slow decline, very broad He and H spectral features make OGLE16aaa similar to other optical/UV Tidal Disruption Events (TDEs). Weak narrow emission lines in the spectrum and archival photometric observations suggest the host galaxy is a weak-line Active Galactic Nucleus (AGN), which has been accreting at higher rate in the past. OGLE16aaa, along with SDSS J0748, seems to form a sub-class of TDEs by weakly or recently active supermassive black holes (SMBHs). This class might bridge the TDEs by quiescent SMBHs and flares observed as "changing-look QSOs", if we interpret the latter as TDEs. If this picture is true, the previously applied requirement for identifying a flare as a TDE that it had to come from an inactive nucleus, could be leading to observational bi...

  12. A Low-Resolution Spectroscopic Exploration of Puzzling OGLE Variable Stars

    CERN Document Server

    Pietrukowicz, P; Angeloni, R; di Mille, F; Soszynski, I; Udalski, A; Germana, C

    2015-01-01

    We present the results of a spectroscopic follow-up of various puzzling variable objects detected in the OGLE-III Galactic disk and bulge fields. The sample includes mainly short-period multi-mode pulsating stars that could not have been unambiguously classified as either delta Sct or beta Cep type stars based on photometric data only, also stars with irregular fluctuations mimicking cataclysmic variables and stars with dusty shells, and periodic variables displaying brightenings in their light curves that last for more than half of the period. The obtained low-resolution spectra show that all observed short-period pulsators are of delta Sct type, the stars with irregular fluctuations are young stellar objects, and the objects with regular brightenings are A type stars or very likely Ap stars with strong magnetic field responsible for the presence of bright caps around magnetic poles on their surface. We also took spectra of objects designated OGLE-GD-DSCT-0058 and OGLE-GD-CEP-0013. An estimated effective tem...

  13. Eclipsing Binary Stars in the OGLE-III Galactic Disk Fields

    CERN Document Server

    Pietrukowicz, P; Soszynski, I; Udalski, A; Poleski, R; Szymanski, M K; Kubiak, M; Pietrzynski, G; Wyrzykowski, L; Ulaczyk, K; Kozlowski, S; Skowron, J

    2013-01-01

    We present the analysis of 11,589 eclipsing binary stars identified in 21 OGLE-III Galactic disk fields toward constellations of Carina, Centaurus, and Musca. All eclipsing binaries but 393 objects are new discoveries. The binaries have out-of-eclipse brightness between I=12.5 and I=21 mag. The completeness of the catalog is estimated at a level of about 75%. Comparison of the orbital period distribution for the OGLE-III disk binaries with systems detected in other recent large-scale Galactic surveys shows the maximum around 0.40 d and an almost flat distribution between 0.5 and 2.5 d, indepedent of population. Among thousands of variables we have found 10 doubly eclipsing objects and one eclipsing-ellipsoidal object, of which 9 are candidates for quadruple systems. We also identify 10 eclipsing subdwarf-B-type binary stars and numerous eclipsing RS Canum-Venaticorum-type variables. All objects reported in this paper are part of the OGLE-III Catalog of Variable Stars.

  14. 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.

  15. 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...

  16. 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.

  17. 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

  18. 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...

  19. 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.

  20. Cytochrome P450c17 (steroid 17. cap alpha. -hydroxylase/17,20 lyase): cloning of human adrenal and testis cDNAs indicates the same gene is expressed in both tissues

    Energy Technology Data Exchange (ETDEWEB)

    Chung, B.; Picado-Leonard, J.; Haniu, M.; Bienkowski, M.; Hall, P.F.; Shively, J.E.; Miller, W.L.

    1987-01-01

    P450c17 is the single enzyme mediating both 17..cap alpha..-hydroxylase (steroid 17..cap alpha..-monooxygenase, EC 1.14.99.9) and 17,20 lyase activities in the synthesis of steroid hormones. It has been suggested that different P450c17 isozymes mediate these activities in the adrenal gland and testis. The authors sequenced 423 of the 509 amino acids (83%) of the porcine adrenal enzyme; based on this partial sequence, a 128-fold degenerate 17-mer was synthesized and used to screen a porcine adrenal cDNA library. This yielded a 380-base cloned cDNA, which in turn was used to isolate several human adrenal cDNAs. The longest of these, lambda hac 17-2, is 1754 base pairs long and includes the full-length coding region, the complete 3'-untranslated region, and 41 bases of the 5'-untranslated region. This cDNA encodes a protein of 508 amino acids having a predicted molecular weight of 57,379.82. High-stringency screening of a human testicular cDNA library yielded a partial clone containing 1303 identical bases. RNA gel blots and nuclease S1-protection experiments confirm that the adrenal and testicular P450c17 mRNAs are indistinguishable. These data indicate that the testis possesses a P450c17 identical to that in the adrenal. The human amino acid sequence is 66.7% homologous to the corresponding regions of the porcine sequence, and the human cDNA and amino acid sequences are 80.1 and 70.3% homologous, respectively, to bovine adrenal P450c17 cDNA. Both comparisons indicate that a central region comprising amino acid residues 160-268 is hypervariable among these species of P450c17.

  1. Construction of Vector Harboring Pectin Lyase C Gene and Its Expression in E. coli%果胶裂解酶基因PelC表达载体的构建及原核表达分析

    Institute of Scientific and Technical Information of China (English)

    邓伟科; 郭安平; 刘恩平; 王炎松; 郭运玲; 孔华; 阳辛凤; 贺立卡

    2009-01-01

    A complete open reading frame of pectin lyase C (PelC) cloned from a pectinase-producing strain BTC105 isolated and collected in the laboratory was constructed on a plasmid pET28a and transferred into E. coli BL21 (DE3) to carry out fuse expression; and flask shaking fermented in LB (Luria-Bertani) , induced with 1 mmol/L IPTG (iso-propyl β-D-1-thiogalactopyranoside). The results showed that the recombinant plasmid pET28a-pelC was constructed successfully and PelC has mainly expressed in E. coli BL21 (DE3). The optimal pH of the enzyme was 5.4, optimal temperature at 50℃, Ca~(2+) stimulated strongly on the enzyme activity, however, Cu~(2+) completely inhibited the activity.%从实验室分离保存的1株产果胶酶的菌株(BTC105)中克隆果胶裂解酶基因(PelC)完整开放阅读框,通过载体构建,将目的基因连接到表达载体pET28a上,转化大肠埃希菌BL21(DE3)进行融合表达,在LB(Luria-Bertani)中进行摇瓶发酵,1 mmol/L IPTG(异丙基-β-D-硫代半乳糖苷)诱导.结果表明,构建了表达载体pET28a-pelC,果胶裂解酶主要在胞内表达,酶活最适pH为5.4,最适温度为50℃,Ca~(2+)对酶活促进作用最为明显,Cu~(2+)完全抑制了酶的活性.

  2. Spitzer Observations of OGLE-2015-BLG-1212 Reveal a New Path to Breaking Strong Microlens Degeneracies

    CERN Document Server

    Bozza, V; Udalski, A; Novati, S Calchi; Bond, I A; Han, C; Hundertmark, M; Poleski, R; Pawlak, M; Szymański, M K; Skowron, J; Mróz, P; Kozłowski, S; Wyrzykowski, Ł; Pietrukowicz, P; Soszyński, I; Ulaczyk, K; Beichman, C; Bryden, G; Carey, S; Fausnaugh, M; Gaudi, B S; Gould, A; Henderson, C B; Pogge, R W; Wibking, B; Yee, J C; Zhu, W; Abe, F; Asakura, Y; Barry, R K; Bennett, D P; Bhattacharya, A; Donachie, M; Freeman, M; Fukui, A; Hirao, Y; Inayama, K; Itow, Y; Koshimoto, N; Li, M C A; Ling, C H; Masuda, K; Matsubara, Y; Muraki, Y; Nagakane, M; Nishioka, T; Ohnishi, K; Oyokawa, H; Rattenbury, N; Saito, T; Sharan, A; Sullivan, D J; Sumi, T; Suzuki, D; Tristram, P J; Wakiyama, Y; Yonehara, A; Choi, J -Y; Park, H; Jung, Y K; Shin, I -G; Albrow, M D; Park, B -G; Kim, S -L; Lee, C -U; Cha, S -M; Kim, D -J; Lee, Y; Dominik, M; Jørgensen, U G; Andersen, M I; Bramich, D M; Burgdorf, M J; Ciceri, S; D'Ago, G; Evans, D F; Jaimes, R Figuera; Gu, S -H; Hinse, T C; Kains, N; Kerins, E; Korhonen, H; Kuffmeier, M; Mancini, L; Popovas, A; Rabus, M; Rahvar, S; Rasmussen, R T; Scarpetta, G; Skottfelt, J; Snodgrass, C; Southworth, J; Surdej, J; Unda-Sanzana, E; von Essen, C; Wang, Y -B; Wertz, O; Maoz, D; Friedmann, M; Kaspi, S

    2016-01-01

    Spitzer microlensing parallax observations of OGLE-2015-BLG-1212 decisively breaks a degeneracy between planetary and binary solutions that is somewhat ambiguous when only ground-based data are considered. Only eight viable models survive out of an initial set of 32 local minima in the parameter space. These models clearly indicate that the lens is a stellar binary system possibly located within the bulge of our Galaxy, ruling out the planetary alternative. We argue that several types of discrete degeneracies can be broken via such space-based parallax observations.

  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. 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.

  5. 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.

  6. OGLE Study of the Sagittarius Dwarf Spheroidal Galaxy and its M54 Globular Cluster

    CERN Document Server

    Hamanowicz, A; Udalski, A; Mroz, P; Soszynski, I; Szymanski, M K; Skowron, J; Poleski, R; Wyrzykowski, L; Kozlowski, S; Pawlak, M; Ulaczyk, K

    2016-01-01

    We use the fundamental-mode RR Lyr-type variable stars (RRab) from OGLE-IV to draw a 3D picture of the central part of the tidally disrupted Sagittarius Dwarf Spheroidal (Sgr dSph) galaxy. We estimate the line-of-sight thickness of the Sgr dSph stream to be 6sigma~6.2 kpc. Based on OGLE-IV observations collected in seasons 2011-2014 we conduct a comprehensive study of stellar variability in the field of the globular cluster M54 (NGC 6715) residing in the core of this dwarf galaxy. Among the total number of 270 detected variables we report the identification of 173 RR Lyr stars, 4 Type II Cepheids, 51 semi-regular variable red giants, 3 SX Phe-type stars, 18 eclipsing binary systems. Seventy-three variables are new discoveries. The distance to the cluster determined from RRab stars is d_M54=26.2+/-0.2_stat+/-1.3_sys kpc. From the location of RRab stars in the period-amplitude (Bailey) diagram we confirm the presence of two old populations, both in the cluster and the Sgr dSph stream.

  7. New Parameters and Transit Timing Studies for OGLE2-TR-L9 b

    CERN Document Server

    Lendl, M; Koppenhoefer, J; Nikolov, N; Henning, Th; Swain, M; Greiner, J

    2010-01-01

    Context: Repeated observations of exoplanet transits allow us to refine the planetary parameters and probe them for any time dependent variations. In particular deviations of the period from a strictly linear ephemeris, transit timing variations (TTVs), can indicate the presence of additional bodies in the planetary system. Aims: Our goal was to reexamine the largely unstudied OGLE2-TR-L9 system with high cadence, multi-color photometry in order to refine the planetary parameters and probe the system for TTVs. Methods: We observed five full transits of OGLE2-TR-L9 with the GROND instrument at the ESO/MPG 2.2 m telescope at La Silla Observatory. GROND is a multichannel imager that allowed us to gather simultaneous light curves in the g', r', i', and z' filters. Results: From our analysis we find that the semi-major axis and the inclination differ from the previously published values. With the newly observed transits, we were able to refine the ephemeris to 2454492.80008(+/- 0.00014) + 2.48553417(+/- 6.4) x 10^...

  8. Microlens OGLE-2005-BLG-169 Implies Cool Neptune-Like Planets are Common

    CERN Document Server

    Gould, A; Anderson, J; Bennett, D P; Bode, M F; Bond, I A; Botzler, C S; Bramich, D M; Burgdorf, M J; Christie, G W; De Poy, D L; Dong, S; Gaudi, B S; Han, C; Horne, K; Kubiak, M; Mao, S; McCormick, J; Paczynski, B; Park, B G; Pietrzynski, G; Pogge, R W; Poindexter, S; Rattenbury, N J; Snodgrass, C; Soszynski, I; Stanek, K Z; Steele, I A; Swaving, S C; Szewczyk, O; Szymanski, M K; Udalski, A; Ulaczyk, K; Wyrzykowski, L; Yock, P C M; Zhou, A Y

    2006-01-01

    We detect a Neptune mass-ratio (q~8e-5) planetary companion to the lens star in the extremely high-magnification (A~800) microlensing event OGLE-2005-BLG-169. If the parent is a main-sequence star, it has mass M~0.5 M_sun implying a planet mass of ~13 M_earth and projected separation of ~2.7 AU. When intensely monitored over their peak, high-magnification events similar to OGLE-2005-BLG-169 have nearly complete sensitivity to Neptune mass-ratio planets with projected separations of 0.6 to 1.6 Einstein radii, corresponding to 1.6--4.3 AU in the present case. Only two other such events were monitored well enough to detect Neptunes, and so this detection by itself suggests that Neptune mass-ratio planets are common. Moreover, another Neptune was recently discovered at a similar distance from its parent star in a low-magnification event, which are more common but are individually much less sensitive to planets. Combining the two detections yields 90% upper and lower frequency limits f=0.37^{+0.30}_{-0.21} over ju...

  9. Modeling Transiting Circumstellar Disks: Characterizing the Newly Discovered Eclipsing Disk System OGLE LMC-ECL-11893

    CERN Document Server

    Scott, Erin L; Pecaut, Mark J; Quillen, Alice C; Moolekamp, Fred; Bell, Cameron P M

    2014-01-01

    We investigate the nature of the unusual eclipsing star OGLE LMC-ECL-11893 (OGLE J05172127-6900558) in the Large Magellanic Cloud recently reported by Dong et al. 2014. The eclipse period for this star is 468 days, and the eclipses exhibit a minimum of ~1.4 mag, preceded by a plateau of ~0.8 mag. Spectra and optical/IR photometry are consistent with the eclipsed star being a lightly reddened B9III star of inferred age ~150 Myr and mass of ~4 solar masses. The disk appears to have an outer radius of ~0.2 AU with predicted temperatures of ~1100-1400 K. We model the eclipses as being due to either a transiting geometrically thin dust disk or gaseous accretion disk around a secondary object; the debris disk produces a better fit. We speculate on the origin of such a dense circumstellar dust disk structure orbiting a relatively old low-mass companion, and on the similarities of this system to the previously discovered EE Cep.

  10. OGLE-2012-BLG-0724Lb: A Saturn-mass Planet around an M-dwarf

    CERN Document Server

    Hirao, Y; Sumi, T; Bennett, D P; Bond, I A; Rattenbury, N; Suzuki, D; Koshimoto, N; Abe, F; Asakura, Y; Bhattacharya, A; Freeman, M; Fukui, A; Itow, Y; Li, M C A; Ling, C H; Masuda, K; Matsubara, Y; Matsuo, T; Muraki, Y; Nagakane, M; Ohnishi, K; Oyokawa, H; Saito, To; Sharan, A; Shibai, H; Sullivan, D J; Tristram, P J; Yonehara, A; Poleski, R; Skowron, J; Mróz, P; Szymański, M K; Kozłowski, S; Pietrukowicz, P; Soszyński, I; Wyzykowski, Ł; Ulaczyk, K

    2016-01-01

    We report the discovery of a planet by the microlensing method, OGLE-2012-BLG-0724Lb. Although the duration of the planetary signal for this event was one of the shortest seen for a planetary event, the anomaly was well covered thanks to high cadence observations taken by the survey groups OGLE and MOA. By analyzing the light curve, this planetary system is found to have a mass ratio $q=(1.58\\pm0.15)\\times10^{-3}$. By conducting a Bayesian analysis, we estimate that the host star is an M-dwarf star with a mass of $M_{\\rm L}=0.29_{-0.16}^{+0.33} \\ M_{\\odot}$ located at $D_{\\rm L}=6.7_{-1.2}^{+1.1} \\ {\\rm kpc}$ away from the Earth and the companion's mass is $m_{\\rm P}=0.47_{-0.26}^{+0.54} \\ M_{\\rm Jup}$. The projected planet-host separation is $a_{\\perp}=1.6_{-0.3}^{+0.4} \\ {\\rm AU}$. Because the lens-source relative proper motion is relatively high, future high resolution images would detect the lens host star and determine the lens properties uniquely. This system is likely a Saturn-mass exoplanet around an ...

  11. 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.

  12. 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

  13. OGLE-2005-BLG-071Lb, the Most Massive M-Dwarf Planetary Companion?

    Energy Technology Data Exchange (ETDEWEB)

    Dong, S; Gould, A; Udalski, A; Anderson, J; Christie, G W; Gaudi, B S; Jaroszynski, M; Kubiak, M; Szymanski, M K; Pietrzynski, G; Soszynski, I; Szewczyk, O; Ulaczyk, K; Wyrzykowski, L; DePoy, D L; Fox, D B; Gal-Yam, A; Han, C; Lepine, S; McCormick, J; Ofek, E; Park, B; Pogge, R W; Abe, F; Bennett, D P; Bond, I A; Britton, T R; Gilmore, A C; Hearnshaw, J B; Itow, Y; Kamiya, K; Kilmartin, P M; Korpela, A; Masuda, K; Matsubara, Y; Motomura, M; Muraki, Y; Nakamura, S; Ohnishi, K; Okada, C; Rattenbury, N; Saito, T; Sako, T; Sasaki, M; Sullivan, D; Sumi, T; Tristram, P J; Yanagisawa, T; Yock, P M; Yoshoika, T; Albrow, M D; Beaulieu, J P; Brillant, S; Calitz, H; Cassan, A; Cook, K H; Coutures, C; Dieters, S; Prester, D D; Donatowicz, J; Fouque, P; Greenhill, J; Hill, K; Hoffman, M; Horne, K; J?rgensen, U G; Kane, S; Kubas, D; Marquette, J B; Martin, R; Meintjes, P; Menzies, J; Pollard, K R; Sahu, K C; Vinter, C; Wambsganss, J; Williams, A; Bode, M; Bramich, D M; Burgdorf, M; Snodgrass, C; Steele, I; Doublier, V; Foelmi, C

    2008-04-18

    We combine all available information to constrain the nature of OGLE-2005-BLG-071Lb, the second planet discovered by microlensing and the first in a high-magnification event. These include photometric and astrometric measurements from Hubble Space Telescope, as well as constraints from higher-order effects extracted from the ground-based light curve, such as microlens parallax, planetary orbital motion and finite-source effects. Our primary analysis leads to the conclusion that the host of Jovian planet OGLE-2005-BLG-071Lb is a foreground M dwarf, with mass M = 0.46 {+-} 0.04M{sub {circle_dot}}, distance D{sub l} = 3.3 {+-} 0.4 kpc, and thick-disk kinematics {nu}{sub LSR} {approx} 103 km s{sup -1}. From the best-fit model, the planet has mass M{sub p} = 3.5 {+-} 0.3 M{sub Jupiter}, lies at a projected separation r{sub {perpendicular}} = 3.6 {+-} 0.2 AU from its host and has an equilibrium temperature of T {approx} 50 K, i.e., similar to Neptune. A degenerate model less favored by {Delta}{sub {chi}}{sup 2} {approx} 4 gives essentially the same planetary mass M{sub p} = 3.3 {+-} 0.3 M{sub Jupiter} with a smaller projected separation, r{sub {perpendicular}} = 2.1 {+-} 0.1 AU, and higher equilibrium temperature T {approx} 68 K. These results from the primary analysis suggest that OGLE-2005-BLG-071Lb is likely to be the most massive planet yet discovered that is hosted by an M dwarf. However, the formation of such high-mass planetary companions in the outer regions of M-dwarf planetary systems is predicted to be unlikely within the core-accretion scenario. There are a number of caveats to this analysis, but these could mostly be resolved by a single astrometric measurement a few years after the event.

  14. 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.

  15. 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...

  16. OGLE-2013-SN-079: A LONELY SUPERNOVA CONSISTENT WITH A HELIUM SHELL DETONATION

    Energy Technology Data Exchange (ETDEWEB)

    Inserra, C.; Sim, S. A.; Smartt, S. J.; Nicholl, M.; Jerkstrand, A.; Chen, T.-W. [Astrophysics Research Centre, School of Mathematics and Physics, Queens University Belfast, Belfast BT7 1NN (United Kingdom); Wyrzykowski, L. [University of Warsaw, Astronomical Observatory, Al. Ujazdowskie 400-478 Warszawa (Poland); Fraser, M.; Blagorodnova, N.; Campbell, H. [Institute of Astronomy, University of Cambridge, Madingley Road, CB3 0HA Cambridge (United Kingdom); Shen, K. J. [Department of Astronomy and Theoretical Astrophysics Center, University of California, Berkeley, CA 94720 (United States); Gal-Yam, A. [Benoziyo Center for Astrophysics, Weizmann Institute of Science, 76100 Rehovot (Israel); Howell, D. A.; Valenti, S. [Las Cumbres Observatory Global Telescope Network, 6740 Cortona Drive, Suite 102 Goleta, CA 93117 (United States); Maguire, K. [European Southern Observatory for Astronomical Research in the Southern Hemisphere (ESO), Karl-Schwarzschild-Str. 2, 85748 Garching b. Munchen (Germany); Mazzali, P.; Bersier, D. [Astrophysics Research Institute, Liverpool John Moores University, Liverpool (United Kingdom); Taubenberger, S.; Benitez-Herrera, S. [Max-Planck-Institut für Astrophysik, Karl-Schwarzschild-Str. 1, 85741 Garching (Germany); Elias-Rosa, N., E-mail: c.inserra@qub.ac.uk [INAF - Osservatorio Astronomico di Padova, Vicolo dell' Osservatorio 5, I-35122 Padova (Italy); and others

    2015-01-20

    We present observational data for a peculiar supernova discovered by the OGLE-IV survey and followed by the Public ESO Spectroscopic Survey for Transient Objects. The inferred redshift of z = 0.07 implies an absolute magnitude in the rest-frame I-band of M{sub I} ∼ –17.6 mag. This places it in the luminosity range between normal Type Ia SNe and novae. Optical and near infrared spectroscopy reveal mostly Ti and Ca lines, and an unusually red color arising from strong depression of flux at rest wavelengths <5000 Å. To date, this is the only reported SN showing Ti-dominated spectra. The data are broadly consistent with existing models for the pure detonation of a helium shell around a low-mass CO white dwarf and ''double-detonation'' models that include a secondary detonation of a CO core following a primary detonation in an overlying helium shell.

  17. OGLE Collection of Star Clusters. New Objects in the Outskirts of the Large Magellanic Cloud

    CERN Document Server

    Sitek, M; Skowron, D M; Udalski, A; Kostrzewa-Rutkowska, Z; Skowron, J; Karczmarek, P; Cieślar, M; Wyrzykowski, Ł; Kozłowski, S; Pietrukowicz, P; Soszyński, I; Mróz, P; Pawlak, M; Poleski, R; Ulaczyk, K

    2016-01-01

    The Magellanic System (MS), consisting of the Large Magellanic Cloud (LMC), the Small Magellanic Cloud (SMC) and the Magellanic Bridge (MBR), contains diverse sample of star clusters. Their spatial distribution, ages and chemical abundances may provide important information about the history of formation of the whole System. We use deep photometric maps derived from the images collected during the fourth phase of The Optical Gravitational Lensing Experiment (OGLE-IV) to construct the most complete catalog of star clusters in the Large Magellanic Cloud using the homogeneous photometric data. In this paper we present the collection of star clusters found in the area of about 225 square degrees in the outer regions of the LMC. Our sample contains 679 visually identified star cluster candidates, 226 of which were not listed in any of the previously published catalogs. The new clusters are mainly young small open clusters or clusters similar to associations.

  18. Bright but slow - Type II supernovae from OGLE-IV & magnitude limited surveys

    CERN Document Server

    Poznanski, Dovi; Wyrzykowski, Lukasz; Blagorodnova, Nadejda

    2015-01-01

    We study a sample of 11 Type II supernovae (SNe) discovered by the OGLE-IV survey. All objects have well sampled I-band light curves, and at least one spectrum. We find that 3 or 4 of the 11 SNe have a declining light curve, making them SNe II-L, while the rest have plateaus that can be as short as 70d, unlike the 100d typically found in nearby galaxies. These SNe are also brighter than found in the local Universe, and show that magnitude limited surveys find SNe that are different than found in nearby galaxies. We discuss this sample in the context of understanding Type II SNe as a class and their suggested use as standard candles.

  19. Binary Source Microlensing Event OGLE-2016-BLG-0733: Interpretation of a Long-Term Asymmetric Perturbation

    Science.gov (United States)

    Jung, Y. K.; Udalski, A.; Yee, J. C.; Sumi, T.; Gould, A.; Han, C.; Albrow, M. D.; Lee, C.-U.; Bennett, D. P.; Suzuki, D.

    2017-01-01

    In the process of analyzing an observed light curve, one often confronts various scenarios that can mimic the planetary signals causing difficulties in the accurate interpretation of the lens system. In this paper, we present the analysis of the microlensing event OGLE-2016-BLG-0733. The light curve of the event shows a long-term asymmetric perturbation that would appear to be due to a planet. From the detailed modeling of the lensing light curve, however, we find that the perturbation originates from the binarity of the source rather than the lens. This result demonstrates that binary sources with roughly equal-luminosity components can mimic long-term perturbations induced by planets with projected separations near the Einstein ring. The result also represents the importance of the consideration of various interpretations in planet-like perturbations and of high-cadence observations for ensuring the unambiguous detection of the planet.

  20. RED NOISE VERSUS PLANETARY INTERPRETATIONS IN THE MICROLENSING EVENT OGLE-2013-BLG-446

    Energy Technology Data Exchange (ETDEWEB)

    Bachelet, E.; Bramich, D. M.; AlSubai, K. [Qatar Environment and Energy Research Institute, Qatar Foundation, P.O. Box 5825, Doha (Qatar); Han, C. [Department of Physics, Chungbuk National University, Cheongju 361-763 (Korea, Republic of); Greenhill, J. [School of Mathematics and Physics, University of Tasmania, Private Bag 37, Hobart, TAS 7001 (Australia); Street, R. A.; Tsapras, Y. [Las Cumbres Observatory Global Telescope Network, 6740 Cortona Drive, Suite 102, Goleta, CA 93117 (United States); Gould, A.; Batista, V. [Department of Astronomy, Ohio State University, 140 W. 18th Ave., Columbus, OH 43210 (United States); D’Ago, G. [Dipartimento di Fisica “E.R. Caianiello,” Università di Salerno, Via Ponte Don Melillo, I-84084-Fisciano (Italy); Dominik, M.; Jaimes, R. Figuera; Horne, K.; Hundertmark, M. [SUPA, School of Physics and Astronomy, University of St Andrews, North Haugh, St Andrews KY16 9SS (United Kingdom); Kains, N. [European Southern Observatory, Karl-Schwarzschild-Str. 2, D-85748 Garching bei München (Germany); Snodgrass, C. [Max Planck Institute for Solar System Research, Justus-von-Liebig-Weg 3, D-37077 Gttingen (Germany); Steele, I. A. [Astrophysics Research Institute, Liverpool John Moores University, Liverpool CH41 1LD (United Kingdom); Albrow, M. D. [Department of Physics and Astronomy, University of Canterbury, Private Bag 4800, Christchurch 8020 (New Zealand); Beaulieu, J.-P. [UPMC-CNRS, UMR 7095, Institut dAstrophysique de Paris, 98bis boulevard Arago, F-75014 Paris (France); Bennett, D. P., E-mail: c.botzler@auckland.ac.nz, E-mail: p.yock@auckland.ac.nz, E-mail: bennett@nd.edu, E-mail: abe@stelab.nagoya-u.ac.jp, E-mail: furusawa@stelab.nagoya-u.ac.jp, E-mail: itow@stelab.nagoya-u.ac.jp [Department of Physics, 225 Nieuwland Science Hall, University of Notre Dame, Notre Dame, IN 46556 (United States); Collaboration: RoboNet collaboration; PLANET collaboration; μFUN collaboration; MOA collaboration; MiNDSTEp collaboration; and others

    2015-10-20

    For all exoplanet candidates, the reliability of a claimed detection needs to be assessed through a careful study of systematic errors in the data to minimize the false positives rate. We present a method to investigate such systematics in microlensing data sets using the microlensing event OGLE-2013-BLG-0446 as a case study. The event was observed from multiple sites around the world and its high magnification (A{sub max} ∼ 3000) allowed us to investigate the effects of terrestrial and annual parallax. Real-time modeling of the event while it was still ongoing suggested the presence of an extremely low-mass companion (∼3M{sub ⨁}) to the lensing star, leading to substantial follow-up coverage of the light curve. We test and compare different models for the light curve and conclude that the data do not favor the planetary interpretation when systematic errors are taken into account.

  1. The OGLE Collection of Variable Stars. Over 45 000 RR Lyrae Stars in the Magellanic System

    CERN Document Server

    Soszyński, I; Szymański, M K; Wyrzykowski, Ł; Ulaczyk, K; Poleski, R; Pietrukowicz, P; Kozłowski, S; Skowron, D; Skowron, J; Mróz, P; Pawlak, M

    2016-01-01

    We present the largest collection of RR Lyrae stars in the Magellanic System and in its foreground. The sample consists of 45 451 RR Lyr stars, of which 39 082 were detected toward the Large Magellanic Cloud and 6369 toward the Small Magellanic Cloud. We provide long-term time-series photometric measurements collected during the fourth phase of the Optical Gravitational Lensing Experiment (OGLE-IV). We discuss several potential astrophysical applications of our collection: investigation of the structure of the Magellanic Clouds and the Galactic halo, studies of the globular clusters in the Magellanic System, analysis of double-mode RR Lyr stars, and searching for RR Lyr stars in eclipsing binary systems.

  2. Difference Image Analysis of the OGLE-II bulge data. II. Microlensing events

    CERN Document Server

    Wozniak, P R; Szymanski, M H; Kubiak, M; Pietrzynski, G; Soszynski, I; Zebrun, K

    2001-01-01

    We present a sample of microlensing events discovered in the Difference Image Analysis (DIA) of the OGLE-II images collected during 3 observing seasons, 1997--1999. 4424 light curves pass our criteria on the presence of a brightening episode on top of a constant baseline. Among those, 512 candidate microlensing events were selected visually. We designed an automated procedure, which unambiguously selects up to 237 best events. Including 8 candidate events recovered by other means, a total of 520 light curves are presented in this work. In addition to microlensing events, the larger sample contains certain types of transients, but is also strongly contaminated by artifacts. All 4424 light curves in the weakly filtered group are available electronically, with the intent of showing the gray zone between microlensing events and variable stars, as well as artifacts, to some extent inevitable in massive data reductions. We welcome suggestions for improving the selection process before the full analysis of complete ...

  3. Red Noise Versus Planetary Interpretations in the Microlensing Event Ogle-2013-BLG-446

    Science.gov (United States)

    Bachelet, E.; Bramich, D. M.; Han, C.; Greenhill, J.; Street, R. A.; Gould, A.; D'Ago, G.; AlSubai, K.; Dominik, M.; Figuera Jaimes, R.; Horne, K.; Hundertmark, M.; Kains, N.; Snodgrass, C.; Steele, I. A.; Tsapras, Y.; RoboNet Collaboration; Albrow, M. D.; Batista, V.; Beaulieu, J.-P.; Bennett, D. P.; Brillant, S.; Caldwell, J. A. R.; Cassan, A.; Cole, A.; Coutures, C.; Dieters, S.; Dominis Prester, D.; Donatowicz, J.; Fouqué, P.; Hill, K.; Marquette, J.-B.; Menzies, J.; Pere, C.; Ranc, C.; Wambsganss, J.; Warren, D.; PLANET Collaboration; de Almeida, L. Andrade; Choi, J.-Y.; DePoy, D. L.; Dong, S.; Hung, L.-W.; Hwang, K.-H.; Jablonski, F.; Jung, Y. K.; Kaspi, S.; Klein, N.; Lee, C.-U.; Maoz, D.; Muñoz, J. A.; Nataf, D.; Park, H.; Pogge, R. W.; Polishook, D.; Shin, I.-G.; Shporer, A.; Yee, J. C.; μFUN Collaboration; Abe, F.; Bhattacharya, A.; Bond, I. A.; Botzler, C. S.; Freeman, M.; Fukui, A.; Itow, Y.; Koshimoto, N.; Ling, C. H.; Masuda, K.; Matsubara, Y.; Muraki, Y.; Ohnishi, K.; Philpott, L. C.; Rattenbury, N.; Saito, To.; Sullivan, D. J.; Sumi, T.; Suzuki, D.; Tristram, P. J.; Yonehara, A.; MOA Collaboration; Bozza, V.; Calchi Novati, S.; Ciceri, S.; Galianni, P.; Gu, S.-H.; Harpsøe, K.; Hinse, T. C.; Jørgensen, U. G.; Juncher, D.; Korhonen, H.; Mancini, L.; Melchiorre, C.; Popovas, A.; Postiglione, A.; Rabus, M.; Rahvar, S.; Schmidt, R. W.; Scarpetta, G.; Skottfelt, J.; Southworth, John; Stabile, An.; Surdej, J.; Wang, X.-B.; Wertz, O.; MiNDSTEp Collaboration

    2015-10-01

    For all exoplanet candidates, the reliability of a claimed detection needs to be assessed through a careful study of systematic errors in the data to minimize the false positives rate. We present a method to investigate such systematics in microlensing data sets using the microlensing event OGLE-2013-BLG-0446 as a case study. The event was observed from multiple sites around the world and its high magnification (Amax ˜ 3000) allowed us to investigate the effects of terrestrial and annual parallax. Real-time modeling of the event while it was still ongoing suggested the presence of an extremely low-mass companion (˜3M⊕) to the lensing star, leading to substantial follow-up coverage of the light curve. We test and compare different models for the light curve and conclude that the data do not favor the planetary interpretation when systematic errors are taken into account.

  4. The lowest mass ratio planetary microlens: OGLE 2016-BLG-1195Lb

    Science.gov (United States)

    Bond, I. A.; Bennett, D. P.; Sumi, T.; Udalski, A.; Suzuki, D.; Rattenbury, N. J.; Bozza, V.; Koshimoto, N.; Abe, F.; Asakura, Y.; Barry, R. K.; Bhattacharya, A.; Donachie, M.; Evans, P.; Fukui, A.; Hirao, Y.; Itow, Y.; Li, M. C. A.; Ling, C. H.; Masuda, K.; Matsubara, Y.; Muraki, Y.; Nagakane, M.; Ohnishi, K.; Ranc, C.; Saito, To.; Sharan, A.; Sullivan, D. J.; Tristram, P. J.; Yamada, T.; Yamada, T.; Yonehara, A.; Skowron, J.; Szymański, M. K.; Poleski, R.; Mróz, P.; Soszyński, I.; Pietrukowicz, P.; Kozłowski, S.; Ulaczyk, K.; Pawlak, M.

    2017-08-01

    We report discovery of the lowest mass ratio exoplanet to be found by the microlensing method in the light curve of the event OGLE 2016-BLG-1195. This planet revealed itself as a small deviation from a microlensing single lens profile from an examination of the survey data. The duration of the planetary signal is ˜2.5 h. The measured ratio of the planet mass to its host star is q = 4.2 ± 0.7 × 10-5. We further estimate that the lens system is likely to comprise a cold ˜3 Earth mass planet in an ˜2 au wide orbit around a 0.2 Solar mass star at an overall distance of 7.1 kpc.

  5. Kynurenine aminotransferase III and glutamine transaminase L are identical enzymes that have cysteine S-conjugate β-lyase activity and can transaminate L-selenomethionine.

    Science.gov (United States)

    Pinto, John T; Krasnikov, Boris F; Alcutt, Steven; Jones, Melanie E; Dorai, Thambi; Villar, Maria T; Artigues, Antonio; Li, Jianyong; Cooper, Arthur J L

    2014-11-01

    Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate β-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate β-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-L-selenocysteine (MSC) and L-selenomethionine (SM) to β-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH3SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the β-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze β-elimination reactions with MSC generating CH3SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH3SeH and/or seleno-keto acid metabolites.

  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. 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.

  8. Ogle-2012-blg-0724lb: A Saturn Mass Planet Around an M-dwarf

    Science.gov (United States)

    Hirao, Y.; Sumi, T.; Bennett, D. P.; Bond, I. A.; Rattenbury, N.; Suzuki, D.; Koshimoto, N.; Abe, F.; Asakura, Y.; Bhattacharya, A.

    2016-01-01

    We report the discovery of a planet by the microlensing method, OGLE-2012-BLG-0724Lb. Although the duration of the planetary signal for this event was one of the shortest seen for a planetary event, the anomaly was well covered thanks to high-cadence observations taken by the survey groups OGLE and MOA. By analyzing the light curve, this planetary system is found to have a mass ratio q = (1.58 +/- 0.15) x 10(exp -3). By conducting a Bayesian analysis, we estimate that the host star is an M dwarf with a mass of M(sub L) = 0.29(+0.33/-0.16) solar mass located at D(sub L) = 6.7(+1.1/-1.2) kpc away from the Earth and the companion's mass is m(sub P) = 0.47(+0.54/-0.26) M(Jup). The projected planet- host separation is a falsum = 1.6(+0.4/-0.3) AU. Because the lens-source relative proper motion is relatively high, future highresolution images would detect the lens host star and determine the lens properties uniquely. This system is likely a Saturn-mass exoplanet around an M dwarf, and such systems are commonly detected by gravitational microlensing. This adds another example of a possible pileup of sub-Jupiters (0.2 less than m(sub P)/M(sub Jup) less than 1) in contrast to a lack of Jupiters (approximately 1-2 M(sub Jup)) around M dwarfs, supporting the prediction by core accretion models that Jupiter-mass or more massive planets are unlikely to form around M dwarfs.

  9. 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.

  10. 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

  11. Difference Image Analysis of the OGLE-II Bulge Data. III. Catalog of 200,000 Candidate Variable Stars

    CERN Document Server

    Wozniak, P R; Szymanski, M H; Kubiak, M; Pietrzynski, G; Soszynski, I; Zebrun, K

    2002-01-01

    We present the first edition of a catalog of variable stars from OGLE-II Galactic Bulge data covering 3 years: 1997-1999. Typically 200-300 I band data points are available in 49 fields between -11 and 11 degrees in galactic longitude, totaling roughly 11 square degrees in sky coverage. Photometry was obtained using the Difference Image Analysis (DIA) software and tied to the OGLE data base with the DoPhot package. The present version of the catalog comprises 221,801 light curves. In this preliminary work the level of contamination by spurious detections is still about 10%. Parts of the catalog have only crude calibration, insufficient for distance determinations. The next, fully calibrated, edition will include the data collected in year 2000. The data is accessible via FTP. Due to the data volume, we also distribute DAT tapes upon request.

  12. Millimagnitude Photometry for Transiting Extrasolar Planetary Candidates III: Accurate Radius and Period for OGLE-TR-111-b

    CERN Document Server

    Minniti, D; D'iaz, R F; Udalski, A; Pietrzynski, G; Gieren, W; Rojo, P; Ru'iz, M T; Zoccali, M; Minniti, Dante; Fern\\'andez, Jos\\'e Miguel; D\\'iaz, Rodrigo F.; Udalski, Andrzej; Pietrzynski, Grzegorz; Gieren, Wolfgang; Rojo, Patricio; Ru\\'iz, Mar\\'ia Teresa; Zoccali, Manuela

    2007-01-01

    We present accurate V-band photometry for a planetary transit of OGLE-TR-111 acquired with VIMOS at the ESO Very Large Telescope. The measurement of this transit allows to refine the planetary radius, obtaining R_p= 1.01 +/- 0.06 R_J. Given the mass of M_p = 0.53 M_J previously measured from radial velocities, we confirm that the density is rho_p= 0.6 +/- 0.2 g/cm^3. We also revise the ephemeris for OGLE-TR-111-b, obtaining an accurate orbital period P= 4.014484 +/- 0.000014 days, and predicting that the next observable transits would occur around December 2006, and after that only in mid-2008. Even though this period is different from previously published values, we cannot yet rule out a constant period.

  13. OGLE-LMC-ECL-11893: The discovery of a long-period eclipsing binary with a circumstellar disk

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Subo [Kavli Institute for Astronomy and Astrophysics, Peking University, Yi He Yuan Road 5, Hai Dian District, Beijing 100871 (China); Katz, Boaz [Institute for Advanced Study, 1 Einstein Drive, Princeton, NJ 08544 (United States); Prieto, Jose L. [Department of Astrophysical Sciences, Princeton University, 4 Ivy Lane, Peyton Hall, Princeton, NJ 08544 (United States); Udalski, Andrzej; Kozlowski, Szymon [Warsaw University Observatory, Al. Ujazdowskie 4, 00-478 Warszawa (Poland); Street, R. A.; Tsapras, Y. [Las Cumbres Observatory Global Telescope Network, 6740 Cortona Drive, suite 102, Goleta, CA 93117 (United States); Bramich, D. M. [Qatar Environment and Energy Research Institute, Qatar Foundation, Tornado Tower, Floor 19, P.O. Box 5825, Doha (Qatar); Hundertmark, M.; Horne, K.; Dominik, M.; Jaimes, R. Figuera [SUPA, School of Physics and Astronomy, University of St Andrews, North Haugh, St Andrews KY16 9SS (United Kingdom); Snodgrass, C. [Max Planck Institute for Solar System Research, Justus-von-Liebig-Weg 3, D-37077 Göttingen (Germany)

    2014-06-10

    We report the serendipitous discovery of a disk-eclipse system OGLE-LMC-ECL-11893. The eclipse occurs with a period of 468 days, a duration of about 15 days, and a deep (up to Δm{sub I} ≈ 1.5), peculiar, and asymmetric profile. A possible origin of such an eclipse profile involves a circumstellar disk. The presence of the disk is confirmed by the H-α line profile from the follow-up spectroscopic observations, and the star is identified as Be/Ae type. Unlike the previously known disk-eclipse candidates, the eclipses of OGLE-LMC-ECL-11893 retain the same shape throughout the span of ∼17 yr (13 orbital periods), indicating no measurable orbital precession of the disk.

  14. 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...

  15. 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

  16. 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

  17. Modification of potato cell wall pectin by the introduction of rhamnogalacturonan lyase and β-galactosidase transgenes and their side effects

    NARCIS (Netherlands)

    Huang, Jie Hong; Kortstee, Anne; Dees, Dianka C.T.; Trindade, Luisa M.; Schols, Henk A.; Gruppen, Harry

    2016-01-01

    Genes encoding pectic enzymes were introduced to wild-type potato Karnico. Cell wall materials were extracted from Karnico and transgenic lines expressing β-galactosidase (β-Gal-14 mutant) or rhamnogalacturonan lyase (RGL-18 mutant). After sequential extraction, β-Gal-14 hot buffer-soluble solids

  18. OGLE2-TR-L9: An extrasolar planet transiting a fast-rotating F3 star

    CERN Document Server

    Snellen, I A G; van der Burg, R F J; Dreizler, S; Greiner, J; de Hoon, M D J; Husser, T O; Kruhler, T; Saglia, R P; Vuijsje, F N

    2008-01-01

    Context: Photometric observations for the OGLE-II microlens monitoring campaign have been taken in the period 1997-2000. All light curves of this campaign have recently been made public. Our analysis of these data has revealed 13 low-amplitude transiting objects among ~15700 stars in three Carina fields towards the galactic disk. One of these objects, OGLE2-TR-L9 (P~2.5 days), turned out to be an excellent transiting planet candidate. Aims: In this paper we report on our investigation of the true nature of OGLE2-TR-L9, by re-observing the photometric transit with the aim to determine the transit parameters at high precision, and by spectroscopic observations, to estimate the properties of the host star, and to determine the mass of the transiting object through radial velocity measurements. Methods: High precision photometric observations have been obtained in g', r', i', and z' band simultaneously, using the new GROND detector, mounted on the MPI/ESO 2.2m telescope at La Silla. Eight epochs of high-dispersio...

  19. Light Curve Solutions of an Eclipsing Binary OGLE-GD-ECL-04451 with a Dramatic Change in Amplitude

    CERN Document Server

    Gang, Li; jianning, Fu

    2016-01-01

    We present light curve solutions of the W UMa-type eclipsing binary OGLE-GD-ECL-04451, observed by both the \\emph{Optical Gravitational Lensing Ex-periment} (\\emph{OGLE}) program in 2006 and the \\emph{Antarctica Survey Telescope} (\\emph{AST3-1}) in 2012 at Dome A. We analyzed this binary system with the Wilson-Devinney(W-D) method 2013 version and derived the mass ratio $q=2.91 \\pm 0.07$, the inclination $i=76.86^\\circ \\pm 0.23^\\circ$, and the light variattion amplitud was $0^m.51$ based on the \\emph{OGLE} data. From the \\emph{AST3-1}'s data, we find that the amplitude dropped to $0^m.44$(2012) and the difference of magnitudes of the two light maxima is $0^m.03$. A hot spot was then added on the surface of the secondary to demonstrate the amplitude change and O'Conell effect of the binary system.

  20. Cloning and Expression Analysis of Tea Hydroperoxide Lyase Gene CsiHPL1%茶树脂氢过氧化物裂解酶基因CsiHPL1的克隆及表达

    Institute of Scientific and Technical Information of China (English)

    辛肇军; 孙晓玲; 张正群; 陈宗懋

    2013-01-01

    根据茶树基因CsiHPL1的cDNA序列设计引物,采用RT-PCR方法从茶树品种龙井43,中克隆了CsiHPL1 序列,并分析了CsiHPL1在生物和非生物胁迫下的诱导表达情况及亚细胞定位.结果表明,CsiHPL1包含一个1476 bp的最大开放阅读框,编码491个氨基酸,预测为13-HPL基因.Real-Time PCR分析结果表明,CsiHPL1的表达受到茶尺蠖取食、机械损伤和茉莉酸的诱导;构建了CsiHPL1与绿色荧光蛋白(GFP)基因的重组表达载体,经农杆菌瞬时转化烟草叶片,利用激光共聚焦显微镜在叶绿体中观察到GFP荧光,表明CsiHPL1编码的蛋白质为叶绿体蛋白质.%Primers were designed according to the cDNA sequence of tea CsiHPLl. RT-PCR method was used to clone CsiHPLl from Longjing 43'. The expression of CsiHPLl under biotic and abiotic stress and subcellular localization were analyzed. The results showed CsiHPLl contains an open reading frame of 1 476 bp which encodes a protein of 491 amino acids. This gene was predicted as a 13-HPL gene. Tea geometrid feeding, wounding and JA treatment up-regulated the expression levels of CsiHPLl. The total sequence of this gene was fused with GFP to construct a binary vector for tobacco transient transformation. Under confocal laser-scanning microscopy, green fluorescent signals were localized in chloroplasts in transgenic tobacco plants, suggesting that the gene encodes a protein targeting to chloroplast.

  1. 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.

  2. 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.

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

    Directory of Open Access Journals (Sweden)

    Fei Wu

    2016-03-01

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

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

    Science.gov (United States)

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

    2016-03-16

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

  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. 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.

  7. 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

  8. 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

  9. The First Circumbinary Planet Found by Microlensing: OGLE-2007-BLG-349L(AB)c

    CERN Document Server

    Bennett, D P; Udalski, A; Gould, A; Tsapras, Y; Kubas, D; Bond, I A; Greenhill, J; Cassan, A; Rattenbury, N J; Boyajian, T S; Luhn, J; Penny, M T; Anderson, J; Abe, F; Bhattacharya, A; Botzler, C S; Donachie, M; Freeman, M; Fukui, A; Hirao, Y; Itow, Y; Koshimoto, N; Li, M C A; Ling, C H; Masuda, K; Matsubara, Y; Muraki, Y; Nagakane, M; Ohnishi, K; Oyokawa, H; Perrott, Y C; Saito, To; Sharan, A; Sullivan, D J; Sumi, T; Suzuki, D; Tristram, P J; Yonehara, A; Yock, P C M; Szymanski, M K; Soszynski, I; Ulaczyk, K; Wyrzykowski, L; Allen, W; DePoy, D; Gal-Yam, A; Gaudi, B S; Han, C; Monard, I A G; Ofek, E; Pogge, R W; Street, R A; Bramich, D M; Dominik, M; Horne, K; Snodgrass, C; Steele, I A; Albrow, M D; Bachelet, E; Batista, V; Beaulieu, J -P; Brillant, S; Caldwell, J A R; Cole, A; Coutures, C; Dieters, S; Prester, D Dominis; Donatowicz, J; Fouque, P; Hundertmark, M; Jorgensen, U G; Kains, N; Kane, S R; Marquette, J -B; Menzies, J; Pollard, K R; Ranc, C; Sahu, K C; Wambsganss, J; Williams, A; Zub, M

    2016-01-01

    We present the analysis of the first circumbinary planet microlensing event, OGLE-2007-BLG-349. This event has a strong planetary signal that is best fit with a mass ratio of $q \\approx 3.4\\times 10^{-4}$, but there is an additional signal due to an additional lens mass, either another planet or another star. We find acceptable light curve fits with two classes of models: 2-planet models (with a single host star) and circumbinary planet models. The light curve also reveals a significant microlensing parallax effect, which constraints the mass of the lens system to be $M_L \\approx 0.7 M_\\odot$. Hubble Space Telescope images resolve the lens and source stars from their neighbors, and indicate excess flux due to the star(s) in the lens system. This is consistent with the predicted flux from the circumbinary models, where the lens mass is shared between two stars, but there is not enough flux to be consistent with the 2-planet, 1-star models. So, only the circumbinary models are consistent with the HST data. They...

  10. Physical parameters and evolutionary route for the LMC interacting binary OGLE 05155332-6925581

    CERN Document Server

    Garrido, Hernán; Djuraŝevic, Gojko; Kołaczkowski, Zbigniew; Niemzcura, Ewa; Mennekens, Nicki

    2012-01-01

    We analyze multicolor light curves and high resolution optical spectroscopy of the eclipsing binary and Double Periodic Variable OGLE 05155332-6925581. According to Mennickent et al., this system shows a significant change in the long non-orbital photometric cycle, a loop in the color-magnitude diagram during this cycle and discrete spectral absorption components that were interpreted as evidence of systemic mass loss. We find that the best fit to the multi-band light curves requires a circumprimary optically thick disc with a radius about twice the radius of the more massive star. The spectroscopy indicates a mass ratio of 0.21+-0.02 and masses for the hot and cool stars of 9.1+-0.5 and 1.9+-0.2 M_sun, respectively. A comparison with synthetic binary-star evolutionary models indicates that the system has an age of 4.76E7 years, is in the phase of rapid mass transfer, the second one in the life of this binary, in a Case-B mass-exchange stage. Donor-subtracted H_alpha profiles show the presence of double emiss...

  11. Mira variables in the Galactic bulge with OGLE-II data

    CERN Document Server

    Matsunaga, N; Nakada, Y

    2005-01-01

    We have extracted a total of 1968 Mira variables from the OGLE-II data base in the Galactic bulge region. Among them, 1960 are associated with 2MASS sources, and 1541 are further identified with MSX point sources. Their photometric properties are compared with those of Mira variables in the Large and Small Magellanic Clouds. We have found that mass-losing stars with circumstellar matter are reddened such that the colour dependence of the absorption coefficient is similar to that of interstellar matter. We also discuss the structure of the bulge. The surface number density of the bulge Mira variables is well correlated with the 2.2-micron surface brightness obtained by the COBE satellite. Using this relation, the total number of Mira variables in the bulge is estimated to be about 600,000. The logP-K relation of the Mira variables gives their space distribution which supports the well-known asymmetry of the bar-like bulge.

  12. A Giant Planet beyond the Snow Line in Microlensing Event OGLE-2011-BLG-0251

    CERN Document Server

    Kains, N; Choi, J -Y; Han, C; Udalski, A; Almeida, L A; Jablonski, F; Tristram, P; Jorgensen, U G; Szymanski, M K; Kubiak, M; Pietrzynski, G; Soszynski, I; Poleski, R; Kozlowski, S; Pietrukowicz, P; Ulaczyk, K; Wyrzykowski, L; Skowron, J; Alsubai, K A; Bozza, V; Browne, P; Burgdorf, M J; Novati, S Calchi; Dodds, P; Dominik, M; Dreizler, S; Fang, X -S; Grundahl, F; Gu, C -H; Hardis, S; Harpsoe, K; Hessman, F V; Hinse, T C; Hornstrup, A; Hundertmark, M; Jessen-Hansen, J; Kerins, E; Liebig, C; Lund, M; Lundkvist, M; Mancini, L; Mathiasen, M; Penny, M T; Rahvar, S; Ricci, D; Sahu, K C; Scarpetta, G; Skottfelt, J; Snodgrass, C; Southworth, J; Surdej, J; Tregloan-Reed, J; Wambsganss, J; Wertz, O; Bajek, D; Bramich, D M; Horne, K; Ipatov, S; Steele, I A; Tsapras, Y; Abe, F; Bennett, D P; Bond, I A; Botzler, C S; Chote, P; Freeman, M; Fukui, A; Furusawa, K; Itow, Y; Ling, C H; Masuda, K; Matsubara, Y; Miyake, N; Muraki, Y; Ohnishi, K; Rattenbury, N; Saito, T; Sullivan, D J; Sumi, T; Suzuki, D; Suzuki, K; Sweatman, W L; Takino, S; Wada, K; Yock, P C M; Allen, W; Batista, V; Chung, S -J; Christie, G; DePoy, D L; Drummond, J; Gaudi, B S; Gould, A; Henderson, C; Jung, Y -K; Koo, J -R; Lee, C -U; McCormick, J; McGregor, D; Munoz, J A; Natusch, T; Ngan, H; Park, H; Pogge, R W; Shin, I -G; Yee, J; Albrow, M D; Bachelet, E; Beaulieu, J -P; Brillant, S; Caldwell, J A R; Cassan, A; Cole, A; Corrales, E; Coutures, Ch; Dieters, S; Prester, D Dominis; Donatowicz, J; Fouque, P; Greenhill, J; Kane, S R; Kubas, D; Marquette, J -B; Martin, R; Meintjes, P; Menzies, J; Pollard, K R; Williams, A; Wouters, D; Zub, M

    2013-01-01

    We present the analysis of the gravitational microlensing event OGLE-2011-BLG-0251. This anomalous event was observed by several survey and follow-up collaborations conducting microlensing observations towards the Galactic Bulge. Based on detailed modelling of the observed light curve, we find that the lens is composed of two masses with a mass ratio q=1.9 x 10^-3. Thanks to our detection of higher-order effects on the light curve due to the Earth's orbital motion and the finite size of source, we are able to measure the mass and distance to the lens unambiguously. We find that the lens is made up of a planet of mass 0.53 +- 0.21,M_Jup orbiting an M dwarf host star with a mass of 0.26 +- 0.11 M_Sun. The planetary system is located at a distance of 2.57 +- 0.61 kpc towards the Galactic Centre. The projected separation of the planet from its host star is d=1.408 +- 0.019, in units of the Einstein radius, which corresponds to 2.72 +- 0.75 AU in physical units. We also identified a competitive model with similar ...

  13. OGLE-2008-BLG-513Lb: The Orbital Solution for a Microlensing Planet

    CERN Document Server

    Yee, J C; Dong, Subo; Greenhill, J; Tsapras, Y; Bond, I A; Gould, A; Kozlowski, S; Fouque, P; Albrow, M D; Han, C; Monard, L A G; McCormick, J; Williams, A; Kains, N; An, J; Dominik, M

    2011-01-01

    The dominant features of the microlensing event OGLE-2008-BLG-513 arise from a 2-body lens with a mass ratio q=0.027+/-0.001. The light curve cannot be adequately described by a static, 2-body lens model, which forces us to consider the orbital motion of the lens system. Including orbital motion improves the fit by Delta chi^2>1000. We model the orbital motion as a Keplerian orbit, and with the additional information from microlens parallax, we are able to place constraints on all eight parameters of the orbit. If our model is correct, this gives us the most complete orbital information of any microlensing planet. We find that the host star is 0.18

  14. OGLE-2011-BLG-0265Lb: a Jovian Microlensing Planet Orbiting an M Dwarf

    CERN Document Server

    Skowron, J; Udalski, A; Han, C; Sumi, T; Shvartzvald, Y; Gould, A; Dominis-Prester, D; Street, R A; Jørgensen, U G; Bennett, D P; Bozza, V; Szymański, M K; Kubiak, M; Pietrzyński, G; Soszyński, I; Poleski, R; Kozłowski, S; Pietrukowicz, P; Ulaczyk, K; Wyrzykowski, Ł; Abe, F; Bhattacharya, A; Bond, I A; Botzler, C S; Freeman, M; Fukui, A; Fukunaga, D; Itow, Y; Ling, C H; Koshimoto, N; Masuda, K; Matsubara, Y; Muraki, Y; Namba, S; Ohnishi, K; Philpott, L C; Rattenbury, N; Saito, T; Sullivan, D J; Suzuki, D; Tristram, P J; Yock, P C M; Maoz, D; Kaspi, S; Friedman, M; Almeida, L A; Batista, V; Christie, G; Choi, J -Y; DePoy, D L; Gaudi, B S; Henderson, C; Hwang, K -H; Jablonski, F; Jung, Y K; Lee, C -U; McCormick, J; Natusch, T; Ngan, H; Park, H; Pogge, R W; Yee, J; Albrow, M D; Bachelet, E; Beaulieu, J -P; Brillant, S; Caldwell, J A R; Cassan, A; Cole, A; Corrales, E; Coutures, Ch; Dieters, S; Donatowicz, J; Fouqué, P; Greenhill, J; Kains, N; Kane, S R; Kubas, D; Marquette, J -B; Martin, R; Menzies, J; Pollard, K R; Ranc, C; Sahu, K C; Wambsganss, J; Williams, A; Wouters, D; Tsapras, Y; Bramich, D M; Horne, K; Hundertmark, M; Snodgrass, C; Steele, I A; Alsubai, K A; Browne, P; Burgdorf, M J; Novati, S Calchi; Dodds, P; Dominik, M; Dreizler, S; Fang, X -S; Gu, C -H; Hardis,; Harpsøe, K; Hessman, F V; Hinse, T C; Hornstrup, A; Jessen-Hansen, J; Kerins, E; Liebig, C; Lund, M; Lundkvist, M; Mancini, L; Mathiasen, M; Penny, M T; Rahvar, S; Ricci, D; Scarpetta, G; Skottfelt, J; Southworth, J; Surdej, J; Tregloan-Reed, J; Wertz, O

    2014-01-01

    We report the discovery of a Jupiter-mass planet orbiting an M-dwarf star that gave rise to the microlensing event OGLE-2011-BLG-0265. Such a system is very rare among known planetary systems and thus the discovery is important for theoretical studies of planetary formation and evolution. High-cadence temporal coverage of the planetary signal combined with extended observations throughout the event allows us to accurately model the observed light curve. The final microlensing solution remains, however, degenerate yielding two possible configurations of the planet and the host star. In the case of the preferred solution, the mass of the planet is $M_{\\rm p}$ = 1.0 $\\pm$ 0.3 $M_{\\rm J}$, and the planet is orbiting a star with a mass $M$ = 0.23 $\\pm$ 0.07 $M_\\odot$. The second possible configuration (2\\sigma away) consists of a planet with $M_{\\rm p}$ = 0.6 $\\pm$ 0.2 $M_{\\rm J}$ and host star with $M$ = 0.15 $\\pm$ 0.06 $M_{\\odot}$. The system is located in the Galactic disk 3-4 kpc towards the Galactic bulge. In...

  15. OGLE-2013-SN-079: a lonely supernova consistent with a helium shell detonation

    CERN Document Server

    Inserra, C; Wyrzykowski, L; Smartt, S J; Fraser, M; Nicholl, M; Shen, K J; Jerkstrand, A; Gal-Yam, A; Howell, D A; Maguire, K; Mazzali, P; Valenti, S; Taubenberger, S; Benitez-Herrera, S; Bersier, D; Blagorodnova, N; Campbell, H; Chen, T -W; Elias-Rosa, N; Hillebrandt, W; Kostrzewa-Rutkowska, Z; Kozlowski, S; Kromer, M; Lyman, J D; Polshaw, J; Ropke, F K; Ruiter, A J; Smith, K; Spiro, S; Sullivan, M; Yaron, O; Young, D; Yuan, F

    2014-01-01

    We present observational data for a peculiar supernova discovered by the OGLE-IV survey and followed by the Public ESO Spectroscopic Survey for Transient Objects. The inferred redshift of z=0.07 implies an absolute magnitude in the rest-frame I-band of M$_{I}\\sim-17.6$ mag. This places it in the luminosity range between normal Type Ia SNe and novae. Optical and near infrared spectroscopy reveal mostly Ti and Ca lines, and an unusually red color arising from strong depression of flux at rest wavelengths <5000 \\AA. To date, this is the only reported SN showing Ti-dominated spectra. Our multi band and bolometric lightcurves, as well as the spectral evolution, are in reasonable agreement with the predictions of models for the pure detonation of a helium shell around a low-mass CO white dwarf and "double-detonation" models that include a secondary detonation of a CO core following a primary detonation in an overlying helium shell.

  16. Black Holes, Neutron Stars and White Dwarf Candidates from Microlensing with OGLE-III

    CERN Document Server

    Wyrzykowski, L; Skowron, J; Rybicki, K A; Mroz, P; Kozlowski, S; Udalski, A; Szymanski, M K; Pietrzynski, G; Soszynski, I; Ulaczyk, K; Pietrukowicz, P; Poleski, R; Pawlak, M; Ilkiewicz, K; Rattenbury, N J

    2015-01-01

    Most stellar remnants so far have been found in binary systems, where they interact with matter from their companions. Isolated neutron stars and black holes are difficult to find as they are dark, yet they are predicted to exist in our Galaxy in vast numbers. We explored the OGLE-III database of 150 million objects observed in years 2001-2009 and found 59 microlensing events exhibiting a parallax effect due to the Earth's motion around the Sun. Combining parallax and brightness measurements from microlensing light curves with expected proper motions in the Milky Way, we identified 15 microlensing events which are consistent with having a white dwarf, neutron star or a black hole lens and we estimated their masses and distances. The most massive of our black hole candidates has 8.3 M_Sun and is at a distance of 2.4 kpc. The distribution of masses of our candidates indicates a continuum in mass distribution with no mass gap between neutron stars and black holes. We also present predictions on how such events w...

  17. Non-radial Pulsations in RR Lyrae Stars from the OGLE Collection

    CERN Document Server

    Netzel, H

    2016-01-01

    RR Lyrae stars are classical pulsating stars. They pulsate mostly in the radial fundamental mode (RRab stars), in the radial first overtone mode (RRc stars), or in both modes simultaneously (RRd stars). Collection of variable stars from the Optical Gravitational Lensing Experiment (OGLE) contains more than 38 000 RR Lyrae stars from the Galactic bulge. We analysed these data for RRc and RRd stars. We have found new members of radial-non-radial double-mode RR Lyrae stars, with characteristic period ratio of the two modes around 0.61. We increased the number of known RR Lyrae stars of this type by a factor of 8. We have also discovered another group of double-mode RR Lyrae stars. They pulsate in the first overtone and in another, unidentified mode, which has period longer than period of the undetected fundamental mode. The period ratios tightly cluster around 0.686. These proceedings are focused on this puzzling group. In particular, we report eight new members of the group.

  18. Discovery of a Gas giant Planet in Microlensing Event OGLE-2014-BLG-1760

    CERN Document Server

    Bhattacharya, A; Bond, I A; Sumi, T; Udalski, A; Street, R; Tsapras, Y; Abe, F; Freeman, M; Fukui, A; Itow, Y; Li, M C A; Ling, C H; Masuda, K; Matsubara, Y; Muraki, Y; Ohnishi, K; Philpott, L C; Rattenbury, N; Saito, T; Sharan, A; Sullivan, D J; Suzuki, D; Tristram, P J; Szymański, M K; Kubiac, M; Pietrzyński, G; Soszyński, I; Poleski, R; Kozlowski, S; Pietrukowicz, P; Ulaczyk, K; Wyrzykowski, L; Bachelet, E; Bramich, D M; Browne, P; Dominik, M; Horne, K; Hundertmark, M; Ipatov, S; Kains, N; Snodgrass, C; Steele, I A

    2016-01-01

    We present the analysis of the planetary microlensing event OGLE-2014-BLG-1760, which shows a strong light curve signal due to the presence of a Jupiter mass-ratio planet. One unusual feature of this event is that the source star is quite blue, with $V-I = 1.48\\pm 0.08$. This is marginally consistent with source star in the Galactic bulge, but it could possibly indicate a young source star in the far side of the disk. Assuming a bulge source, we perform a Bayesian analysis assuming a standard Galactic model, and this indicates that the planetary system resides in or near the Galactic bulge at $D_L = 6.9 \\pm 1.1 $ kpc. It also indicates a host star mass of $M_* = 0.51 \\pm 0.44 M_\\odot$, a planet mass of $m_p = 180 \\pm 110 M_\\oplus$, and a projected star-planet separation of $a_\\perp = 1.7\\pm 0.3\\,$AU. The lens-source relative proper motion is $\\mu_{\\rm rel} = 6.5\\pm 1.1$ mas/yr. The lens (and stellar host star) is predicted to be very faint, so it is most likely that it can detected only when the lens and sour...

  19. Spectroscopic characterisation of microlensing events Towards a new interpretation of OGLE-2011-BLG-0417

    CERN Document Server

    Santerne, A; Ayala, B Rojas; Boisse, I; Schlawin, E; Almenara, J -M; Batista, V; Bennett, D; Díaz, R F; Figueira, P; James, D J; Herter, T; Lillo-Box, J; Marquette, J B; Ranc, C; Santos, N C; Sousa, S G

    2016-01-01

    The microlensing event OGLE-2011-BLG-0417 is an exceptionally bright lens binary that was predicted to present radial velocity variation at the level of several km/s. Pioneer radial velocity follow-up observations with the UVES spectrograph at the ESO - VLT of this system clearly ruled out the large radial velocity variation, leaving a discrepancy between the observation and the prediction. In this paper, we further characterise the microlensing system by analysing its spectral energy distribution (SED) derived using the UVES spectrum and new observations with the ARCoIRIS (CTIO) near-infrared spectrograph and the Keck adaptive optics instrument NIRC2 in the J, H, and Ks bands. We determine the mass and distance of the stars independently from the microlensing modelling. We find that the SED is compatible with a giant star in the Galactic bulge and a foreground star with a mass of 0.94+/-0.09Msun at a distance of 1.07+/-0.24kpc. We find that this foreground star is likely the lens. Its parameters are not comp...

  20. A New Non-Planetary Interpretation of the Microlensing Event OGLE-2013-BLG-0723

    CERN Document Server

    Han, Cheongho; Udalski, Andrzej; Jung, Youn Kil

    2016-01-01

    Recently, the discovery of a Venus-mass planet orbiting a brown-dwarf host in a binary system was reported from the analysis of the microlensing event OGLE-2013-BLG-0723. We reanalyze the event considering the possibility of other interpretations. From this, we find a new solution where the lens is composed of 2 bodies in contrast to the 3-body solution of the previous analysis. The new solution better explains the observed light curve than the previous solution with $\\Delta\\chi^2\\sim 202$, suggesting that the new solution is a correct model for the event. From the estimation of the physical parameters based on the new interpretation, we find that the lens system is composed of two low-mass stars with $\\sim 0.2\\ M_\\odot$ and $\\sim 0.1\\ M_\\odot$ and located at a distance $\\sim 3$ kpc. The fact that the physical parameters correspond to those of the most common lens population located at a distance with a large lensing probability further supports the likelihood of the new interpretation. Considering that two d...

  1. OGLE-2015-BLG-0196: Ground-based Gravitational Microlens Parallax Confirmed By Space-Based Observation

    CERN Document Server

    Han, C; Gould, A; Zhu, Wei; Szymański, M K; Soszyński, I; Skowron, J; Mróz, P; Poleski, R; Pietrukowicz, P; Kozłowski, S; Ulaczyk, K; Pawlak, M; Yee, J C; Beichman, C; Novati, S Calchi; Carey, S; Bryden, C; Fausnaugh, M; Gaudi, B S; Henderson, Calen B; Shvartzvald, Y; Wibking, B

    2016-01-01

    In this paper, we present the analysis of the binary gravitational microlensing event OGLE-2015-BLG-0196. The event lasted for almost a year and the light curve exhibited significant deviations from the lensing model based on the rectilinear lens-source relative motion, enabling us to measure the microlens parallax. The ground-based microlens parallax is confirmed by the data obtained from space-based microlens observations using the {\\it Spitzer} telescope. By additionally measuring the angular Einstein radius from the analysis of the resolved caustic crossing, the physical parameters of the lens are determined up to the two-fold degeneracy: $u_00$ solutions caused by the well-known "ecliptic" degeneracy. It is found that the binary lens is composed of two M dwarf stars with similar masses $M_1=0.38\\pm 0.04\\ M_\\odot$ ($0.50\\pm 0.05\\ M_\\odot)$ and $M_2=0.38\\pm 0.04\\ M_\\odot$ ($0.55\\pm 0.06\\ M_\\odot$) and the distance to the lens is $D_{\\rm L}=2.77\\pm 0.23$ kpc ($3.30\\pm 0.29$ kpc). Here the physical parameter...

  2. A compound heterozwous mutation in CYP17A1 gene in a female subject with partial 17α-hydroxylase/17,20 lyase deficiency%复合杂合突变致部分性17α-羟化酶/17,20碳链裂解酶缺陷症的临床和遗传分析

    Institute of Scientific and Technical Information of China (English)

    姜艳; 张妲; 聂敏; 肖新华; 郁琦; 陆召麟

    2011-01-01

    Objective To explore the clinical and molecular genetic characteristics of a Chinese female patient with partial 17α- hydroxylase/17, 20 lyase deficiency ( 17OHD), a rare type of congenital adrenal hyperplasia. Methods Her clinical features and laboratory data were collected. Genomic DNA was extracted from leukocytes of peripheral blood of her and her mother. All eight exons of CYP17A1 gene,including flanking regions of introns, were amplified by PCR. The mutations of CYP17A1 gene were identified by direct sequencing or cloning and sequencing the amplified DNA fragments. Results The patient presented with hypertension, hypokalemia and irregular menstruation. DNA sequencing results demonstrated a compound heterozygous mutation in CYP17A1 gene. One allele of her had the deletion of phenylalanine (TTC) at either codon 53 or 54 and the other allele contained a base transversion at codon 329 (TAC/AA) and leading to a missense mutation of tyrosine to lysine and the open reading frame shift following this codon to produce a truncated enzyme with 417 amino acids and without activity site. Her mother was a heterozygous carrier of the latter allele. Conclusion The partial 17OHD in this patient is caused by a compound heterozygous mutation in CYP17A1 gene.%目的 通过对1例部分性17α-羟化酶/17,20碳链裂解酶缺陷症(17OHD)的女性患者及其母亲的临床特点和基因突变研究,初步探讨部分性17OHD患者临床表现的基因分子生物学机制。方法 收集患者临床资料,提取患者及其母亲的外周血白细胞DNA,PCR扩增CYP17A1基因的8个外显子及内含子边界,测序确定CYP17A1基因的突变位点。结果 患者临床表现为高血压及轻度低钾血症,有不规律月经,CYP17A1基因序列分析发现患者为复合杂合突变,其中1个等位基因突变为编码P45Oc17蛋白的53或54位苯丙氨酸的密码子TTC缺失,即Delp. 53/54F;另1个等位基因突变为编码329位氨基酸的密码子

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. Phenylalanine ammonia-lyase (PAL) gene activity in response to ...

    African Journals Online (AJOL)

    Yomi

    2012-01-03

    Jan 3, 2012 ... Shetty, 1998). Tyrosine changes through a series of chemical reaction ... All tubes were placed under cool white florescent light at intensity of 52 to 66 µmol/m2/s, ..... Enzymatic regulation of photosynthetic. CO2 fixation in C3 ...

  8. VizieR Online Data Catalog: OGLE-III SMC massive stars VI light curves (Kourniotis+, 2014)

    Science.gov (United States)

    Kourniotis, M.; Bonanos, A. Z.; Soszynski, I.; Poleski, R.; Krikelis, G.; Udalski, A.; Szymanski, M. K.; Kubiak, M.; Pietrzynski, G.; Wyrzykowski, L.; Ulaczyk, K.; Kozlowski, S.; Pietrukowicz, P.

    2014-01-01

    We used the photometry provided by the OGLE-III monitoring survey to study the variability of 4646 massive stars with known spectral types in the SMC. Based on the standard deviation of the light curves, we classified the stars into constant, low-amplitude and high amplitude variables. We searched the low- and high-amplitude variables for periodic signals using the Analysis of Variance method (Schwarzenberg-Czerny, 1989MNRAS.241..153S). The following tables present newly discovered and known EBs, ellipsoidal variables, Cepheids and other periodic stars. We also present the irregular variables, which have stochastic, high-amplitude variability. (12 data files).

  9. The Optical Gravitational Lensing Experiment (OGLE-II). Difference Image Analysis of the Bulge Data.

    Science.gov (United States)

    Wozniak, P. R.

    2000-12-01

    During 1997-1999 observing seasons (mid March to mid December) the OGLE-II project collected more than 11,000 2Kx8K frames (over 370 GB of pixel data) of the Galactic Bulge using 1.3m Warsaw Telescope at the Las Campanas Observatory, Chile. Each of the 49 fields has roughly 200-300 measurements in I band. The fields span the range approximately from -10 to 10 deg in galactic longitude. I present a complete reanalysis of this data set using the optimal image subtraction method developed by Alard and Lupton (1998) and Alard (1999). Databases of difference measurements contain about 100,000 variable objects. This information is supplemented with colors from DoPhot photometry. Noise properties of our difference light curves are exceptionally good for this kind of massive monitoring program. The nongaussian tail in the distribution of residuals is totally negligible for usual applications. For faint stars the measurement errors are only 1.15 times photon noise. The difference photometry is always at least a factor of 2 better than results from DoPhot. Systematic effects due to blending are greatly relieved, the most important difference being the unbiased value of the variable light centroid. We discovered 512 microlensing events (compared to 214 from DoPhot photometry, Udalski et al. 2000). 305 of those were found fully algorithmically and have good quality light curves making them very well suited for optical depth determination. In the nearest future we plan to obtain an upper limit on the number of jupiters around microlenses as these should manifest themselves in the nongaussian tail of the residual distribution. Next possibilities include much better and larger extinction maps of the bulge and studies of the galactic bar. With 300-500 events we should be able to study the depth of the lens/source populations (Stanek 1996).

  10. The Initial Mass Function of the Inner Galaxy Measured from OGLE-III Microlensing Timescales

    Science.gov (United States)

    Wegg, Christopher; Gerhard, Ortwin; Portail, Matthieu

    2017-07-01

    We use the timescale distribution of ˜3000 microlensing events measured by the OGLE-III survey, together with accurate new made-to-measure dynamical models of the Galactic bulge/bar region, to measure the IMF in the inner Milky Way. The timescale of each event depends on the mass of the lensing object, together with the relative distances and velocities of the lens and source. The dynamical model statistically provides these distances and velocities, allowing us to constrain the lens mass function, and thereby infer the IMF. Parameterizing the IMF as a broken power-law, we find slopes in the main-sequence {α }{ms}=1.31+/- 0.10{| }{stat}+/- 0.10{| }{sys}, and brown dwarf region {α }{bd}=-0.7+/- 0.9{| }{stat}+/- 0.8{| }{sys}, where we use a fiducial 50% binary fraction, and the systematic uncertainty covers the range of binary fractions 0%-100%. Similarly, for a log-normal IMF we conclude {M}c=(0.17+/- 0.02{| }{stat}+/- 0.01{| }{sys}) {\\text{}}{M}⊙ and {σ }m=0.49+/- 0.07{| }{stat}+/- 0.06{| }{sys}. These values are very similar to a Kroupa or Chabrier IMF, respectively, showing that the IMF in the bulge is indistinguishable from that measured locally, despite the lenses lying in the inner Milky Way where the stars are mostly ˜10 Gyr old and formed on a fast α-element enhanced timescale. This therefore constrains models of IMF variation that depend on the properties of the collapsing gas cloud.

  11. OGLE-2013-BLG-1761Lb: A Massive Planet around an M/K Dwarf

    Science.gov (United States)

    Hirao, Y.; Udalski, A.; Sumi, T.; Bennett, D. P.; Koshimoto, N.; Bond, I. A.; Rattenbury, N. J.; Suzuki, D.; and; Abe, F.; Asakura, Y.; Barry, R. K.; Bhattacharya, A.; Donachie, M.; Evans, P.; Fukui, A.; Itow, Y.; Li, M. C. A.; Ling, C. H.; Masuda, K.; Matsubara, Y.; Matsuo, T.; Muraki, Y.; Nagakane, M.; Ohnishi, K.; Ranc, C.; Saito, To.; Sharan, A.; Shibai, H.; Sullivan, D. J.; Tristram, P. J.; Yamada, T.; Yamada, T.; Yonehara, A.; MOA Collaboration; Poleski, R.; Skowron, J.; Mróz, P.; Szymański, M. K.; Kozłowski, S.; Pietrukowicz, P.; Soszyński, I.; Wyrzykowski, Ł.; Ulaczyk, K.; OGLE Collaboration

    2017-07-01

    We report the discovery and the analysis of the planetary microlensing event, OGLE-2013-BLG-1761. There are some degenerate solutions in this event because the planetary anomaly is only sparsely sampled. However, the detailed light-curve analysis ruled out all stellar binary models and shows the lens to be a planetary system. There is the so-called close/wide degeneracy in the solutions with the planet/host mass ratio of q ˜ (7.0 ± 2.0) × 10-3 and q ˜ (8.1 ± 2.6) × 10-3 with the projected separation in Einstein radius units of s = 0.95 (close) and s = 1.18 (wide), respectively. The microlens parallax effect is not detected, but the finite source effect is detected. Our Bayesian analysis indicates that the lens system is located DL=6.9-1.2+1.0 kpc away from us and the host star is an M/K dwarf with a mass of ML=0.33-0.19+0.32 M⊙ orbited by a super-Jupiter mass planet with a mass of mP=2.7-1.5+2.5 MJup at the projected separation of a\\perp=1.8-0.5+0.5 au. The preference of the large lens distance in the Bayesian analysis is due to the relatively large observed source star radius. The distance and other physical parameters may be constrained by the future high-resolution imaging by large ground telescopes or HST. If the estimated lens distance is correct, then this planet provides another sample for testing the claimed deficit of planets in the Galactic bulge.

  12. Discovery of a Gas Giant Planet in Microlensing Event OGLE-2014-BLG-1760

    Science.gov (United States)

    Bhattacharya, A.; Bennett, D. P.; Bond, I. A.; Sumi, T.; Udalski, A.; Street, R.; Tsapras, Y.; Abe, F.; Freeman, M.; Fukui, A.; Hirao, Y.; Itow, Y.; Koshimoto, N.; Li, M. C. A.; Ling, C. H.; Masuda, K.; Matsubara, Y.; Muraki, Y.; Nagakane, M.; Ohnishi, K.; Rattenbury, N.; Saito, T.; Sharan, A.; Sullivan, D. J.; Suzuki, D.; Tristram, P. J.; MOA Collaboration; Skowron, J.; Szymański, M. K.; Soszyński, I.; Poleski, R.; Mróz, P.; Kozlowski, S.; Pietrukowicz, P.; Ulaczyk, K.; Wyrzykowski, L.; OGLE Collaboration; Bachelet, E.; Bramich, D. M.; D'Ago, G.; Dominik, M.; Figuera Jaimes, R.; Horne, K.; Hundertmark, M.; Kains, N.; Menzies, J.; Schmidt, R.; Snodgrass, C.; Steele, I. A.; Wambsganss, J.; ROBONET Collaboration

    2016-11-01

    We present the analysis of the planetary microlensing event OGLE-2014-BLG-1760, which shows a strong light-curve signal due to the presence of a Jupiter mass ratio planet. One unusual feature of this event is that the source star is quite blue, with V-I=1.48+/- 0.08. This is marginally consistent with a source star in the Galactic bulge, but it could possibly indicate a young source star on the far side of the disk. Assuming a bulge source, we perform a Bayesian analysis assuming a standard Galactic model, and this indicates that the planetary system resides in or near the Galactic bulge at {D}L=6.9+/- 1.1 {kpc}. It also indicates a host-star mass of {M}* ={0.51}-0.28+0.44{M}⊙ , a planet mass of {m}{{p}}={0.56}-0.26+0.34{M}J, and a projected star-planet separation of {a}\\perp ={1.75}-0.33+0.34 au. The lens-source relative proper motion is {μ }{rel}=6.5+/- 1.1 mas yr-1. The lens (and stellar host star) is estimated to be very faint compared to the source star, so it is most likely that it can be detected only when the lens and source stars start to separate. Due to the relatively high relative proper motion, the lens and source will be resolved to about ˜46 mas in 6-8 yr after the peak magnification. So, by 2020-2022, we can hope to detect the lens star with deep, high-resolution images.

  13. OGLE-2013-BLG-1761Lb: A Massive Planet around an MK Dwarf

    Science.gov (United States)

    Hirao, Y.; Udalski, A.; Sumi, T.; Bennett, D. P.; Koshimoto, N.; Bond, I. A.; Rattenbury, N. J.; Suzuki, D.; Abe, F.; Asakura, Y.; hide

    2017-01-01

    We report the discovery and the analysis of the planetary microlensing event, OGLE-2013-BLG-1761. There are some degenerate solutions in this event because the planetary anomaly is only sparsely sampled. However, the detailed light curve analysis ruled out all stellar binary models and shows the lens to be a planetary system. There is the so-called close wide degeneracy in the solutions with the planet host mass ratio of q approx.(7.0+/-2.0) x 10(exp -3) and q approx.(8.1+/-2.6) x 10(exp -3) with the projected separation in Einstein radius units of s = 0.95 (close) and s = 1.18(wide), respectively. The microlens parallax effect is not detected, but the finite source effect is detected. Our Bayesian analysis indicates that the lens system is located -D(sub L) = 6.9(+ 1.0 -1.2)kpc away from us and the host star is an M/K dwarf with amass of M(sub L) = 0.33(+ 0.32- 1.9)Stellar Mass orbited by a super-Jupiter mass planet with a mass of m(sub p) = 2.7(+ 2.5 - 1.5) M(sub Jup) at the projected separation of a(sub l) = 1.8(+ 0.5 -0.5)au. The preference of the large lens distance in the Bayesian analysis is due to the relatively large observed source star radius. The distance and other physical parameters may be constrained by the future high-resolution imaging by large ground telescopes or HST. If the estimated lens distance is correct, then this planet provides another sample for testing the claimed deficit of planets in the Galactic bulge.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  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.

    2015-01-01

    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

  20. 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.

  1. 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.

  2. SPITZER AS A MICROLENS PARALLAX SATELLITE: MASS MEASUREMENT FOR THE OGLE-2014-BLG-0124L PLANET AND ITS HOST STAR

    Energy Technology Data Exchange (ETDEWEB)

    Udalski, A.; Skowron, J.; Kozłowski, S.; Poleski, R.; Pietrukowicz, P.; Pietrzyński, G.; Szymański, M. K.; Mróz, P.; Soszyński, I.; Ulaczyk, K.; Wyrzykowski, Ł. [Warsaw University Observatory, Al. Ujazdowskie 4, 00-478 Warszawa (Poland); Yee, J. C. [Harvard-Smithsonian Center for Astrophysics, 60 Garden St., Cambridge, MA 02138 (United States); Gould, A.; Zhu, W.; Pogge, R. W. [Department of Astronomy, Ohio State University, 140 W. 18th Ave., Columbus, OH 43210 (United States); Carey, S. [Spitzer Science Center, MS 220-6, California Institute of Technology, Pasadena, CA (United States); Han, C. [Department of Physics, Chungbuk National University, Cheongju 371-763 (Korea, Republic of); Calchi Novati, S. [NASA Exoplanet Science Institute, MS 100-22, California Institute of Technology, Pasadena, CA 91125 (United States)

    2015-02-01

    We combine Spitzer and ground-based observations to measure the microlens parallax vector π{sub E}, and thus the mass and distance of OGLE-2014-BLG-0124L, making it the first microlensing planetary system with a space-based parallax measurement. The planet and star have masses of m ∼ 0.5 M {sub jup} and M ∼ 0.7 M {sub ☉} and are separated by a ∼ 3.1 AU in projection. The main source of uncertainty in all of these numbers (approximately 30%, 30%, and 20%) is the relatively poor measurement of the Einstein radius θ{sub E}, rather than uncertainty in π{sub E}, which is measured with 2.5% precision. This compares to 22% based on OGLE data alone, implying that the Spitzer data provide not only a substantial improvement in the precision of the π{sub E} measurement, but also the first independent test of a ground-based π{sub E} measurement.

  3. OGLE-2012-BLG-0950Lb: The Possible First Planet Mass Measurement from Only Microlens Parallax and Lens Flux

    CERN Document Server

    Koshimoto, N; Beaulieu, J P; Sumi, T; Bennett, D P; Bond, I A; Rattenbury, N; Fukui, A; Batista, V; Marquette, J B; Brillant, S; Abe, F; Asakura, Y; Bhattacharya, A; Freeman, M; Hirao, Y; Itow, Y; Li, M C A; Ling, C H; Masuda, K; Matsubara, Y; Matsuo, T; Muraki, Y; Ohnishi, K; Oyokawa, H; Saito, To; Sharan, A; Shibai, H; Sullivan, D J; Suzuki, D; Tristram, P J; Yonehara, A; Kozlowski, S; Pietrukowicz, P; Poleski, R; Skowron, J; Soszynski, I; Szymanski, M K; Ulaczyk, K; Wyrzykowski, L

    2016-01-01

    We report the discovery of a microlensing planet OGLE-2012-BLG-0950Lb with the planet/host mass ratio of $q = 2 \\times 10^{-4}$. A long term distortion detected in both MOA and OGLE light curve can be explained by the microlens parallax due to the Earth's orbital motion around the Sun. Although the finite source effect is not detected, we obtain the lens flux by the high resolution Keck AO observation. Combining the microlens parallax and the lens flux reveal the nature of the lens: a planet with mass of $M_{p} = 35^{+17}_{-9} M_{Earth}$ is orbiting around a M-dwarf with mass of $M_{h} = 0.56^{+0.12}_{-0.16} M_{Sun}$ with a planet-host projected separation of $r_{proj} =2.7^{+0.6}_{-0.7}$ AU located at $D_{L} = 3.0^{+0.8}_{-1.1}$ kpc from us. This is the first mass measurement from only microlens parallax and the lens flux without the finite source effect. The long term distortion can also be explained by the source orbital motion (xallarap) which is suspicious but not ruled out. These models can be distingui...

  4. Period-Luminosity relations derived from the OGLE-III First-overtone mode Cepheids in the Magellanic Clouds

    CERN Document Server

    Bhardwaj, Anupam; Kanbur, Shashi M; Singh, Harinder P

    2016-01-01

    We present multi-band Period-Luminosity (PL) relations for first-overtone mode Cepheids in the Small Magellanic Cloud (SMC). We derive optical band PL relations and the Wesenheit function using $VI$ mean magnitudes from the Optical Gravitational Lensing Experiment (OGLE-III) survey. We cross-match OGLE-III first-overtone mode Cepheids to the 2MASS and SAGE-SMC catalogs to derive PL relations at near-infrared ($JHK_s$) and mid-infrared ($3.6~\\&~4.5\\mu\\mathrm{m}$) wavelengths. We test for possible non-linearities in these PL relations using robust statistical tests and find a significant break only in the optical-band PL relations at 2.5 days for first-overtone mode Cepheids. We do not find statistical evidence for a non-linearity in these PL relations at 1 day. The multi-band PL relations for fundamental-mode Cepheids in the SMC also exhibit a break at 2.5 days. We suggest that the period break around 2.5 days is related to sharp changes in the light curve parameters for SMC Cepheids. We also derive new op...

  5. Period-Luminosity Relations Derived From the OGLE-III Fundamental Mode Cepheids II: The Small Magellanic Cloud Cepheids

    CERN Document Server

    Ngeow, Chow-Choong; Bhardwaj, Anupam; Singh, Harinder P

    2015-01-01

    In this paper we present multi-band period-luminosity (P-L) relations for fundamental mode Cepheids in the SMC. The optical VI-band mean magnitudes for these SMC Cepheids were taken from the third phase of the Optical Gravitational Lensing Experiment (OGLE-III) catalog. We also matched the OGLE-III SMC Cepheids to 2MASS and SAGE-SMC catalog to derive mean magnitudes in the JHK-bands and the four {\\it Spitzer} IRAC bands, respectively. All photometry was corrected for extinction by adopting the Zaritsky's extinction map. Cepheids with periods smaller than $\\sim2.5$ days were removed from the sample. In addition to the extinction corrected P-L relations in nine filters from optical to infrared, we also derived the extinction-free Wesenheit function for these Cepheids. We tested the nonlinearity of these SMC P-L relations (except the $8.0\\mu\\mathrm{m}$-band P-L relation) at 10 days: none of the P-L relations show statistically significant evidence of nonlinearity. When compared to the P-L relations in the LMC, t...

  6. Physical parameters and the projection factor of the classical Cepheid in the binary system OGLE-LMC-CEP-0227

    CERN Document Server

    Pilecki, B; Pietrzyński, G; Gieren, W; Thompson, I B; Freedman, W L; Scowcroft, V; Madore, B F; Udalski, A; Soszyński, I; Konorski, P; Smolec, R; Nardetto, N; Bono, G; Moroni, P G Prada; Storm, J; Gallenne, A

    2013-01-01

    A novel method of analysis of double-lined eclipsing binaries containing a radially pulsating star is presented. The combined pulsating-eclipsing light curve is built up from a purely eclipsing light curve grid created using an existing modeling tool. For every pulsation phase the instantaneous radius and surface brightness are taken into account, being calculated from the disentangled radial velocity curve of the pulsating star and from its out-of-eclipse pulsational light curve and the light ratio of the components, respectively. The best model is found using the Markov Chain Monte Carlo method. The method is applied to the eclipsing binary Cepheid OGLE-LMC-CEP-0227 (P_puls = 3.80 d, P_orb = 309 d). We analyze a set of new spectroscopic and photometric observations for this binary, simultaneously fitting OGLE V-band, I-band and Spitzer 3.6 {\\mu}m photometry. We derive a set of fundamental parameters of the system significantly improving the precision comparing to the previous results obtained by our group. ...

  7. Spitzer as Microlens Parallax Satellite: Mass Measurement for the OGLE-2014-BLG-0124L Planet and its Host Star

    CERN Document Server

    Udalski, A; Gould, A; Carey, S; Zhu, W; Skowron, J; Kozłowski, S; Poleski, R; Pietrukowicz, P; Pietrzyński, G; Szymański, M K; Mróz, P; Soszyński, I; Ulaczyk, K; Wyrzykowski, Ł; Han, C; Novati, S Calchi; Pogge, R W

    2014-01-01

    We combine Spitzer and ground-based observations to measure the microlens parallax vector ${\\mathbf \\pi}_{\\rm E}$, and so the mass and distance of OGLE-2014-BLG-0124L, making it the first microlensing planetary system with a space-based parallax measurement. The planet and star have masses $m \\sim 0.5\\,M_{\\rm jup}$ and $M\\sim 0.7\\,M_\\odot$ and are separated by $a_\\perp\\sim 3.1$ AU in projection. The main source of uncertainty in all these numbers (approximately 30%, 30%, and 20%) is the relatively poor measurement of the Einstein radius $\\theta_{\\rm E}$, rather than uncertainty in $\\pi_{\\rm E}$, which is measured with 2.5% precision. This compares to 22% based on OGLE data alone, implying that the Spitzer data provide not only a substantial improvement in the precision of the $\\pi_{\\rm E}$ measurement but also the first independent test of a ground-based ${\\mathbf \\pi}_{\\rm E}$ measurement.

  8. Spitzer as Microlens Parallax Satellite: Mass and Distance Measurements of Binary Lens System OGLE-2014-BLG-1050L

    CERN Document Server

    Zhu, Wei; Gould, A; Dominik, M; Bozza, V; Han, C; Yee, J C; Novati, S Calchi; Beichman, C A; Carey, S; Poleski, R; Skowron, J; Kozlowski, S; Mroz, P; Pietrukowicz, P; Pietrzynski, G; Szymanski, M K; Soszynski, I; Ulaczyk, K; Wyrzykowski, L; Han, C; Gaudi, B S; Pogge, R W; DePoy, D L; Jung, Y K; Choi, J -Y; Hwang, K -H; Shin, I -G; Park, H; Jeong, J

    2015-01-01

    We report the first mass and distance measurement of a caustic-crossing binary system OGLE-2014-BLG-1050L using the space-based microlens parallax method. \\emph{Spitzer} captured the second caustic-crossing of the event, which occurred $\\sim$10 days before that seen from Earth. Due to the coincidence that the source-lens relative motion was almost parallel to the direction of the binary-lens axis, the four-fold degeneracy, which was known before only to occur in single-lens events, persists in this case, leading to either a lower-mass (0.2 $M_\\odot$ and 0.07 $M_\\odot$) binary at $\\sim$1.1 kpc or a higher-mass (0.9 $M_\\odot$ and 0.35 $M_\\odot$) binary at $\\sim$3.5 kpc. However, the latter solution is strongly preferred for reasons including blending and lensing probability. OGLE-2014-BLG-1050L demonstrates the power of microlens parallax in probing stellar and substellar binaries.

  9. OGLE-2012-BLG-0950Lb: The First Planet Mass Measurement from Only Microlens Parallax and Lens Flux

    Science.gov (United States)

    Koshimoto, N.; Udalski, A.; Beaulieu, J. P.; Sumi, T.; Bennett, D. P.; Bond, I. A.; Rattenbury, N.; Fukui, A.; Batista, V.; Marquette, J. B.; Brillant, S.; and; Abe, F.; Asakura, Y.; Bhattacharya, A.; Donachie, M.; Freeman, M.; Hirao, Y.; Itow, Y.; Li, M. C. A.; Ling, C. H.; Masuda, K.; Matsubara, Y.; Matsuo, T.; Muraki, Y.; Nagakane, M.; Ohnishi, K.; Oyokawa, H.; Saito, To.; Sharan, A.; Shibai, H.; Sullivan, D. J.; Suzuki, D.; Tristram, P. J.; Yonehara, A.; MOA Collaboration; Kozłowski, S.; Pietrukowicz, P.; Poleski, R.; Skowron, J.; Soszyński, I.; Szymański, M. K.; Ulaczyk, K.; Wyrzykowski, Ł.; OGLE Collaboration

    2017-01-01

    We report the discovery of a microlensing planet OGLE-2012-BLG-0950Lb with a planet/host mass ratio of q≃ 2× {10}-4. A long term distortion detected in both MOA and OGLE light curve can be explained by the microlens parallax due to the Earth’s orbital motion around the Sun. Although the finite source effect is not detected, we obtain the lens flux by the high resolution Keck AO observation. Combining the microlens parallax and the lens flux reveal the nature of the lens: a planet with mass of {M}{{p}}={35}-9+17{M}\\oplus is orbiting around an M-dwarf with mass of {M}{host}={0.56}-0.16+0.12{M}ȯ with a planet-host projected separation of {r}\\perp ={2.7}-0.7+0.6 au located at {D}{{L}}={3.0}-1.1+0.8 kpc from us. This is the first mass measurement from only microlens parallax and the lens flux without the finite source effect. In the coming space observation-era with Spitzer, K2, Euclid, and WFIRST, we expect many such events for which we will not be able to measure any finite source effect. This work demonstrates an ability of mass measurements in such events.

  10. Faint-Source-Star Planetary Microlensing: The Discovery of the Cold Gas-Giant Planet OGLE-2014-BLG-0676Lb

    Science.gov (United States)

    Rattenbury, N. J.; Bennett, D. P.; Sumi, T.; Koshimoto, N.; Bond, I. A.; Udalski, A.; Shvartzvald, Y.; Maoz, D.; Jorgensen, U. G.; Barry, R.; hide

    2016-01-01

    We report the discovery of a planet OGLE-2014-BLG-0676Lb via gravitational microlensing. Observations for the lensing event were made by the following groups: Microlensing Observations in Astrophysics; Optical Gravitational Lensing Experiment; Wise Observatory; RoboNETLas Cumbres Observatory Global Telescope; Microlensing Network for the Detection of Small Terrestrial Exoplanets; and -FUN. All analyses of the light-curve data favoura lens system comprising a planetary mass orbiting a host star. The most-favoured binary lens model has a mass ratio between the two lens masses of (4.78 +/- 0.13) 10(exp -3). Subject to some important assumptions, a Bayesian probability density analysis suggests the lens system comprises a 3.09(+1.02/-1.12) MJ planet orbiting a 0.62(+0.20/-0.22) solar mass host star at a deprojected orbital separation of 4.40(+2.16/-1.46) au. The distance to the lens system is 2.22(+0.96/-0.83) kpc. Planet OGLE-2014-BLG-0676Lb provides additional data to the growing number of cool planets discover redusing gravitational microlensing against which planetary formation theories may be tested. Most of the light in the baseline of this event is expected to come from the lens and thus high-resolution imaging observations could confirm our planetary model interpretation.

  11. Constraints on the tidal dissipation factor of a main sequence star: The case of OGLE-TR-56b

    Science.gov (United States)

    Carone, Ludmila; Pätzold, Martin

    2007-04-01

    The planet OGLE-TR-56b is the extrasolar giant planet closest to its host star. This planet and its star exchange extreme tidal forces. This leads to a reduction of the planetary orbit and a spin-up of the stellar rotation. The tidal migration rate depends crucially on the ratio of the tidal dissipation factor Q* and the stellar love number kof the star, which is only poorly known and estimates range within 5×1051.5×109no observable influence by tidal forces on the planet's orbit within the lifetime for the star can be found. A lower limit for the possible values of the parameter Q*/kfor the G-type star OGLE-TR-56 was found by studying the evolution of possible tidal interaction into the future and in the past. This study demonstrates that on the basis of conservative model assumptions, a considerable but unrealistic spin-up of the star can be expected if Q*/k<2×107, which is not in agreement with observed stellar rotation periods. From a statistical analysis based on a Monte-Carlo tidal evolution simulation, the Q*/k parameter can be constrained to the range 2×107

  12. 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.

  13. 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.

  14. 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.

  15. 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).

  16. 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).

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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

  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

  3. 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...

  4. 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

  5. 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.

  6. 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...

  7. The Transit Light Curve (TLC) Project. II. Two Transits of the Exoplanet OGLE-TR-111b

    CERN Document Server

    Winn, J N; Fuentes, C I; Winn, Joshua N.; Holman, Matthew J.; Fuentes, Cesar I.

    2006-01-01

    As part of our ongoing effort to measure exoplanet sizes and transit times with greater accuracy, we present I band observations of two transits of OGLE-TR-111b. The photometry has an accuracy of 0.15-0.20% and a cadence of 1-2 minutes. We derive a planetary radius of 1.067 +/- 0.054 Jupiter radii and a stellar radius of 0.831 +/- 0.031 solar radii. The uncertainties are dominated by errors in the photometry, rather than by systematic errors arising from uncertainties in the limb darkening function or the stellar mass. Both the stellar radius and the planetary radius are in agreement with theoretical expectations. The transit times are accurate to within 30 seconds, and allow us to refine the estimate of the mean orbital period: 4.0144479 +/- 0.0000041 days.

  8. 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

  9. The Optical Gravitational Lensing Experiment: Analysis of the Bulge RR Lyrae Population from the OGLE-III Data

    Science.gov (United States)

    Pietrukowicz, P.; Udalski, A.; Soszyński, I.; Nataf, D. M.; Wyrzykowski, Ł.; Poleski, R.; Kozłowski, S.; Szymański, M. K.; Kubiak, M.; Pietrzyński, G.; Ulaczyk, K.

    2012-05-01

    We have analyzed the data on 16,836 RR Lyrae (RR Lyr) variables observed toward the Galactic bulge during the third phase of the Optical Gravitational Lensing Experiment (OGLE-III), which took place in 2001-2009. Using these standard candles, we show that the ratio of total-to-selective extinction toward the bulge is given by RI = AI /E(V - I) = 1.080 ± 0.007 and is independent of color. We demonstrate that the bulge RR Lyr stars form a metal-uniform population, slightly elongated in its inner part. The photometrically derived metallicity distribution is sharply peaked at [Fe/H] = -1.02 ± 0.18, with a dispersion of 0.25 dex. In the inner regions (|l| < 3°, |b| < 4°) the RR Lyr tend to follow the barred distribution of the bulge red clump giants. The distance to the Milky Way center inferred from the bulge RR Lyr is R 0 = 8.54 ± 0.42 kpc. We report a break in the mean density distribution at a distance of ~0.5 kpc from the center indicating its likely flattening. Using the OGLE-III data, we assess that (4-7) × 104 type ab RR Lyr variables should be detected toward the bulge area of the ongoing near-IR VISTA Variables in the Via Lactea (VVV) survey, where the uncertainty partially results from the unknown RR Lyr spatial density distribution within 0.2 kpc from the Galactic center.

  10. Machine learning techniques to select Be star candidates. An application in the OGLE-IV Gaia south ecliptic pole field

    Science.gov (United States)

    Pérez-Ortiz, M. F.; García-Varela, A.; Quiroz, A. J.; Sabogal, B. E.; Hernández, J.

    2017-09-01

    Context. Optical and infrared variability surveys produce a large number of high quality light curves. Statistical pattern recognition methods have provided competitive solutions for variable star classification at a relatively low computational cost. In order to perform supervised classification, a set of features is proposed and used to train an automatic classification system. Quantities related to the magnitude density of the light curves and their Fourier coefficients have been chosen as features in previous studies. However, some of these features are not robust to the presence of outliers and the calculation of Fourier coefficients is computationally expensive for large data sets. Aims: We propose and evaluate the performance of a new robust set of features using supervised classifiers in order to look for new Be star candidates in the OGLE-IV Gaia south ecliptic pole field. Methods: We calculated the proposed set of features on six types of variable stars and also on a set of Be star candidates reported in the literature. We evaluated the performance of these features using classification trees and random forests along with the K-nearest neighbours, support vector machines, and gradient boosted trees methods. We tuned the classifiers with a 10-fold cross-validation and grid search. We then validated the performance of the best classifier on a set of OGLE-IV light curves and applied this to find new Be star candidates. Results: The random forest classifier outperformed the others. By using the random forest classifier and colours criteria we found 50 Be star candidates in the direction of the Gaia south ecliptic pole field, four of which have infrared colours that are consistent with Herbig Ae/Be stars. Conclusions: Supervised methods are very useful in order to obtain preliminary samples of variable stars extracted from large databases. As usual, the stars classified as Be stars candidates must be checked for the colours and spectroscopic characteristics

  11. 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.

  12. Millimagnitude Photometry for Transiting Extrasolar Planetary Candidates: II. Transits of OGLE-TR-113-b in the Optical and Near-IR

    CERN Document Server

    Díaz, Rodrigo F; Fernández, José Miguel; Gallardo, José; Gieren, Wolfgang; Ivanov, Valentin D; Mauas, Pablo; Minniti, Dante; Pietrzynski, Grzegorz; Pérez, Felipe; Ruíz, María Teresa; Udalski, Andrzej; Zoccali, Manuela

    2007-01-01

    We present precise V and Ks-band transit photometry for the planetary host star OGLE-TR-113. Using the Ks-band photometry, we confirm the dwarf nature of OGLE-TR-113, and obtain new estimates for its effective temperature, distance and reddening. We employ the V-band photometry to obtain planetary and orbit parameters from the transit fit, a= (0.0232 \\pm 0.0038) AU, orbital period P= (1.4324752 \\pm 0.0000015) days, i= 86.7 - 90, R_p= (1.09 \\pm 0.09) R_J. These values are in excellent agreement with previous works. Assuming a mass M_p= (1.32 \\pm 0.19) M_J for the planet we obtain its mean density \\rho= (1.26 \\pm 0.50) g cm^{-3}, also in agreement with previous works. The transit observed in the Ks-band has a larger scatter and we find its amplitude to be consistent with that in the V-band. In this way, we find an independent confirmation of the planetary nature of OGLE-TR-113b.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1995-12-18

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

  14. 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.

  15. 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.

  16. 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...

  17. Lactoylglutathione lyase, a critical enzyme in methylglyoxal detoxification, contributes to survival of Salmonella in the nutrient rich environment

    Science.gov (United States)

    Chakraborty, Sangeeta; Gogoi, Mayuri; Chakravortty, Dipshikha

    2015-01-01

    Glyoxalase I which is synonymously known as lactoylglutathione lyase is a critical enzyme in methylglyoxal (MG) detoxification. We assessed the STM3117 encoded lactoylglutathione lyase (Lgl) of Salmonella Typhimurium, which is known to function as a virulence factor, due in part to its ability to detoxify methylglyoxal. We found that STM3117 encoded Lgl isomerises the hemithioacetal adduct of MG and glutathione (GSH) into S-lactoylglutathione. Lgl was observed to be an outer membrane bound protein with maximum expression at the exponential growth phase. The deletion mutant of S. Typhimurium (Δlgl) exhibited a notable growth inhibition coupled with oxidative DNA damage and membrane disruptions, in accordance with the growth arrest phenomenon associated with typical glyoxalase I deletion. However, growth in glucose minimal medium did not result in any inhibition. Endogenous expression of recombinant Lgl in serovar Typhi led to an increased resistance and growth in presence of external MG. Being a metalloprotein, Lgl was found to get activated maximally by Co2+ ion followed by Ni2+, while Zn2+ did not activate the enzyme and this could be attributed to the geometry of the particular protein-metal complex attained in the catalytically active state. Our results offer an insight on the pivotal role of the virulence associated and horizontally acquired STM3117 gene in non-typhoidal serovars with direct correlation of its activity in lending survival advantage to Salmonella spp. PMID:25517857

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

    Directory of Open Access Journals (Sweden)

    Zhang Xiuguo

    2011-04-01

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

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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....

  4. 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.

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

    Directory of Open Access Journals (Sweden)

    Dany J V Beste

    2011-07-01

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

  6. 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 ...

  7. 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

  8. 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

  9. 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...

  10. 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.

  11. 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...

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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).

  17. Pectin lyase overproduction by Penicillium griseoroseum mutants resistant to catabolite repression.

    Science.gov (United States)

    Lima, Juliana Oliveira; Pereira, Jorge Fernando; Araújo, Elza Fernandes de; Queiroz, Marisa Vieira de

    2017-02-09

    Expression of pectinolytic genes is regulated by catabolic repression limiting the production of pectin lyase (PL) if the natural inducer, pectin, is missing from the growth medium. Here, we report the isolation of Penicillium griseoroseum mutants resistant to 2-deoxy-d-glucose (DG) that show resistance to catabolite repression and overproduce PL. Three spontaneous and nine UV-induced mutants were obtained. Some mutants produced sectors (segments morphologically different) that were also studied. The mutants were analyzed for pectinases production on pectinase-agar plates and five mutants and two sectors showing larger clearing zones than the wild type were selected for quantitative assay. Although PL production higher than the wild type has been found, phenotype instability was observed for most of the mutants and, after transfers to nonselective medium, the DG resistance was no longer present. Only mutants M03 and M04 were stable maintaining the DG-resistance phenotype. When growing for 120h in liquid medium containing glucose with or without pectin, both mutants showed higher PL production. In the presence of glucose as sole carbon source, the mutant M03 produced 7.8-fold more PL than the wild type. Due its phenotypic stability and PL overproduction, the mutant M03 presents potential for industrial applications.

  18. Mutations underlying 3-Hydroxy-3-Methylglutaryl CoA Lyase deficiency in the Saudi population

    Directory of Open Access Journals (Sweden)

    Rashed Mohammed S

    2006-12-01

    Full Text Available Abstract Background 3-Hydroxy-3-Methylglutaric aciduria (3HMG, McKusick: 246450 is an autosomal recessive branched chain organic aciduria caused by deficiency of the enzyme 3-Hydroxy-3-Methylglutaryl CoA lyase (HL, HMGCL, EC 4.1.3.4. HL is encoded by HMGCL gene and many mutations have been reported. 3HMG is commonly observed in Saudi Arabia. Methods We utilized Whole Genome Amplification (WGA, PCR and direct sequencing to identify mutations underlying 3HMG in the Saudi population. Two patients from two unrelated families and thirty-four 3HMG positive dried blood spots (DBS were included. Results We detected the common missense mutation R41Q in 89% of the tested alleles (64 alleles. 2 alleles carried the frame shift mutation F305fs (-2 and the last two alleles had a novel splice site donor IVS6+1G>A mutation which was confirmed by its absence in more than 100 chromosomes from the normal population. All mutations were present in a homozygous state, reflecting extensive consanguinity. The high frequency of R41Q is consistent with a founder effect. Together the three mutations described account for >94% of the pathogenic mutations underlying 3HMG in Saudi Arabia. Conclusion Our study provides the most extensive genotype analysis on 3HMG patients from Saudi Arabia. Our findings have direct implications on rapid molecular diagnosis, prenatal and pre-implantation diagnosis and population based prevention programs directed towards 3HMG.

  19. Phenylalanine ammonia-lyase modified with polyethylene glycol: potential therapeutic agent for phenylketonuria.

    Science.gov (United States)

    Ikeda, K; Schiltz, E; Fujii, T; Takahashi, M; Mitsui, K; Kodera, Y; Matsushima, A; Inada, Y; Schulz, G E; Nishimura, H

    2005-11-01

    Phenylketonuria (PKU) is an autosomal recessive genetic disease caused by the defects in the phenylalanine hydroxylase (PAH) gene. Individuals homozygous for defective PAH alleles show elevated levels of systemic phenylalanine and should be under strict dietary control to reduce the risk of neuronal damage associated with high levels of plasma phenylalanine. Researchers predict that plant phenylalanine ammonia-lyase (PAL), which converts phenylalanine to nontoxic t-cinnamic acid, will be an effective therapeutic enzyme for the treatment of PKU. The problems of this potential enzyme therapy have been the low stability in the circulation and the antigenicity of the plant enzyme. Recombinant PAL originated from parsley (Petroselinum crispum) chemically conjugated with activated PEG2 [2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine] showed greatly enhanced stability in the circulation and was effective in reducing the plasma concentration of phenylalanine in the circulation of mice. PEG-PAL conjugate will be an effective therapeutic enzyme for the treatment of PKU.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. First simultaneous microlensing observations by two space telescopes: $Spitzer$ & $Swift$ reveal a brown dwarf in event OGLE-2015-BLG-1319

    CERN Document Server

    Shvartzvald, Y; Udalski, A; Gould, A; Sumi, T; Street, R A; Novati, S Calchi; Hundertmark, M; Bozza, V; Beichman, C; Bryden, G; Carey, S; Drummond, J; Fausnaugh, M; Gaudi, B S; Henderson, C B; Tan, T G; Wibking, B; Pogge, R W; Yee, J C; Zhu, W; Tsapras, Y; Bachelet, E; Dominik, M; Bramich, D M; Cassan, A; Jaimes, R Figuera; Horne, K; Ranc, C; Schmidt, R; Snodgrass, C; Wambsganss, J; Steele, I A; Menzies, J; Mao, S; Poleski, R; Pawlak, M; Szymański, M K; Skowron, J; Mróz, P; Kozłowski, S; Wyrzykowski, Ł; Pietrukowicz, P; Soszyński, I; Ulaczyk, K; Abe, F; Asakura, Y; Barry, R K; Bennett, D P; Bhattacharya, A; Bond, I A; Freeman, M; Hirao, Y; Itow, Y; Koshimoto, N; Li, M C A; Ling, C H; Masuda, K; Fukui, A; Matsubara, Y; Muraki, Y; Nagakane, M; Nishioka, T; Ohnishi, K; Oyokawa, H; Rattenbury, N J; Saito, To; Sharan, A; Sullivan, D J; Suzuki, D; Tristram, P J; Yonehara, A; Jørgensen, U G; Burgdorf, M J; Ciceri, S; D'Ago, G; Evans, D F; Hinse, T C; Kains, N; Kerins, E; Korhonen, H; Mancini, L; Popovas, A; Rabus, M; Rahvar, S; Scarpetta, G; Skottfelt, J; Southworth, J; Peixinho, N; Verma, P; Sbarufatti, B; Kennea, J A; Gehrels, N

    2016-01-01

    Simultaneous observations of microlensing events from multiple locations allow for the breaking of degeneracies between the physical properties of the lensing system, specifically by exploring different regions of the lens plane and by directly measuring the "microlens parallax". We report the discovery of a 30-55$M_J$ brown dwarf orbiting a K dwarf in microlensing event OGLE-2015-BLG-1319. The system is located at a distance of $\\sim$5 kpc toward the Galactic bulge. The event was observed by several ground-based groups as well as by $Spitzer$ and $Swift$, allowing the measurement of the physical properties. However, the event is still subject to an 8-fold degeneracy, in particular the well-known close-wide degeneracy, and thus the projected separation between the two lens components is either $\\sim$0.25 AU or $\\sim$45 AU. This is the first microlensing event observed by $Swift$, with the UVOT camera. We study the region of microlensing parameter space to which $Swift$ is sensitive, finding that while for thi...

  2. Mass measurement of a single unseen star and detection efficiency to low mass planets for OGLE 2007-BLG-050

    CERN Document Server

    Batista, V; Gould, A; Beaulieu, J P; Cassan, A; Christie, G W; Han, C; Udalski, A; Allen, W; De Poy, D L; Gal-Yam, A; Gaudi, B S; Johnson, B; Kaspi, S; Lee, C U; Maoz, D; McCormick, J; McGreer, I; Monard, B; Natusch, T; Ofek, E; Park, B -G; Pogge, R W; Polishook, D; Shporer, A; Albrow, M D; Bennett, D P; Brillant, S; Bode, M; Bramich, D M; Burgdorf, M; Caldwell, J A R; Calitz, H; Cole, A; Cook, K H; Coutures, Ch; Dieters, S; Dominik, M; Prester, D Dominis; Donatowicz, J; Fouqué, P; Greenhill, J; Hoffman, M; Horne, K; Jørgensen, U G; Kains, N; Kane, S; Kubas, D; Marquette, J B; Martin, R; Meintjes, P; Menzies, J; Pollard, K R; Sahu, K C; Snodgrass, C; Steele, I; Tsapras, Y; Wambsganss, J; Williams, A; Zub, M; Wyrzykowski, Ł; Kubiak, M; Szymański, M K; Pietrzyński, G; Soszyński, I; Szewczyk, O; Ulaczyk, K; Abe, F; Bond, I A; Fukui, A; Furusawa, K; Hearnshaw, J B; Holderness, S; Itow, Y; Kamiya, K; Kilmartin, P M; Korpela, A; Lin, W; Ling, C H; Masuda, K; Matsubara, Y; Miyake, N; Muraki, Y; Nagaya, M; Ohnishi, K; Okumura, T; Perrott, Y C; Rattenbury, N; Saito, To; Sako, T; Skuljan, L; Sullivan, D; Sumi, T; Sweatman, W L; Tristram, P J; Yock, P C M

    2009-01-01

    We analyze OGLE-2007-BLG-050, a high magnification microlensing event ($A\\sim 432$) whose peak occurred on 2 May, 2007, with pronounced finite-source and parallax effects. We compute planet detection efficiencies for this event in order to determine its sensitivity to the presence of planets around the lens star. Both finite-source and parallax effects permit a measurement of the angular Einstein radius $\\theta_{\\rm E}=0.48\\pm 0.01$ mas and the parallax $\\pi_{\\rm E}=0.12\\pm 0.03$, leading to an estimate of the lens mass $M=0.50\\pm0.14 M_{\\odot}$ and its distance to the observer $D_L=5.5\\pm0.4$ \\rm{kpc}. This is only the second determination of a reasonably precise ($<30%$) mass estimate for an isolated unseen object, using any method. This allows us to calculate the planetary detection efficiency in physical units $(r_\\perp,m_p)$, where $r_\\perp$ is the projected planet-star separation and $m_p$ is the planet mass. When computing planet detection efficiency, we did not find any planetary signature and our ...

  3. Revisiting the microlensing event OGLE 2012-BLG-0026: A solar mass star with two cold giant planets

    CERN Document Server

    Beaulieu, J P; Batista, V; Fukui, A; Marquette, J -B; Brillant, S; Cole, A A; Rogers, L A; Sumi, T; Abe, F; Bhattacharya, A; Koshimoto, N; Suzuki, D; Tristram, P J; Han, C; Gould, A; Pogge, R; Yee, J

    2016-01-01

    Two cold, gas giant planets orbiting a G-type main sequence star in the galactic disk have previously been discovered in the high magnification microlensing event OGLE-2012-BLG-0026 (Han et al. 2013). Here we present revised host star flux measurements and a refined model for the two-planet system using additional light curve data. We performed high angular resolution adaptive optics imaging with the Keck and Subaru telescopes at two epochs while the source star was still amplified. We detected the lens flux, $H=16.39 \\pm 0.08$. The lens, a disk star, is brighter than predicted from the modeling in the original study. We revisited the light curve modeling using additional photometric data from the B\\&C telescope in New Zealand and CTIO 1.3m H band light curve. We then include the Keck and Subaru adaptive optic observation constraints. The system is composed of a $\\sim 4-9$ Gyr lens star of $\\rm M_{lens} = 1.06 \\pm 0.05~\\,M_\\odot$ at a distance of $\\rm D_{lens} = 4.0 \\pm 0.3~$kpc, orbited by two giant plan...

  4. A possible binary system of a stellar remnant in the high magnification gravitational microlensing event OGLE-2007-BLG-514

    CERN Document Server

    Miyake, N; Sumi, T; Bennett, D P; Dong, S; Street, R A; Greenhill, J; Bond, I A; Gould, A; Kubiak, M; Szymanski, M K; Pietrzynski, G; Soszynski, I; Ulaczyk, K; Wyrzykowski, L; Abe, F; Fukui, A; Furusawa, K; Holderness, S; Itow, Y; Korpela, A; Ling, C H; Masuda, K; Matsubara, Y; Muraki, Y; Nagayama, T; Ohnishi, K; Rattenbury, N; Saito, To; Sako, T; Sullivan, D J; Sweatman, W L; Tristram, P J; Yock, P C M; Allen, W; Christie, G W; DePoy, D L; Gaudi, B S; Han, C; Lee, C -U; McCormick, J; Monard, B; Natusch, T; Park, B -G; Pogge, R W; Allan, A; Bode, M; Bramich, D M; Clay, N; Dominik, M; Horne, K D; Kains, N; Mottram, C; Snodgrass, C; Steele, I; Tsapras, Y; Albrow, M D; Batista, V; Beaulieu, J P; Brillant, S; Burgdorf, M; Caldwell, J A R; Cassan, A; Cole, A; Cook, K H; Coutures, Ch; Dieters, S; Prester, D Dominis; Donatowicz, J; Fouque, P; Jorgensen, U G; Kane, S; Kubas, D; Marquette, J B; Martin, R; Menzies, J; Pollard, K R; Sahu, K C; Wambsganss, J; Williams, A; Zub, M

    2012-01-01

    We report the extremely high magnification (A > 1000) binary microlensing event OGLE-2007-BLG-514. We obtained good coverage around the double peak structure in the light curve via follow-up observations from different observatories. The binary lens model that includes the effects of parallax (known orbital motion of the Earth) and orbital motion of the lens yields a binary lens mass ratio of q = 0.321 +/- 0.007 and a projected separation of s = 0.072 +/- 0.001$ in units of the Einstein radius. The parallax parameters allow us to determine the lens distance D_L = 3.11 +/- 0.39 kpc and total mass M_L=1.40 +/- 0.18 M_sun; this leads to the primary and secondary components having masses of M_1 = 1.06 +/- 0.13 M_sun and M_2 = 0.34 +/- 0.04 M_sun, respectively. The parallax model indicates that the binary lens system is likely constructed by the main sequence stars. On the other hand, we used a Bayesian analysis to estimate probability distributions by the model that includes the effects of xallarap (possible orbi...

  5. A Super-Jupiter orbiting a late-type star: A refined analysis of microlensing event OGLE-2012-BLG-0406

    CERN Document Server

    Tsapras, Y; Street, R A; Han, C; Bozza, V; Gould, A; Dominik, M; Beaulieu, J -P; Udalski, A; Jørgensen, U G; Sumi, T; Bramich, D M; Browne, P; Horne, K; Hundertmark, M; Ipatov, S; Kains, N; Snodgrass, C; Steele, I A; Alsubai, K A; Andersen, J M; Novati, S Calchi; Damerdji, Y; Diehl, C; Elyiv, A; Giannini, E; Hardis, S; Harpsøe, K; Hinse, T C; Juncher, D; Kerins, E; Korhonen, H; Liebig, C; Mancini, L; Mathiasen, M; Penny, M T; Rabus, M; Rahvar, S; Scarpetta, G; Skottfelt, J; Southworth, J; Surdej, J; Tregloan-Reed, J; Vilela, C; Kozłowski, J Wambsganss S; Kubiak, M; Pietrukowicz, P; Pietrzyński, G; Poleski, R; Skowron, J; Soszyński, I; Szymański, M K; Ulaczyk, K; Albrow, Łukasz Wyrzykowski M D; Bachelet, E; Barry, R; Batista, V; Bhattacharya, A; Brillant, S; Caldwell, J A R; Cassan, A; Cole, A; Corrales, E; Coutures, Ch; Dieters, S; Prester, D Dominis; Donatowicz, J; Fouqué, P; Greenhill, J; Kane, S R; Kubas, D; Marquette, J -B; Martin, R; Menzies, J; Pollard, K R; Williams, A; Wouters, D; Christie, G; DePoy, D L; Dong, S; Drummond, J; Gaudi, B S; Henderson, C B; Hwang, K H; Jung, Y K; Kavka, A; Koo, J -R; Lee, C -U; Maoz, D; Monard, L A G; Natusch, T; Ngan, H; Park, H; Pogge, R W; Porritt, I; Shin, I -G; Shvartzvald, Y; Tan, T G; Yee, J C; Abe, F; Bennett, D P; Bond, I A; Botzler, C S; Freeman, M; Fukui, A; Fukunaga, D; Itow, Y; Koshimoto, N; Ling, C H; Masuda, K; Matsubara, Y; Muraki, Y; Namba, S; Ohnishi, K; Rattenbury, N J; Saito, To; Sullivan, D J; Sweatman, W L; Suzuki, D; Tristram, P J; Tsurumi, N; Wada, K; Yamai, N; Yonehara, P C M Yock A

    2013-01-01

    We present a detailed analysis of survey and follow-up observations of microlensing event OGLE-2012-BLG-0406 based on data obtained from 10 different observatories. Intensive coverage of the lightcurve, especially the perturbation part, allowed us to accurately measure the parallax effect and lens orbital motion. Combining our measurement of the lens parallax with the angular Einstein radius determined from finite-source effects, we estimate the physical parameters of the lens system. We find that the event was caused by a $2.73\\pm 0.43\\ M_{\\rm J}$ planet orbiting a $0.44\\pm 0.07\\ M_{\\odot}$ early M-type star. The distance to the lens is $4.97\\pm 0.29$\\ kpc and the projected separation between the host star and its planet at the time of the event is $3.45\\pm 0.26$ AU. We find that the additional coverage provided by follow-up observations, especially during the planetary perturbation, leads to a more accurate determination of the physical parameters of the lens.

  6. Double-mode radial-non-radial RR Lyrae stars in the OGLE photometry of the Galactic bulge

    CERN Document Server

    Netzel, H; Moskalik, P

    2014-01-01

    Non-radial modes are excited in classical pulsators, both in Cepheids and in RR Lyrae stars. Firm evidence come from the first overtone pulsators, in which additional shorter period mode is detected with characteristic period ratio falling in between 0.60 and 0.65. In the case of first overtone Cepheids three separate sequences populated by nearly 200 stars are formed in the Petersen diagram, i.e. the diagram of period ratio versus longer period. In the case of first overtone RR Lyrae stars (RRc stars) situation is less clear. A dozen or so such stars are known which form a clump in the Petersen diagram without any obvious structure. Interestingly, all first overtone RR Lyrae stars for which precise space-borne photometry is available show the additional mode, which suggests that its excitation is common. Motivated by these results we searched for non-radial modes in the OGLE-III photometry of RRc stars from the Galactic bulge. We report the discovery of 147 stars, members of a new group of double-mode, radia...

  7. A super-jupiter orbiting a late-type star: A refined analysis of microlensing event OGLE-2012-BLG-0406

    Energy Technology Data Exchange (ETDEWEB)

    Tsapras, Y.; Street, R. A. [Las Cumbres Observatory Global Telescope Network, 6740 Cortona Drive, Suite 102, Goleta, CA 93117 (United States); Choi, J.-Y.; Han, C. [Department of Physics, Chungbuk National University, Cheongju 361-763 (Korea, Republic of); Bozza, V. [Dipartimento di Fisica " E. R. Caianiello," Università di Salerno, Via Giovanni Paolo II n. 132, I-84084 Fisciano (Italy); Gould, A. [Department of Astronomy, Ohio State University, 140 West 18th Avenue, Columbus, OH 43210 (United States); Dominik, M.; Browne, P.; Horne, K.; Hundertmark, M. [SUPA, School of Physics and Astronomy, University of St Andrews, North Haugh, St Andrews KY16 9SS (United Kingdom); Beaulieu, J.-P. [UPMC-CNRS, UMR7095, Institut d' Astrophysique de Paris, 98bis boulevard Arago, F-75014 Paris (France); Udalski, A. [Warsaw University Observatory, Al. Ujazdowskie 4, 00-478 Warszawa (Poland); Jørgensen, U. G. [Niels Bohr Institute, Astronomical Observatory, Juliane Maries vej 30, DK-2100 Copenhagen (Denmark); Sumi, T. [Department of Earth and Space Science, Osaka University, Osaka 560-0043 (Japan); Bramich, D. M.; Kains, N. [European Southern Observatory, Karl-Schwarzschild-Str. 2, D-85748 Garching bei München (Germany); Ipatov, S.; Alsubai, K. A. [Qatar Foundation, P.O. Box 5825, Doha (Qatar); Snodgrass, C. [Max Planck Institute for Solar System Research, Max-Planck-Str. 2, D-37191 Katlenburg-Lindau (Germany); Steele, I. A. [Astrophysics Research Institute, Liverpool John Moores University, Liverpool CH41 1LD (United Kingdom); Collaboration: RoboNet Collaboration; MiNDSTEp Collaboration; OGLE Collaboration; PLANET Collaboration; μFUN Collaboration; MOA Collaboration; and others

    2014-02-10

    We present a detailed analysis of survey and follow-up observations of microlensing event OGLE-2012-BLG-0406 based on data obtained from 10 different observatories. Intensive coverage of the light curve, especially the perturbation part, allowed us to accurately measure the parallax effect and lens orbital motion. Combining our measurement of the lens parallax with the angular Einstein radius determined from finite-source effects, we estimate the physical parameters of the lens system. We find that the event was caused by a 2.73 ± 0.43 M {sub J} planet orbiting a 0.44 ± 0.07 M {sub ☉} early M-type star. The distance to the lens is 4.97 ± 0.29 kpc and the projected separation between the host star and its planet at the time of the event is 3.45 ± 0.26 AU. We find that the additional coverage provided by follow-up observations, especially during the planetary perturbation, leads to a more accurate determination of the physical parameters of the lens.

  8. Optical Gravitational Lensing Experiment. OGLE-1999-BUL-32 the Longest Ever Microlensing Event -- Evidence for a Stellar Mass Black Hole?

    CERN Document Server

    Mao, S; Wozniak, P R; Udalski, A; Kubiak, M; Pietrzynski, G; Soszynski, I; Zebrun, K; Mao, Shude; Smith, Martin C.

    2002-01-01

    We describe the discovery of the longest microlensing event ever observed, OGLE-1999-BUL-32, also independently identified by the MACHO collaboration as MACHO-99-BLG-22. This unique event has an Einstein radius crossing time of 641 days. The high quality data obtained with difference image analysis shows a small but significant parallax signature. This parallax effect allows one to determine the Einstein radius projected onto the observer plane as rE^hat ~ 29.2AU. The transverse velocity projected onto the observer plane is about 79km/s. We argue that the lens is likely to be have a mass of at least a few solar masses, i.e., it could be a stellar black hole. The black hole hypothesis can be tested using the astrometric microlensing signature with the soon-to-be installed Advanced Camera for Surveys on board the Hubble Space Telescope. Deep X-ray and radio images may also be useful for revealing the nature of the object.

  9. On the Possible Properties of Small and Cold Extrasolar Planets: Is OGLE-2005-BLG-390Lb Entirely Frozen?

    CERN Document Server

    Ehrenreich, D; Beaulieu, J P; Grasset, O; Ehrenreich, David; Etangs, Alain Lecavelier Des; Beaulieu, Jean-Philippe; Grasset, Olivier

    2006-01-01

    Extrasolar planets as light as a few Earths are now being detected. Such planets are likely not gas or ice giants. Here, we present a study on the possible properties of the small and cold extrasolar planets, applied to the case of the recently discovered planet OGLE-2005-BLG-390Lb (Beaulieu et al. 2006). This planet (5.5[+5.5/-2.7] Earth masses) orbits 2.6[+1.5/-0.6]-astronomical units away from an old M-type star of the Galactic Bulge. The planet should be entirely frozen given the low surface temperature (35 to 47 K). However, depending on the rock-to-ice mass ratio in the planet, the radiogenic heating could be sufficient to make the existence of liquid water within an icy crust possible. This possibility is estimated as a function of the planetary mass and the illumination received from the parent star, both being strongly related by the observational constraints. The results are presented for water-poor and water-rich planets. We find that no oceans can be present in any cases at 9-10 Gyr, a typical age...

  10. Confirmation of the OGLE-2005-BLG-169 planet signature and characteristics with lens-source proper motion detection

    CERN Document Server

    Batista, V; Bennett, D P; Gould, A; Marquette, J -B; Fukui, A; Bhattacharya, A

    2015-01-01

    We present Keck NIRC2 high angular resolution adaptive optics observations of the microlensing event OGLE-2005-BLG-169, taken 8.21 years after the discovery of this planetary system. For the first time for a microlensing planetary event, the source and the lens are completely resolved, providing a precise measurement of their heliocentric relative proper motion, $\\mu_{\\rm{rel},\\rm{helio}}=7.44 \\pm 0.17$ mas yr$^{-1}$. This confirms and refines the initial model presented in the discovery paper and rules out a range of solutions that were allowed by the microlensing light curve. This is also the first time that parameters derived from a microlensing planetary signal are confirmed, both with Keck measurements, presented in this paper, and independent measurements obtained with the Hubble Space Telescope in I, V and B bands, presented in a companion paper. Hence, this new measurement of $\\mu_{\\rm{rel},\\rm{helio}}$, as well as the measured brightness of the lens in H band, enabled the mass and distance of the sys...

  11. Twenty-One New Light Curves of OGLE-TR-56b: New System Parameters and Limits on Timing Variations

    CERN Document Server

    Adams, E R; Elliot, J L; Seager, S; Osip, D J; Holman, M J; Winn, J N; Hoyer, S; Rojo, P

    2011-01-01

    Although OGLE-TR-56b was the second transiting exoplanet discovered, only one light curve, observed in 2006, has been published besides the discovery data. We present twenty-one light curves of nineteen different transits observed between July 2003 and July 2009 with the Magellan Telescopes and Gemini South. The combined analysis of the new light curves confirms a slightly inflated planetary radius relative to model predictions, with R_p = 1.378 +/- 0.090 R_J. However, the values found for the transit duration, semimajor axis, and inclination values differ significantly from the previous result, likely due to systematic errors. The new semimajor axis and inclination, a = 0.01942 +/- 0.00015 AU and i = 73.72 +/- 0.18 degrees, are smaller than previously reported, while the total duration, T_14 = 7931 +/- 38 s, is 18 minutes longer. The transit midtimes have errors from 23 s to several minutes, and no evidence is seen for transit midtime or duration variations. Similarly, no change is seen in the orbital period...

  12. The Frequency of Snowline-Region Planets from Four Years of OGLE-MOA-Wise Second-Generation Microlensing

    Science.gov (United States)

    Shvartzvald, Y.; Maoz, D.; Udalski, A.; Sumi, T.; Friedmann, M.; Kaspi, S.; Poleski, R.; Szymanski, M. K.; Skowron, J.; Kozlowski, S.; hide

    2016-01-01

    We present a statistical analysis of the first four seasons from a second-generation microlensing survey for extrasolar planets, consisting of near-continuous time coverage of 8 deg to the 2nd power of the Galactic bulge by the Optical Gravitational Lens Experiment (OGLE), Microlensing Observations in Astrophysics (MOA), and Wise microlensing surveys. During this period, 224 microlensing events were observed by all three groups. Over 12% of the events showed a deviation from single-lens microlensing, and for approx. 1/3 of those the anomaly is likely caused by a planetary companion. For each of the 224 events, we have performed numerical ray-tracing simulations to calculate the detection efficiency of possible companions as a function of companion-to-host mass ratio and separation. Accounting for the detection efficiency, we find that 55 +34 -22%of microlensed stars host a snowline planet. Moreover, we find that Neptune-mass planets are approx.10 times more common than Jupiter-mass planets. The companion-to-host mass-ratio distribution shows a deficit at q approx. 10 (exp -2), separating the distribution into two companion populations, analogous to the stellar-companion and planet populations, seen in radial-velocity surveys around solar-like stars. Our survey, however, which probes mainly lower mass stars, suggests a minimum in the distribution in the super-Jupiter mass range, and a relatively high occurrence of brown-dwarf companions.

  13. 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...

  14. 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.

  15. 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.

  16. 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...

  17. 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.

  18. 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.

  19. CYP74B24 is the 13-hydroperoxide lyase involved in biosynthesis of green leaf volatiles in tea (Camellia sinensis).

    Science.gov (United States)

    Ono, Eiichiro; Handa, Taiki; Koeduka, Takao; Toyonaga, Hiromi; Tawfik, Moataz M; Shiraishi, Akira; Murata, Jun; Matsui, Kenji

    2016-01-01

    Green leaf volatiles (GLVs) are C6-aliphatic aldehydes/alcohols/acetates, and biosynthesized from the central precursor fatty acid 13-hydroperoxides by 13-hydroperoxide lyases (HPLs) in various plant species. While GLVs have been implicated as defense compounds in plants, GLVs give characteristic grassy note to a bouquet of aroma in green tea, which is manufactured from young leaves of Camellia sinensis. Here we identify three HPL-related genes from C. sinensis via RNA-Sequencing (RNA-Seq) in silico, and functionally characterized a candidate gene, CYP74B24, as a gene encoding tea HPL. Recombinant CYP74B24 protein heterologously expressed in Escherichia coli specifically produced (Z)-3-hexenal from 13-HPOT with the optimal pH 6.0 in vitro. CYP74B24 gene was expressed throughout the aerial organs in a rather constitutive manner and further induced by mechanical wounding. Constitutive expression of CYP74B24 gene in intact tea leaves might account for low but substantial and constitutive formation of a subset of GLVs, some of which are stored as glycosides. Our results not only provide novel insights into the biological roles that GLVs play in tea plants, but also serve as basis for the improvement of aroma quality in tea manufacturing processes.

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

    Science.gov (United States)

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

    2014-06-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2014-01-01

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

  2. Revisiting the Microlensing Event OGLE 2012-BLG-0026: A Solar Mass Star with Two Cold Giant Planets

    Science.gov (United States)

    Beaulieu, J.-P.; Bennett, D. P.; Batista, V.; Fukui, A.; Marquette, J.-B.; Brillant, S.; Cole, A. A.; Rogers, L. A.; Sumi, T.; Abe, F.

    2016-01-01

    Two cold gas giant planets orbiting a G-type main-sequence star in the galactic disk were previously discovered in the high-magnification microlensing event OGLE-2012-BLG-0026. Here, we present revised host star flux measurements and a refined model for the two-planet system using additional light curve data. We performed high angular resolution adaptive optics imaging with the Keck and Subaru telescopes at two epochs while the source star was still amplified. We detected the lens flux, H = 16.39 +/- 0.08. The lens, a disk star, is brighter than predicted from the modeling in the original study. We revisited the light curve modeling using additional photometric data from the B and C telescope in New Zealand and CTIO 1.3 m H-band light curve. We then include the Keck and Subaru adaptive optic observation constraints. The system is composed of an approximately 4-9 Gyr lens star of M(sub lens) = 1.06 +/- 0.05 solar mass at a distance of D(sub lens) = 4.0 +/- 0.3 kpc, orbited by two giant planets of 0.145 +/- 0.008 M(sub Jup) and 0.86 +/- 0.06 M(sub Jup), with projected separations of 4.0 +/- 0.5 au and 4.8 +/- 0.7 au, respectively. Because the lens is brighter than the source star by 16 +/- 8% in H, with no other blend within one arcsec, it will be possible to estimate its metallicity using subsequent IR spectroscopy with 8-10 m class telescopes. By adding a constraint on the metallicity it will be possible to refine the age of the system.

  3. Discovery of the distant cool sub-Neptune mass planet OGLE 2005-BLG-390Lb by microlensing

    Energy Technology Data Exchange (ETDEWEB)

    Beaulieu, J P; Bennett, D P; Fouque, P; Williams, A; Dominik, M; Jorgensen, U G; Kubas, D; Cassan, A; Coutures, C; Greenhill, J; Hill, K; Menzies, J; Sackett, P D; Albrow, M; Brillant, S; Caldwell, J R; Calitz, J J; Cook, K H; Corrales, E; Desort, M; Dieters, S; Dominis, D; Donatowicz, J; Hoffman, M; Kane, S; Marquette, J B; Martin, R; Meintjes, P; Pollard, K; Sahu, K; Vinter, C; Wambsganss, J; Woller, K; Horne, K; Steele, I; Bramich, D M; Burgdorf, M; Snodgrass, C; Bode, M; Udalski, A; Szymanski, M K; Kubiak, M; Wieckowski, T; Pietrzynski, G; Soszynski, I; Szewczyk, O; Wyrzykowski, L; Paczynski, B; Abe, F; Bond, I A; Britton, T R; Gilmore, A C; Hearnshaw, J B; Itow, Y; Kamiya, K; Kilmartin, P M; Korpela, A V; Masuda, K; Matsubara, Y; Motomura, M; Muraki, Y; Nakamura, S; Okada, C; Ohnishi, K; Rattenbury, N J; Sako, T; Sato, S; Sasaki, M; Sekiguchi, T; Sullivan, D J; Tristram, P J; Yock, P M; Yoskioka, T

    2005-11-07

    The favoured theoretical explanation for planetary systems formation is the core-accretion model in which solid planetesimals accumulate to build up planetary cores, which then accrete nebular gas if they are sufficiently massive. Around M-dwarf stars, the most common stars of our Galaxy, this model favours the formation of Earth- to Neptune-mass planets in a few million years with orbital sizes of 1 to 10 AU, which is consistent with the small number of detections of giant planets with M-dwarf host stars. More than 170 extrasolar planets have been discovered so far with a wide range of masses and orbital periods, but planets of Neptune's mass or less have not previously been detected at separations of more than 0.15 AU from normal stars. Here we report the discovery of a 5.5{sub -2.7}{sup +5.5} Earthmass planetary companion at a separation of 2.6{sub -0.6}{sup +1.5}AU from a 0.22{sub -0.11}{sup +0.21} M{sub e} M-dwarf star, which is the lens star for gravitational microlensing event OGLE 2005-BLG-390. This is the lowest mass ever reported for an extrasolar planet orbiting a main sequence star, although the error bars overlap those for the mass of GJ876d. Our detection suggests that such cool, sub-Neptune mass planets may be common than gas giant planets, as predicted by the core accretion theory.

  4. Revisiting the Microlensing Event OGLE 2012-BLG-0026: A Solar Mass Star with Two Cold Giant Planets

    Science.gov (United States)

    Beaulieu, J.-P.; Bennett, D. P.; Batista, V.; Fukui, A.; Marquette, J.-B.; Brillant, S.; Cole, A. A.; Rogers, L. A.; Sumi, T.; Abe, F.

    2016-01-01

    Two cold gas giant planets orbiting a G-type main-sequence star in the galactic disk were previously discovered in the high-magnification microlensing event OGLE-2012-BLG-0026. Here, we present revised host star flux measurements and a refined model for the two-planet system using additional light curve data. We performed high angular resolution adaptive optics imaging with the Keck and Subaru telescopes at two epochs while the source star was still amplified. We detected the lens flux, H = 16.39 +/- 0.08. The lens, a disk star, is brighter than predicted from the modeling in the original study. We revisited the light curve modeling using additional photometric data from the B and C telescope in New Zealand and CTIO 1.3 m H-band light curve. We then include the Keck and Subaru adaptive optic observation constraints. The system is composed of an approximately 4-9 Gyr lens star of M(sub lens) = 1.06 +/- 0.05 solar mass at a distance of D(sub lens) = 4.0 +/- 0.3 kpc, orbited by two giant planets of 0.145 +/- 0.008 M(sub Jup) and 0.86 +/- 0.06 M(sub Jup), with projected separations of 4.0 +/- 0.5 au and 4.8 +/- 0.7 au, respectively. Because the lens is brighter than the source star by 16 +/- 8% in H, with no other blend within one arcsec, it will be possible to estimate its metallicity using subsequent IR spectroscopy with 8-10 m class telescopes. By adding a constraint on the metallicity it will be possible to refine the age of the system.

  5. 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...

  6. 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

  7. 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...

  8. 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.

  9. Fourier decomposition and frequency analysis of the pulsating stars with P < 1day in the OGLE database. II. Multiperiodic RR Lyrae variables in the Galactic Bulge

    CERN Document Server

    Moskalik, P

    2003-01-01

    We present the results of a systematic search for multiperiodic pulsators among the Galactic Bulge RR Lyrae stars of the OGLE-1 sample. We identify one "canonical" double-mode variable (RRd star) pulsating in two radial modes. In 38 stars we detect secondary periodicities very close to the primary pulsation frequency. This type of multiperiodic variables constitute ~23% of RRab and ~5% of RRc population of the Bulge. With the observed period ratios of 0.95-1.02 the secondary periods must correspond to nonradial modes of oscillation. Their beating with the primary (radial) pulsation leads to a long-term amplitude and phase modulation, known as the Blazhko effect. The Blazhko RRab variables occur more frequently in the Galactic Bulge than in the LMC. The opposite tendency is seen in case of the RRd stars. The differences of incidence rates are most likely caused by different metallicity of the two populations. We discuss pulsation properties of the OGLE-1 Blazhko stars and compare them with predictions of theor...

  10. OGLE-2015-BLG-0479LA,B: Binary Gravitational Microlens Characterized by Simultaneous Ground-based and Space-based Observation

    CERN Document Server

    Han, C; Gould, A; Zhu, Wei; Street, R A; Yee, J C; Beichman, C; Bryden, C; Novati, S Calchi; Carey, S; Fausnaugh, M; Gaudi, B S; Henderson, Calen B; Shvartzvald, Y; Wibking, B; Szymański, M K; Soszyński, I; Skowron, J; Mróz, P; Poleski, R; Pietrukowicz, P; Kozłowski, S; Ulaczyk, K; Wyrzykowski, Ł; Pawlak, M; Tsapras, Y; Hundertmark, M; Bachelet, E; Dominik, M; Bramich, D M; Cassan, A; Jaimes, R Figuera; Horne, K; Ranc, C; Schmidt, R; Snodgrass, C; Wambsganss, J; Steele, I A; Menzies, J; Mao, S; Bozza, V; Jørgensen, U G; Alsubai, K A; Ciceri, S; D'Ago, G; Haugbølle, T; Hessman, F V; Hinse, T C; Juncher, D; Korhonen, H; Mancini, L; Popovas, A; Rabus, M; Rahvar, S; Scarpetta, G; Skottfelt, J; Southworth, J; Starkey, D; Surdej, J; Wertz, O; Zarucki, M; Pogge, R W; DePoy, D L

    2016-01-01

    We present a combined analysis of the observations of the gravitational microlensing event OGLE-2015-BLG-0479 taken both from the ground and by the {\\it Spitzer Space Telescope}. The light curves seen from the ground and from space exhibit a time offset of $\\sim 13$ days between the caustic spikes, indicating that the relative lens-source positions seen from the two places are displaced by parallax effects. From modeling the light curves, we measure the space-based microlens parallax. Combined with the angular Einstein radius measured by analyzing the caustic crossings, we determine the mass and distance of the lens. We find that the lens is a binary composed of two G-type stars with masses $\\sim 1.0\\ M_\\odot$ and $\\sim 0.9\\ M_\\odot$ located at a distance $\\sim 3$ kpc. In addition, we are able to constrain the complete orbital parameters of the lens thanks to the precise measurement of the microlens parallax derived from the joint analysis. In contrast to the binary event OGLE-2014-BLG-1050, which was also ob...

  11. Frequency of Hot Jupiters and Very Hot Jupiters from the OGLE-III Transit Surveys toward the Galactic Bulge and Carina

    Science.gov (United States)

    Gould, A.; Dorsher, S.; Gaudi, B. S.; Udalski, A.

    2006-03-01

    We derive the frequencies of hot Jupiters (HJs) with 3-5 day periods and very hot Jupiters (VHJs) with 1-3 day periods by comparing the planets actually detected in the OGLE-III survey with those predicted by our models. The models are constructed following Gould and Morgan (2003) by populating the line of sight with stars drawn from the Hipparcos Catalogue. Using these, we demonstrate that the number of stars with sensitivity to HJs and VHJs is only 5-16% of those in the OGLE-III fields satisfying the spectroscopic-follow-up limit of V_max HJs and (1/710)(1^+1.10_-0.54) for VHJs. The HJ rate is statistically indistinguishable from that found in radial velocity (RV) studies. However, we note that magnitude-limited RV samples are heavily biased toward metal-rich (hence, planet-bearing) stars, while transit surveys are not, and therefore we expect that more sensitive transit surveys should find a deficit of HJs as compared to RV surveys. The detection of three transiting VHJs, all with periods less than 2 days, is marginally consistent with the complete absence of such detections in RV surveys. The planets detected are consistent with being uniformly distributed between 1.00 and 1.25 Jovian radii, but there are too few in the sample to map this distribution in detail.

  12. Double-mode radial-non-radial RR Lyrae stars. OGLE-IV photometry of two high cadence fields in the Galactic bulge

    CERN Document Server

    Netzel, H; Moskalik, P

    2015-01-01

    We analyse the OGLE-IV photometry of the first overtone and double-mode RR Lyrae stars (RRc/RRd) in the two fields towards the Galactic bulge observed with high cadence. In 27 per cent of RRc stars we find additional non-radial mode, with characteristic period ratio, P x /P 1O \\in (0.6, 0.64). It strongly corroborates the conclusion arising from the analysis of space photometry of RRc stars, that this form of pulsation must be common. In the Petersen diagram the stars form three sequences. In 20 stars we find two or three close secondary modes simultaneously. The additional modes are clearly non-stationary. Their amplitude and/or phase vary in time. As a result, the patterns observed in the frequency spectra of these stars may be very complex. In some stars the additional modes split into doublets, triplets or appear as a more complex bands of increased power. Subharmonics of additional modes are detected in 20 per cent of stars. They also display a complex structure. Including our previous study of the OGLE-...

  13. 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.

  14. 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

  15. TPR domain of NrfG mediates complex formation between heme lyase and formate-dependent nitrite reductase in Escherichia coli O157:H7.

    Science.gov (United States)

    Han, Dohyun; Kim, Kyunggon; Oh, Jongkil; Park, Jungeun; Kim, Youngsoo

    2008-02-15

    Escherichia coli synthesize C-type cytochromes only during anaerobic growth in media supplemented with nitrate and nitrite. The reduction of nitrate to ammonium in the periplasm of Escherichia coli involves two separate periplasmic enzymes, nitrate reductase and nitrite reductase. The nitrite reductase involved, NrfA, contains cytochrome C and is synthesized coordinately with a membrane-associated cytochrome C, NrfB, during growth in the presence of nitrite or in limiting nitrate concentrations. The genes NrfE, NrfF, and NrfG are required for the formate-dependent nitrite reduction pathway, which involves at least two C-type cytochrome proteins, NrfA and NrfB. The NrfE, NrfF, and NrfG genes (heme lyase complex) are involved in the maturation of a special C-type cytochrome, apocytochrome C (apoNrfA), to cytochrome C (NrfA) by transferring a heme to the unusual heme binding motif of the Cys-Trp-Ser-Cys-Lys sequence in apoNrfA protein. Thus, in order to further investigate the roles of NrfG in the formation of heme lyase complex (NrfEFG) and in the interaction between heme lyase complex and formate-dependent nitrite reductase (NrfA), we determined the crystal structure of NrfG at 2.05 A. The structure of NrfG showed that the contact between heme lyase complex (NrfEFG) and NrfA is accomplished via a TPR domain in NrfG which serves as a binding site for the C-terminal motif of NrfA. The portion of NrfA that binds to TPR domain of NrfG has a unique secondary motif, a helix followed by about a six-residue C-terminal loop (the so called "hook conformation"). This study allows us to better understand the mechanism of special C-type cytochrome assembly during the maturation of formate-dependent nitrite reductase, and also adds a new TPR binding conformation to the list of TPR-mediated protein-protein interactions.

  16. 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.

  17. 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...

  18. 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

  19. 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

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  1. Characterization of an Alginate Lyase, FlAlyA, from Flavobacterium sp. Strain UMI-01 and Its Expression in Escherichia coli

    Science.gov (United States)

    Inoue, Akira; Takadono, Kohei; Nishiyama, Ryuji; Tajima, Kenji; Kobayashi, Takanori; Ojima, Takao

    2014-01-01

    A major alginate lyase, FlAlyA, was purified from the periplasmic fraction of an alginate-assimilating bacterium, Flavobacterium sp. strain UMI-01. FlAlyA showed a single band of ~30 kDa on SDS-PAGE and exhibited the optimal temperature and pH at 55 °C and pH 7.7, respectively. Analyses for substrate preference and reaction products indicated that FlAlyA was an endolytic poly(mannuronate) lyase (EC 4.2.2.3). A gene fragment encoding the amino-acid sequence of 288 residues for FlAlyA was amplified by inverse PCR. The N-terminal region of 21 residues except for the initiation Met in the deduced sequence was predicted as the signal peptide and the following region of six residues was regarded as propeptide, while the C-terminal region of 260 residues was regarded as the polysaccharide-lyase-family-7-type catalytic domain. The entire coding region for FlAlyA was subjected to the pCold I—Escherichia coli BL21(DE3) expression system and ~eight times higher yield of recombinant FlAlyA (recFlAlyA) than that of native FlAlyA was achieved. The recFlAlyA recovered in the periplasmic fraction of E. coli had lost the signal peptide region along with the N-terminal 3 residues of propeptide region. This suggested that the signal peptide of FlAlyA could function in part in E. coli. PMID:25153766

  2. Characterization of an Alginate Lyase, FlAlyA, from Flavobacterium sp. Strain UMI-01 and Its Expression in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Akira Inoue

    2014-08-01

    Full Text Available A major alginate lyase, FlAlyA, was purified from the periplasmic fraction of an alginate-assimilating bacterium, Flavobacterium sp. strain UMI-01. FlAlyA showed a single band of ~30 kDa on SDS-PAGE and exhibited the optimal temperature and pH at 55 °C and pH 7.7, respectively. Analyses for substrate preference and reaction products indicated that FlAlyA was an endolytic poly(mannuronate lyase (EC 4.2.2.3. A gene fragment encoding the amino-acid sequence of 288 residues for FlAlyA was amplified by inverse PCR. The N-terminal region of 21 residues except for the initiation Met in the deduced sequence was predicted as the signal peptide and the following region of six residues was regarded as propeptide, while the C-terminal region of 260 residues was regarded as the polysaccharide-lyase-family-7-type catalytic domain. The entire coding region for FlAlyA was subjected to the pCold I—Escherichia coli BL21(DE3 expression system and ~eight times higher yield of recombinant FlAlyA (recFlAlyA than that of native FlAlyA was achieved. The recFlAlyA recovered in the periplasmic fraction of E. coli had lost the signal peptide region along with the N-terminal 3 residues of propeptide region. This suggested that the signal peptide of FlAlyA could function in part in E. coli.

  3. 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.

  4. A POSSIBLE BINARY SYSTEM OF A STELLAR REMNANT IN THE HIGH-MAGNIFICATION GRAVITATIONAL MICROLENSING EVENT OGLE-2007-BLG-514

    Energy Technology Data Exchange (ETDEWEB)

    Miyake, N.; Abe, F.; Furusawa, K.; Itow, Y. [Solar-Terrestrial Environment Laboratory, Nagoya University, Nagoya 464-8601 (Japan); Udalski, A.; Kubiak, M.; Szymanski, M. K.; Pietrzynski, G.; Soszynski, I.; Ulaczyk, K.; Wyrzykowski, L. [Warsaw University Observatory, Al. Ujazdowskie 4, 00-478 Warszawa (Poland); Sumi, T. [Department of Earth and Space Science, Osaka University, Osaka 560-0043 (Japan); Bennett, D. P. [Department of Physics, University of Notre Dame, 225 Nieuwland Science Hall, Notre Dame, IN 46556 (United States); Dong, S.; Gould, A. [Department of Astronomy, Ohio State University, 140 West 18th Avenue, Columbus, OH 43210 (United States); Street, R. A. [Las Cumbres Observatory, 6740B Cortona Drive, Suite 102, Goleta, CA 93117 (United States); Greenhill, J. [School of Maths and Physics, University of Tasmania, Private bag 37, GPO Hobart, Tasmania 7001 (Australia); Bond, I. A. [Institute for Information and Mathematical Sciences, Massey University, Private Bag 102-904, Auckland 1330 (New Zealand); Fukui, A. [Okayama Astrophysical Observatory, National Astronomical Observatory of Japan, Okayama 719-0232 (Japan); Holderness, S., E-mail: nmiyake@stelab.nagoya-u.ac.jp [Computer Science Department, University of Auckland, Auckland (New Zealand); Collaboration: OGLE Collaboration; MOA Collaboration; muFUN Collaboration; RoboNet Collaboration; PLANET Collaboration; and others

    2012-06-20

    We report the extremely high-magnification (A > 1000) binary microlensing event OGLE-2007-BLG-514. We obtained good coverage around the double peak structure in the light curve via follow-up observations from different observatories. The binary lens model that includes the effects of parallax (known orbital motion of the Earth) and orbital motion of the lens yields a binary lens mass ratio of q = 0.321 {+-} 0.007 and a projected separation of s = 0.072 {+-} 0.001 in units of the Einstein radius. The parallax parameters allow us to determine the lens distance D{sub L} = 3.11 {+-} 0.39 kpc and total mass M{sub L} = 1.40 {+-} 0.18 M{sub Sun }; this leads to the primary and secondary components having masses of M{sub 1} = 1.06 {+-} 0.13 M{sub Sun} and M{sub 2} = 0.34 {+-} 0.04 M{sub Sun }, respectively. The parallax model indicates that the binary lens system is likely constructed by the main-sequence stars. On the other hand, we used a Bayesian analysis to estimate probability distributions by the model that includes the effects of xallarap (possible orbital motion of the source around a companion) and parallax (q = 0.270 {+-} 0.005, s = 0.083 {+-} 0.001). The primary component of the binary lens is relatively massive, with M{sub 1} = 0.9{sup +4.6}{sub -0.3} M{sub Sun} and it is at a distance of D{sub L} = 2.6{sup +3.8}{sub -0.9} kpc. Given the secure mass ratio measurement, the companion mass is therefore M{sub 2} = 0.2{sup +1.2}{sub -0.1} M{sub Sun }. The xallarap model implies that the primary lens is likely a stellar remnant, such as a white dwarf, a neutron star, or a black hole.

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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).

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  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 hi

  18. 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.

  19. 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.

  20. Pectate lyase pollen allergens: sensitization profiles and cross-reactivity pattern.

    Directory of Open Access Journals (Sweden)

    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

  1. 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.

  2. 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.

  3. Hydrogen sulfide generation in mammals: the molecular biology of cystathionine-β- synthase (CBS) and cystathionine-γ-lyase (CSE).

    Science.gov (United States)

    Renga, Barbara

    2011-04-01

    Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) are two key enzymes involved in the synthesis of hydrogen sulphide (H(2)S). CBS catalyzes the pyridoxal 5'-phosphate (PLP)-dependent conversion of homocysteine in Cystathionine whilst CSE the pyridoxal 5'-phosphate (PLP)-dependent synthesis of L-cysteine from Cystathionine. In mammals, CBS gene transcription is poorly investigated and the activity of the enzyme is highly regulated. In fact, the CBS enzyme contains a heme cofactor that functions as a redox sensor and utilizes S-adenosylmethionine (SAM) as an allosteric activator. Impaired CBS activity causes hyperhomocystinuria and hyperhomocysteinemia, both risk factors for cardiovascular diseases. Murine CSE gene regulation is well characterized but little is known about the human counterpart and there is no information regarding the enzyme activity regulation. Recently it has been demonstrated that CSE transcription is regulated by the nuclear receptor Farnesoid X Receptor (FXR). Mutations that decrease the activity of CSE cause cystathioninuria, hypercystathioninemia and increase the risk of developing atherosclerosis and bladder cancer. This review focuses on the recent aspects of the molecular regulation of both CBS and CSE and highlights the possibility that members of the nuclear receptors superfamily might be involved in the regulation of hydrogen sulphide metabolism.

  4. Role of the Pseudomonas fluorescens alginate lyase (AlgL) in clearing the periplasm of alginates not exported to the extracellular environment.

    Science.gov (United States)

    Bakkevig, Karianne; Sletta, Håvard; Gimmestad, Martin; Aune, Randi; Ertesvåg, Helga; Degnes, Kristin; Christensen, Bjørn Erik; Ellingsen, Trond E; Valla, Svein

    2005-12-01

    Alginate is an industrially widely used polysaccharide produced by brown seaweeds and as an exopolysaccharide by bacteria belonging to the genera Pseudomonas and Azotobacter. The polymer is composed of the two sugar monomers mannuronic acid and guluronic acid (G), and in all these bacteria the genes encoding 12 of the proteins essential for synthesis of the polymer are clustered in the genome. Interestingly, 1 of the 12 proteins is an alginate lyase (AlgL), which is able to degrade the polymer down to short oligouronides. The reason why this lyase is associated with the biosynthetic complex is not clear, but in this paper we show that the complete lack of AlgL activity in Pseudomonas fluorescens in the presence of high levels of alginate synthesis is toxic to the cells. This toxicity increased with the level of alginate synthesis. Furthermore, alginate synthesis became reduced in the absence of AlgL, and the polymers contained much less G residues than in the wild-type polymer. To explain these results and other data previously reported in the literature, we propose that the main biological function of AlgL is to degrade alginates that fail to become exported out of the cell and thereby become stranded in the periplasmic space. At high levels of alginate synthesis in the absence of AlgL, such stranded polymers may accumulate in the periplasm to such an extent that the integrity of the cell is lost, leading to the observed toxic effects.

  5. Molecular Characterization of a Recombinant Zea mays Phenylalanine Ammonia-Lyase (ZmPAL2) and Its Application in trans-Cinnamic Acid Production from L-Phenylalanine.

    Science.gov (United States)

    Zang, Ying; Jiang, Ting; Cong, Ying; Zheng, Zhaojuan; Ouyang, Jia

    2015-06-01

    Phenylalanine ammonia-lyase (PAL) is one of the most extensively studied enzymes with its crucial role in secondary phenylpropanoid metabolism of plants. Recently, its demand has been increased for aromatic chemical production, but its applications in trans-cinnamic acid production were not much explored. In the present study, a putative PAL gene from Zea mays designated as ZmPAL2 was expressed and characterized in Escherichia coli BL21 (DE3). The recombinant ZmPAL2 exhibited a high PAL activity (7.14 U/mg) and a weak tyrosine ammonia-lyase activity. The optimal temperature of ZmPAL2 was 55 °C, and the thermal stability results showed that about 50 % of enzyme activity remained after a treatment at 60 °C for 6 h. The recombinant ZmPAL2 is a good candidate for the production of trans-cinnamic acid. The vitro conversion indicated that the recombinant ZmPAL2 could effectively catalyze the L-phenylalanine to trans-cinnamic acid, and the trans-cinnamic acid concentration can reach up to 5 g/l.

  6. OGLE 2008--BLG--290: An accurate measurement of the limb darkening of a Galactic Bulge K Giant spatially resolved by microlensing

    CERN Document Server

    Fouque, P; Dong, S; Gould, A; Udalski, A; Albrow, M D; Batista, V; Beaulieu, J -P; Bennett, D P; Bond, I A; Bramich, D M; Novati, S Calchi; Cassan, A; Coutures, C; Dieters, S; Dominik, M; Prester, D Dominis; Greenhill, J; Horne, K; Jorgensen, U G; Kozlowski, S; Kubas, D; Lee, C -H; Marquette, J -B; Mathiasen, M; Menzies, J; Monard, L A G; Nishiyama, S; Papadakis, I; Street, R; Sumi, T; Williams, A; Yee, J C; Brillant, S; Caldwell, J A R; Cole, A; Cook, K H; Donatowicz, J; Kains, N; Kane, S R; Martin, R; Pollard, K R; Sahu, K C; Tsapras, Y; Wambsganss, J; Zub, M; DePoy, D L; Gaudi, B S; Han, C; Lee, C -U; Park, B -G; Pogge, R W; Kubiak, M; Szymanski, M K; Pietrzynski, G; Soszynski, I; Szewczyk, O; Ulaczyk, K; Wyrzykowski, L; Abe, F; Fukui, A; Furusawa, K; Gilmore, A C; Hearnshaw, J B; Itow, Y; ~Kamiya, K; Kilmartin, P M; Korpela, A V; Lin, W; Ling, C H; Masuda, K; Matsubara, Y; Miyake, N; Muraki, Y; Nagaya, M; Ohnishi, K; Okumura, T; Perrott, Y; Rattenbury, N J; Saito, To; Sako, T; Sato, S; Skuljan, L; Sullivan, D; Sweatman, W; Tristram, P J; Yock, P C M; Allan, A; Bode, M F; Burgdorf, M J; Clay, N; Fraser, S N; Hawkins, E; Kerins, E; Lister, T A; Mottram, C J; Saunders, E S; Snodgrass, C; Steele, I A; Wheatley, P J; Anguita, T; Bozza, V; Harpsoe, K; Hinse, T C; Hundertmark, M; Kjaergaard, P; Liebig, C; Mancini, L; Masi, G; Rahvar, S; Ricci, D; Scarpetta, G; Southworth, J; Surdej, J; Thone, C C; Riffeser, A; ~Seitz, S; Bender, R

    2015-01-01

    Gravitational microlensing is not only a successful tool for discovering distant exoplanets, but it also enables characterization of the lens and source stars involved in the lensing event. In high magnification events, the lens caustic may cross over the source disk, which allows a determination of the angular size of the source and additionally a measurement of its limb darkening. When such extended-source effects appear close to maximum magnification, the resulting light curve differs from the characteristic Paczynski point-source curve. The exact shape of the light curve close to the peak depends on the limb darkening of the source. Dense photometric coverage permits measurement of the respective limb-darkening coefficients. In the case of microlensing event OGLE 2008-BLG-290, the K giant source star reached a peak magnification of about 100. Thirteen different telescopes have covered this event in eight different photometric bands. Subsequent light-curve analysis yielded measurements of linear limb-darke...

  7. The Orbital and Physical Parameters, and the Distance of the Eclipsing Binary System OGLE-LMC-ECL-25658 in the Large Magellanic Cloud

    Science.gov (United States)

    Elgueta, S. S.; Graczyk, D.; Gieren, W.; Pietrzyński, G.; Thompson, I. B.; Konorski, P.; Pilecki, B.; Villanova, S.; Udalski, A.; Soszyński, I.; Suchomska, K.; Karczmarek, P.; Górski, M.; Wielgórski, P.

    2016-08-01

    We present an analysis of a new detached eclipsing binary, OGLE-LMC-ECL-25658, in the Large Magellanic Cloud (LMC). The system consists of two late G-type giant stars on an eccentric orbit with an orbital period of ˜200 days. The system shows total eclipses and the components have similar temperatures, making it ideal for a precise distance determination. Using multi-color photometric and high resolution spectroscopic data, we have performed an analysis of light and radial velocity curves simultaneously using the Wilson-Devinney code. We derived orbital and physical parameters of the binary with a high precision of \\lt 1%. The masses and surface metallicities of the components are virtually the same and equal to 2.23+/- 0.02 {M}⊙ and [{Fe}/{{H}}]\\=\\-0.63+/- 0.10 dex. However, their radii and rates of rotation show a distinct trace of differential stellar evolution. The distance to the system was calculated using an infrared calibration between V-band surface brightness and (V-K) color, leading to a distance modulus of (m-M)\\=\\18.452+/- 0.023 (statistical) ± 0.046 (systematic). Because OGLE-LMC-ECL-25658 is located relatively far from the LMC barycenter, we applied a geometrical correction for its position in the LMC disk using the van der Marel et al. model of the LMC. The resulting barycenter distance to the galaxy is {d}{{LMC}}\\=\\50.30+/- 0.53 (stat.) kpc, and is in perfect agreement with the earlier result of Pietrzyński et al.

  8. 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...

  9. Perturbation of formate pathway for hydrogen production by expressions of formate hydrogen lyase and its transcriptional activator in wild Enterobacter aerogenes and its mutants

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Yuan; Zhao, Hongxin; Zhang, Chong; Lai, Qiheng; Xing, Xin-Hui [Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084 (China)

    2009-06-15

    To examine perturbation effects of formate pathway on hydrogen productivity in Enterobacter aerogenes (Ea), formate dehydrogenase FDH-H gene (fdhF) and formate hydrogen lyase activator protein FHLA gene (fhlA) originated from Escherichia coli, were overexpressed in the wild strain Ea, its hycA-deleted mutant (A) by knockout the formate hydrogen lyase repressor and hybO-deleted mutant (O) by knockout of the uptake hydrogenase, respectively. Overexpression of fdhF and fhlA promoted cell growth and volumetric hydrogen production rates of all the strains, and the hydrogen production per gram cell dry weight (CDW) for Ea, A and O was increased by 38.5%, 21.8% and 5.25%, respectively. The fdhF and fhlA overexpression improved the hydrogen yield per mol glucose of strains Ea and A, but declined that of strain O. The increase of hydrogen yield of the strain Ea with fdhF and fhlA expression was mainly attributed to the increase of formate pathway, while for the mutant A, the improved hydrogen yield with fdhF and fhlA expression was mainly due to the increase of NADH pathway. Analysis of the metabolites and ratio of ethanol-to-acetate showed that the cellular redox state balance and energy level were also changed for these strains by fdhF and fhlA expression. These findings demonstrated that the hydrogen production was not only dependent on the hydrogenase genes, but was also affected by the regulation of the whole metabolism. Therefore, fdhF and fhlA expression in different strains of E. aerogenes could exhibit different perturbation effects on the metabolism and the hydrogen productivity. (author)

  10. 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

  11. Cinical treatment of 3-hydroxy-3-methylglutaric aciduria and a novel mutation on 3-hydroxy-3-methylglutaryl-coenzyme A lyase gene%3-羟基-3-甲基戊二酸尿症患儿的临床诊治及3-羟基-3-甲基戊二酰裂解酶基因新突变分析

    Institute of Scientific and Technical Information of China (English)

    艾力克木·阿不都玩克; 古力米热·布然江; 李溪远; 杨艳玲

    2016-01-01

    产前诊断亦至关重要。%Objective To investigate the clinical manifestations and biochemical characteristics of 3-hydroxy-3-methylglutaric aciduria,and discuss results of 3-hydroxy-3-methylglutaryl-coenzyme A lyase (HMGCL)gene mutation.Methods The clinical data of a boy with 3-hydroxy-3-methylglutaric aciduria who was treated at Peking University First Hospital on December 7th 2014 was collected into this study. The admission examination, auxiliary examination, diagnosis and treatment, genedetection and follow-up of this boy were retrospectively analyzed.The study protocol was approved by the Ethical Review Board of Investigation in Human Being of Peking University First Hospital. Informed consents were obtained from patient′s parents.Results The boy complained of unexplained diarrhea,vomiting,repeated hematemesis,fever 2 d and coma 3 h.The admission examination and auxiliary examination results showed increased white blood cell count,liver damage,electrolyte imbalance,low blood sugar,blood clotting abnormalities,low-protein,high ammonia,metabolic acidosis.Cranial MRI result showed that abnormal signal could be found in bilateral lateral ventricle and double fronto-temporal parietal cortex. The final diagnosis of the boy was 3-hydroxy-3- methylglutaric aciduria.By limiting protein intake,intravenous infusion of glucose and L-carnitine treatment,the symptom alleviated gradually.Genetic analysis results showed that exon region of HMGCL was found two heterozygous mutation point:c.509G> T and c.348 + 1 G> C,where c.509G> T was heterozygous mutation from the mother,c.348 + 1 G > C emerged in child homozygous mutation,the mutation site of c.348 + 1G> C splice site mutation caused amino acid changes p.Cys170Phe,splicing (cysteine phenylalanine,shear mutation).After discharge,the six months follow-up results showed as follow. Psychomotor developed slightly behind the health children,but no significant setback.The boy lived in good general condition,and biochemical

  12. 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.

  13. 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.

  14. Cystathionine γ-lyase, an enzyme related to the reverse transsulfuration pathway, is functional in Leishmania spp.

    Science.gov (United States)

    Giordana, Lucila; Mantilla, Brian Suárez; Santana, Marianela; Silber, Ariel M; Nowicki, Cristina

    2014-01-01

    Leishmania parasites seem capable of producing cysteine by de novo biosynthesis, similarly to bacteria, some pathogenic protists, and plants. In Leishmania spp., cysteine synthase (CS) and cystathionine β-synthase (CBS) are expected to participate in this metabolic process. Moreover, the reverse transsulfuration pathway (RTP) is also predicted to be operative in this trypanosomatid because CBS also catalyzes the condensation of serine with homocysteine, and a gene encoding a putative cystathionine γ-lyase (CGL) is present in all the sequenced genomes. Our results show that indeed, Leishmania major CGL is able to rescue the wild-type phenotype of a Saccharomyces cerevisiae CGL-null mutant and is susceptible to inhibition by an irreversible CGL inhibitor, DL-propargylglycine (PAG). In Leishmania promastigotes, CGL and CS are cytosolic enzymes. The coexistence of de novo synthesis with the RTP is extremely rare in most living organisms; however, despite this potentially high redundancy in cysteine production, PAG arrests the proliferation of L. major promastigotes with an IC50 of approximately 65 μM. These findings raise new questions regarding the biological role of CGL in these pathogens and indicate the need for understanding the molecular mechanism of PAG action in vivo to identify the potential targets affected by this drug.

  15. 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.

  16. 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.

  17. 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.

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

    Science.gov (United States)

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

    2015-08-05

    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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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...

  4. A Catalase-related Hemoprotein in Coral Is Specialized for Synthesis of Short-chain Aldehydes: DISCOVERY OF P450-TYPE HYDROPEROXIDE LYASE ACTIVITY IN A CATALASE.

    Science.gov (United States)

    Teder, Tarvi; Lõhelaid, Helike; Boeglin, William E; Calcutt, Wade M; Brash, Alan R; Samel, Nigulas

    2015-08-07

    In corals a catalase-lipoxygenase fusion protein transforms arachidonic acid to the allene oxide 8R,9-epoxy-5,9,11,14-eicosatetraenoic acid from which arise cyclopentenones such as the prostanoid-related clavulones. Recently we cloned two catalase-lipoxygenase fusion protein genes (a and b) from the coral Capnella imbricata, form a being an allene oxide synthase and form b giving uncharacterized polar products (Lõhelaid, H., Teder, T., Tõldsepp, K., Ekins, M., and Samel, N. (2014) PloS ONE 9, e89215). Here, using HPLC-UV, LC-MS, and NMR methods, we identify a novel activity of fusion protein b, establishing its role in cleaving the lipoxygenase product 8R-hydroperoxy-eicosatetraenoic acid into the short-chain aldehydes (5Z)-8-oxo-octenoic acid and (3Z,6Z)-dodecadienal; these primary products readily isomerize in an aqueous medium to the corresponding 6E- and 2E,6Z derivatives. This type of enzymatic cleavage, splitting the carbon chain within the conjugated diene of the hydroperoxide substrate, is known only in plant cytochrome P450 hydroperoxide lyases. In mechanistic studies using (18)O-labeled substrate and incubations in H2(18)O, we established synthesis of the C8-oxo acid and C12 aldehyde with the retention of the hydroperoxy oxygens, consistent with synthesis of a short-lived hemiacetal intermediate that breaks down spontaneously into the two aldehydes. Taken together with our initial studies indicating differing gene regulation of the allene oxide synthase and the newly identified catalase-related hydroperoxide lyase and given the role of aldehydes in plant defense, this work uncovers a potential pathway in coral stress signaling and a novel enzymatic activity in the animal kingdom.

  5. THE ARAUCARIA PROJECT: A STUDY OF THE CLASSICAL CEPHEID IN THE ECLIPSING BINARY SYSTEM OGLE LMC562.05.9009 IN THE LARGE MAGELLANIC CLOUD

    Energy Technology Data Exchange (ETDEWEB)

    Gieren, Wolfgang; Pilecki, Bogumił; Pietrzyński, Grzegorz; Graczyk, Dariusz; Górski, Marek; Taormina, Mónica; Gallenne, Alexandre, E-mail: wgieren@astro-udec.cl, E-mail: pilecki@astrouw.edu.pl, E-mail: pietrzyn@astrouw.edu.pl [Universidad de Concepción, Departamento de Astronomía, Casilla 160-C, Concepción (Chile); and others

    2015-12-10

    We present a detailed study of the classical Cepheid in the double-lined, highly eccentric eclipsing binary system OGLE-LMC562.05.9009. The Cepheid is a fundamental mode pulsator with a period of 2.988 days. The orbital period of the system is 1550 days. Using spectroscopic data from three 4–8-m telescopes and photometry spanning 22 years, we were able to derive the dynamical masses and radii of both stars with exquisite accuracy. Both stars in the system are very similar in mass, radius, and color, but the companion is a stable, non-pulsating star. The Cepheid is slightly more massive and bigger (M{sub 1} = 3.70 ± 0.03 M{sub ⊙}, R{sub 1} = 28.6 ± 0.2 R{sub ⊙}) than its companion (M{sub 2} = 3.60 ± 0.03 M{sub ⊙}, R{sub 2} = 26.6 ± 0.2 R{sub ⊙}). Within the observational uncertainties both stars have the same effective temperature of 6030 ± 150 K. Evolutionary tracks place both stars inside the classical Cepheid instability strip, but it is likely that future improved temperature estimates will move the stable giant companion just beyond the red edge of the instability strip. Within current observational and theoretical uncertainties, both stars fit on a 205 Myr isochrone arguing for their common age. From our model, we determine a value of the projection factor of p = 1.37 ± 0.07 for the Cepheid in the OGLE-LMC562.05.9009 system. This is the second Cepheid for which we could measure its p-factor with high precision directly from the analysis of an eclipsing binary system, which represents an important contribution toward a better calibration of Baade-Wesselink methods of distance determination for Cepheids.

  6. 1株产褐藻胶裂解酶海洋细菌的分离鉴定及其酶学性质%Isolation and identification of an alginate lyase-producing marine bacterium strain and its enzymological characteristics

    Institute of Scientific and Technical Information of China (English)

    汤海青; 欧昌荣; 郑晓冬

    2013-01-01

    Alginate polysaccharide produced by marine brown algae or certain Gram-negative bacteria is a linear anionic binary copolymerof β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues.Alginate lyase (EC 4.2.2.3 ; EC 4.2.2.11),also known as alginase or alginate depolymerase,catalyzes the degradation of alginate at the non-reducing terminus by β-elimination mechanism,forming unsaturated oligosaccharides with double bonds.Since the original description of alginate lyases about 50 years ago,more than 50 enzymes have been characterized from a variety of algae,marine invertebrates,terrestrial and marine microorganisms.As a tool enzyme to degrade alginate biologically,alginate lyase has been explored and utilized in broad fields.It has been reported that alginate lyase or its crude extract can play a pivotal role in decomposing brown seaweeds,producing alginate oligosaccharides,analyzing the fine structure of alginate,preparing protoplasts of seaweed,and reducing viscosity of alginate biofilm built up in the lungs of cystic fibrosis sufferers.To elucidate the unique properties of alginate lyase,great efforts have so far been made around enzyme production,purification,characterization,and gene recombinant and so on.In the present study,a wild marine strain capable of producing alginate lyase was screened and studied in order to develop a potential overproducing strain using a low-cost culture medium.Marine microorganisms producing alginate lyase were screened by agar plate using alginate as only carbon source.The morphological,biochemical and physiological characteristics and 16S rRNA gene were analyzed to identify the taxonomic position of strain QZ-4.The enzymological characteristics of alginate lyase from the strain QZ-4 were determined by ultraviolet (UV) method,including the optimal temperature,pH,thermal and pH stability,metal ions and ethylene diamine tetraacetic acid (EDTA) tolerance,etc.The results showed that the strain QZ-4 was a Gram-negative rod

  7. 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.

  8. 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.

  9. 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.

  10. The Araucaria Project: A study of the classical Cepheid in the eclipsing binary system OGLE LMC562.05.9009 in the Large Magellanic Cloud

    CERN Document Server

    Gieren, Wolfgang; Pietrzynski, Grzegorz; Graczyk, Dariusz; Udalski, Andrzej; Soszynki, Igor; Thompson, Ian B; Moroni, Pier Giorgio Prada; Smolec, Radoslaw; Konorski, Piotr; Gorski, Marek; Karczmarek, Paulina; Suchomska, Ksenia; Taormina, Monica; Gallenne, Alexandre; Storm, Jesper; Bono, Giuseppe; Catelan, Marcio; Szymanski, Michal; Kozlowski, Szymon; Pietrukowicz, Pawel; Wyrzykowski, Lukasz; Poleski, Radoslaw; Skowron, Jan; Minniti, Dante; Ulaczyk, K; Mroz, P; Pawlak, M; Nardetto, Nicolas

    2015-01-01

    We present a detailed study of the classical Cepheid in the double-lined, highly eccentric eclipsing binary system OGLE-LMC562.05.9009. The Cepheid is a fundamental mode pulsator with a period of 2.988 days. The orbital period of the system is 1550 days. Using spectroscopic data from three 4-8-m telescopes and photometry spanning 22 years, we were able to derive the dynamical masses and radii of both stars with exquisite accuracy. Both stars in the system are very similar in mass, radius and color, but the companion is a stable, non-pulsating star. The Cepheid is slightly more massive and bigger (M_1 = 3.70 +/- 0.03M_sun, R_1 = 28.6 +/- 0.2R_sun) than its companion (M_2 = 3.60 +/- 0.03M_sun, R_2 = 26.6 +/- 0.2R_sun). Within the observational uncertainties both stars have the same effective temperature of 6030 +/- 150K. Evolutionary tracks place both stars inside the classical Cepheid instability strip, but it is likely that future improved temperature estimates will move the stable giant companion just beyond...

  11. OGLE-2015-BLG-0051/KMT-2015-BLG-0048Lb: a Giant Planet Orbiting a Low-mass Bulge Star Discovered by High-cadence Microlensing Surveys

    CERN Document Server

    Han, C; Gould, A; Bozza, V; Jung, Y K; Albrow, M D; Kim, S -L; Lee, C -U; Cha, S -M; Kim, D -J; Lee, Y; Park, B -G; Shin, I -G; Szymański, M K; Soszyński, I; Skowron, J; Mróz, P; Poleski, R; Pietrukowicz, P; Kozłowski, S; Ulaczyk, K; Wyrzykowski, Ł; Pawlak, M

    2016-01-01

    We report the discovery of an extrasolar planet detected from the combined data of a microlensing event OGLE-2015-BLG-0051/KMT-2015-BLG-0048 acquired by two microlensing surveys. Despite that the short planetary signal occurred in the very early Bulge season during which the lensing event could be seen for just about an hour, the signal was continuously and densely covered. From the Bayesian analysis using models of the mass function, matter and velocity distributions combined with the information of the angular Einstein radius, it is found that the host of the planet is located in the Galactic bulge. The planet has a mass $0.72_{-0.07}^{+0.65}\\ M_{\\rm J}$ and it is orbiting a low-mass M-dwarf host with a projected separation $d_\\perp=0.73 \\pm 0.08$ AU. The discovery of the planet demonstrates the capability of the current high-cadence microlensing lensing surveys in detecting and characterizing planets.

  12. OGLE-2012-BLG-0563Lb: a Saturn-mass Planet around an M Dwarf with the Mass Constrained by Subaru AO imaging

    CERN Document Server

    Fukui, A; Sumi, T; Bennett, D P; Bond, I A; Han, C; Suzuki, D; Beaulieu, J -P; Batista, V; Udalski, A; Street, R A; Tsapras, Y; Hundertmark, M; Abe, F; Freeman, M; Itow, Y; Ling, C H; Koshimoto, N; Masuda, K; Matsubara, Y; Muraki, Y; Ohnishi, K; Philpott, L C; Rattenbury, N; Saito, T; Sullivan, D J; Tristram, P J; Yonehara, A; Choi, J -Y; Christie, G W; DePoy, D L; Dong, Subo; Drummond, J; Gaudi, B S; Hwang, K -H; Kavka, A; Lee, C U; McCormick, J; Natusch, T; Ngan, H; Park, H; Pogge, R W; Shin, I-G; Tan, T -G; Yee, J C; Szymański, M K; Pietrzyński, G; Soszyński, I; Poleski, R; Kozłowski, S; Pietrukowicz, P; Ulaczyk, K; Bramich, Ł Wyrzykowski D M; Browne, P; Dominik, M; Horne, K; Ipatov, S; Kains, N; Snodgrass, C; Steele, I A

    2015-01-01

    We report the discovery of a microlensing exoplanet OGLE-2012-BLG-0563Lb with the planet-star mass ratio ~1 x 10^{-3}. Intensive photometric observations of a high-magnification microlensing event allow us to detect a clear signal of the planet. Although no parallax signal is detected in the light curve, we instead succeed at detecting the flux from the host star in high-resolution JHK'-band images obtained by the Subaru/AO188 and IRCS instruments, allowing us to constrain the absolute physical parameters of the planetary system. With the help of a spectroscopic information of the source star obtained during the high-magnification state by Bensby et al. (2013), we find that the lens system is located at 1.3^{+0.6}_{-0.8} kpc from us, and consists of an M dwarf (0.34^{+0.12}_{-0.20} M_sun) orbited by a Saturn-mass planet (0.39^{+0.14}_{-0.23} M_Jup) at the projected separation of 0.74^{+0.26}_{-0.42} AU (close model) or 4.3^{+1.5}_{-2.5} AU (wide model). The probability of contamination in the host star's flux...

  13. OGLE-2012-BLG-0455/MOA-2012-BLG-206: Microlensing event with ambiguity in planetary interpretations caused by incomplete coverage of planetary signal

    CERN Document Server

    Park, H; Gould, A; Udalski, A; Sumi, T; Fouqué, P; Choi, J -Y; Christie, G; Depoy, D L; Dong, Subo; Gaudi, B S; Hwang, K -H; Jung, Y K; Kavka, A; Lee, C -U; Monard, L A G; Natusch, T; Ngan, H; Pogge, R W; Shin, I -G; Yee, J C; Szymański, M K; Kubiak, M; Soszyński, I; Pietrzyński, G; Poleski, R; Ulaczyk, K; Pietrukowicz, P; Kozłowski, S; Skowron, J; Wyrzykowski, Ł; Abe, F; Bennett, D P; Bond, I A; Botzler, C S; Chote, P; Freeman, M; Fukui, A; Fukunaga, D; Harris, P; Itow, Y; Koshimoto, N; Ling, C H; Masuda, K; Matsubara, Y; Muraki, Y; Namba, S; Ohnishi, K; Rattenbury, N J; Saito, To; Sullivan, D J; Sweatman, W L; Suzuki, D; Tristram, P J; Wada, K; Yamai, N; Yock, P C M; Yonehara, A

    2014-01-01

    Characterizing a microlensing planet is done from modeling an observed lensing light curve. In this process, it is often confronted that solutions of different lensing parameters result in similar light curves, causing difficulties in uniquely interpreting the lens system, and thus understanding the causes of different types of degeneracy is important. In this work, we show that incomplete coverage of a planetary perturbation can also result in degenerate solutions even for events where the planetary signal is detected with a high level of statistical significance. We demonstrate the degeneracy for an actually observed event OGLE-2012-BLG-0455/MOA-2012-BLG-206. The peak of this high-magnification event $(A_{\\rm max}\\sim400)$ exhibits very strong deviation from a point-lens model with $\\Delta\\chi^{2}\\gtrsim4000$. From detailed modeling of the light curve, we find that the deviation can be explained by four distinct solutions, i.e., two very different sets of solutions, each with a two-fold degeneracy. While th...

  14. OGLE-2015-BLG-0051/KMT-2015-BLG-0048Lb: A Giant Planet Orbiting a Low-mass Bulge Star Discovered by High-cadence Microlensing Surveys

    Science.gov (United States)

    Han, C.; Udalski, A.; Gould, A.; Bozza, V.; Jung, Y. K.; Albrow, M. D.; Kim, S.-L.; Lee, C.-U.; Cha, S.-M.; Kim, D.-J.; Lee, Y.; Park, B.-G.; Shin, I.-G.; KMTNet Collaboration; Szymański, M. K.; Soszyński, I.; Skowron, J.; Mróz, P.; Poleski, R.; Pietrukowicz, P.; Kozłowski, S.; Ulaczyk, K.; Wyrzykowski, Ł.; Pawlak, M.; OGLE Collaboration

    2016-10-01

    We report the discovery of an extrasolar planet detected from the combined data of a microlensing event OGLE-2015-BLG-0051/KMT-2015-BLG-0048 acquired by two microlensing surveys. Despite the fact that the short planetary signal occurred in the very early Bulge season during which the lensing event could be seen for just about an hour, the signal was continuously and densely covered. From the Bayesian analysis using models of the mass function, and matter and velocity distributions, combined with information on the angular Einstein radius, it is found that the host of the planet is located in the Galactic bulge. The planet has a mass {0.72}-0.07+0.65 {M}{{J}} and it is orbiting a low-mass M-dwarf host with a projected separation {d}\\perp =0.73+/- 0.08 {{au}}. The discovery of the planet demonstrates the capability of the current high-cadence microlensing lensing surveys in detecting and characterizing planets.

  15. Orbital and physical parameters, and the distance of the eclipsing binary system OGLE-LMC-ECL-25658 in the Large Magellanic Cloud

    CERN Document Server

    Elgueta, S S; Gieren, W; Pietrzynski, G; Thompson, I B; Konorski, P; Pilecki, B; Villanova, S; Udalski, A; Soszynski, I; Suchomska, K; Karczmarek, P; Gorski, M; Wielgorski, P

    2016-01-01

    We present an analysis of a new detached eclipsing binary, OGLE-LMC-ECL-25658, in the Large Magellanic Cloud. The system consists of two late G-type giant stars on an eccentric orbit and orbital period of ~200 days. The system shows total eclipses and the components have similar temperatures, making it ideal for a precise distance determination. Using multi-color photometric and high resolution spectroscopic data, we have performed an analysis of light and radial velocity curves simultaneously using the Wilson Devinney code. We derived orbital and physical parameters of the binary with a high precision of < 1 %. The masses and surface metallicities of the components are virtually the same and equal to 2.23 +/- 0.02 M_sun and [Fe/H] = -0.63 +/- 0.10 dex. However their radii and rates of rotation show a distinct trace of differential stellar evolution. The distance to the system was calculated using an infrared calibration between V-band surface brightness and (V-K) color, leading to a distance modulus of (m...

  16. 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.

  17. 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...

  18. 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...

  19. 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.

  20. 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.

  1. 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...

  2. Formate hydrogen lyase mediates stationary-phase deacidification and increases survival during sugar fermentation in acetoin-producing enterobacteria

    Directory of Open Access Journals (Sweden)

    Bram eVivijs

    2015-02-01

    Full Text Available Two fermentation types exist in the Enterobacteriaceae family. Mixed-acid fermenters produce substantial amounts of lactate, formate, acetate and succinate, resulting in lethal medium acidification. On the other hand, 2,3-butanediol fermenters switch to the production of the neutral compounds acetoin and 2,3-butanediol and even deacidify the environment after an initial acidification phase, thereby avoiding cell death. We equipped three mixed-acid fermenters (Salmonella Typhimurium, S. Enteritidis and Shigella flexneri with the acetoin pathway from Serratia plymuthica to investigate the mechanisms of deacidification. Acetoin production caused attenuated acidification during exponential growth in all three bacteria, but stationary-phase deacidification was only observed in Escherichia coli and Salmonella, suggesting that it was not due to the consumption of protons accompanying acetoin production. To identify the mechanism, 34 transposon mutants of acetoin-producing E. coli that no longer deacidified the culture medium were isolated. The mutations mapped to 16 genes, all involved in formate metabolism. Formate is an end product of mixed-acid fermentation that can be converted to H2 and CO2 by the formate hydrogen lyase (FHL complex, a reaction that consumes protons and thus can explain medium deacidification. When hycE, encoding the large subunit of hydrogenase 3 that is part of the FHL complex, was deleted in acetoin-producing E. coli, deacidification capacity was lost. Metabolite analysis in E. coli showed that introduction of the acetoin pathway reduced lactate and acetate production, but increased glucose consumption and formate and ethanol production. Analysis of a hycE mutant in S. plymuthica confirmed that medium deacidification in this organism is also mediated by FHL. These findings improve our understanding of the physiology and function of fermentation pathways in Enterobacteriaceae.

  3. pH regulation of pectate lyase secretion modulates the attack of Colletotrichum gloeosporioides on avocado fruits.

    Science.gov (United States)

    Yakoby, N; Kobiler, I; Dinoor, A; Prusky, D

    2000-03-01

    Growth of Colletotrichum gloeosporioides in pectolytic enzyme-inducing medium (PEIM) increased the pH of the medium from 3. 8 to 6.5. Pectate lyase (PL) secretion was detected when the pH reached 5.8, and the level of secretion increased up to pH 6.5. PL gene (pel) transcript production began at pH 5.0 and increased up to pH 5.7. PL secretion was never detected when the pH of the inducing medium was lower than 5.8 or when C. gloeosporioides hyphae were transferred from PL-secreting conditions at pH 6.5 to pH 3.8. This behavior differed from that of polygalacturonase (PG), where pg transcripts and protein secretion were detected at pH 5.0 and continued up to 5.7. Under in vivo conditions, the pH of unripe pericarp of freshly harvested avocado (Persea americana cv. Fuerte) fruits, resistant to C. gloeosporioides attack, was 5.2, whereas in ripe fruits, when decay symptoms were expressed, the pericarp pH had increased to 6.3. Two avocado cultivars, Ardit and Ettinger, which are resistant to C. gloeosporioides attack, had pericarp pHs of less than 5.5, which did not increase during ripening. The present results suggest that host pH regulates the secretion of PL and may affect C. gloeosporioides pathogenicity. The mechanism found in avocado may have equivalents in other post-harvest pathosystems and suggests new approaches for breeding against and controlling post-harvest diseases.

  4. Overexpression of hydroxynitrile lyase in cassava roots elevates protein and free amino acids while reducing residual cyanogen levels.

    Directory of Open Access Journals (Sweden)

    Narayanan N Narayanan

    Full Text Available Cassava is the major source of calories for more than 250 million Sub-Saharan Africans, however, it has the lowest protein-to-energy ratio of any major staple food crop in the world. A cassava-based diet provides less than 30% of the minimum daily requirement for protein. Moreover, both leaves and roots contain potentially toxic levels of cyanogenic glucosides. The major cyanogen in cassava is linamarin which is stored in the vacuole. Upon tissue disruption linamarin is deglycosylated by the apolplastic enzyme, linamarase, producing acetone cyanohydrin. Acetone cyanohydrin can spontaneously decompose at pHs >5.0 or temperatures >35°C, or is enzymatically broken down by hydroxynitrile lyase (HNL to produce acetone and free cyanide which is then volatilized. Unlike leaves, cassava roots have little HNL activity. The lack of HNL activity in roots is associated with the accumulation of potentially toxic levels of acetone cyanohydrin in poorly processed roots. We hypothesized that the over-expression of HNL in cassava roots under the control of a root-specific, patatin promoter would not only accelerate cyanogenesis during food processing, resulting in a safer food product, but lead to increased root protein levels since HNL is sequestered in the cell wall. Transgenic lines expressing a patatin-driven HNL gene construct exhibited a 2-20 fold increase in relative HNL mRNA levels in roots when compared with wild type resulting in a threefold increase in total root protein in 7 month old plants. After food processing, HNL overexpressing lines had substantially reduced acetone cyanohydrin and cyanide levels in roots relative to wild-type roots. Furthermore, steady state linamarin levels in intact tissues were reduced by 80% in transgenic cassava roots. These results suggest that enhanced linamarin metabolism contributed to the elevated root protein levels.

  5. Overexpression of hydroxynitrile lyase in cassava roots elevates protein and free amino acids while reducing residual cyanogen levels.

    Science.gov (United States)

    Narayanan, Narayanan N; Ihemere, Uzoma; Ellery, Claire; Sayre, Richard T

    2011-01-01

    Cassava is the major source of calories for more than 250 million Sub-Saharan Africans, however, it has the lowest protein-to-energy ratio of any major staple food crop in the world. A cassava-based diet provides less than 30% of the minimum daily requirement for protein. Moreover, both leaves and roots contain potentially toxic levels of cyanogenic glucosides. The major cyanogen in cassava is linamarin which is stored in the vacuole. Upon tissue disruption linamarin is deglycosylated by the apolplastic enzyme, linamarase, producing acetone cyanohydrin. Acetone cyanohydrin can spontaneously decompose at pHs >5.0 or temperatures >35°C, or is enzymatically broken down by hydroxynitrile lyase (HNL) to produce acetone and free cyanide which is then volatilized. Unlike leaves, cassava roots have little HNL activity. The lack of HNL activity in roots is associated with the accumulation of potentially toxic levels of acetone cyanohydrin in poorly processed roots. We hypothesized that the over-expression of HNL in cassava roots under the control of a root-specific, patatin promoter would not only accelerate cyanogenesis during food processing, resulting in a safer food product, but lead to increased root protein levels since HNL is sequestered in the cell wall. Transgenic lines expressing a patatin-driven HNL gene construct exhibited a 2-20 fold increase in relative HNL mRNA levels in roots when compared with wild type resulting in a threefold increase in total root protein in 7 month old plants. After food processing, HNL overexpressing lines had substantially reduced acetone cyanohydrin and cyanide levels in roots relative to wild-type roots. Furthermore, steady state linamarin levels in intact tissues were reduced by 80% in transgenic cassava roots. These results suggest that enhanced linamarin metabolism contributed to the elevated root protein levels.

  6. 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.

  7. GUS Gene Expression Driven by A Citrus Promoter in Transgenic Tobacco and 'Valencia' Sweet Orange

    Science.gov (United States)

    The objective of this work was the transformation of tobacco and ‘Valencia’ sweet orange with the GUS gene driven by the citrus phenylalanine ammonia-lyase (PAL) gene promoter (CsPP). Transformation was accomplished by co-cultivation of tobacco and ‘Valencia’ sweet orange explants with Agrobacteriu...

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. [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.

  15. 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.

  16. 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.

  17. 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.

  18. OGLE-2012-BLG-0455/MOA-2012-BLG-206: Microlensing event with ambiguity in planetary interpretations caused by incomplete coverage of planetary signal

    Energy Technology Data Exchange (ETDEWEB)

    Park, H.; Han, C.; Choi, J.-Y.; Hwang, K.-H.; Jung, Y. K.; Shin, I.-G. [Department of Physics, Institute for Astrophysics, Chungbuk National University, Cheongju 371-763 (Korea, Republic of); Gould, A.; Gaudi, B. S.; Kavka, A.; Pogge, R. W. [Department of Astronomy, The Ohio State University, 140 West 18th Avenue, Columbus, OH 43210 (United States); Udalski, A. [Warsaw University Observatory, Al. Ujazdowskie 4, 00-478 Warszawa (Poland); Sumi, T. [Department of Earth and Space Science, Osaka University, Osaka 560-0043 (Japan); Fouqué, P. [IRAP, CNRS, Université de Toulouse, F-31400 Toulouse (France); Christie, G.; Natusch, T.; Ngan, H. [Auckland Observatory, Auckland (New Zealand); Depoy, D. L. [Department of Physics and Astronomy, Texas A and M University, College Station, TX 77843 (United States); Dong, Subo [Kavli Institute for Astronomy and Astrophysics, Peking University, Yi He Yuan Road 5, Hai Dian District, Beijing 100871 (China); Lee, C.-U. [Korea Astronomy and Space Science Institute, 776 Daedukdae-ro, Yuseong-gu, Daejeon 305-348 (Korea, Republic of); Monard, L. A. G. [Kleinkaroo Observatory, Calitzdorp, and Bronberg Observatory, Pretoria (South Africa); Collaboration: μFUN Collaboration; OGLE Collaboration; MOA Collaboration; and others

    2014-05-20

    Characterizing a microlensing planet is done by modeling an observed lensing light curve. In this process, it is often confronted that solutions of different lensing parameters result in similar light curves, causing difficulties in uniquely interpreting the lens system, and thus understanding the causes of different types of degeneracy is important. In this work, we show that incomplete coverage of a planetary perturbation can result in degenerate solutions even for events where the planetary signal is detected with a high level of statistical significance. We demonstrate the degeneracy for an actually observed event OGLE-2012-BLG-0455/MOA-2012-BLG-206. The peak of this high-magnification event (A {sub max} ∼ 400) exhibits very strong deviation from a point-lens model with Δχ{sup 2} ≳ 4000 for data sets with a total of 6963 measurements. From detailed modeling of the light curve, we find that the deviation can be explained by four distinct solutions, i.e., two very different sets of solutions, each with a twofold degeneracy. While the twofold (so-called close/wide) degeneracy is well understood, the degeneracy between the radically different solutions is not previously known. The model light curves of this degeneracy differ substantially in the parts that were not covered by observation, indicating that the degeneracy is caused by the incomplete coverage of the perturbation. It is expected that the frequency of the degeneracy introduced in this work will be greatly reduced with the improvement of the current lensing survey and follow-up experiments and the advent of new surveys.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  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. 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.

  5. 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.

  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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. Phenylalanine ammonia-lyase and cell wall peroxidase are cooperatively involved in the extensive formation of ferulate network in cell walls of developing rice shoots.

    Science.gov (United States)

    Wakabayashi, Kazuyuki; Soga, Kouichi; Hoson, Takayuki

    2012-02-15

    The relationship between the formation of cell wall-bound ferulic acid (FA) and diferulic acid (DFA) and the change in activities of phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) was studied in rice shoots. The length and the fresh mass of shoots increased during the growth period from day 4 to 6, while coleoptiles ceased elongation growth on day 5. The amounts of FA and DFA isomers as well as cell wall polysaccharides continued to increase during the whole period. The activities of PAL and CW-PRX greatly increased in the same manner during the period. There were close correlations between the PAL activity and ferulate content or between the CW-PRX activity and DFA content. The expression levels of investigated genes for PAL and putative CW-PRX showed good accordance with the activities of these enzymes. These results suggest that increases in PAL and CW-PRX activities are cooperatively involved in the formation of ferulate network in cell walls of rice shoots and that investigated genes may be, at least in part, associated with the enzyme activities. The substantial increase in such network probably causes the maturation of cell walls and thus the cessation of elongation growth of coleoptiles.

  15. Comparative expression of wild-type and highly soluble mutant His103Leu of hydroxynitrile lyase from Manihot esculenta in prokaryotic and eukaryotic expression systems.

    Science.gov (United States)

    Dadashipour, Mohammad; Fukuta, Yasuhisa; Asano, Yasuhisa

    2011-05-01

    Low protein solubility and inclusion body formation represent big challenges in production of recombinant proteins in Escherichia coli. We have recently reported functional expression of hydroxynitrile lyase from Manihot esculenta, MeHNL, in E. coli with high in vivo solubility and activity using directed evolution. As a part of attempts to clarify the mechanism of this phenomenon, we have described the possibility of expression of the highly active and soluble mutant MeHNL-His103Leu as well as wild-type enzyme in several expression systems. Methylotrophic yeast Pichia pastoris, protozoan host Leishmania tarentolae and two cell-free translations, including an E. coli lysate (WakoPURE system) and wheat germ translation system were used to compare expression profiles of the genes. Two distinguishable protein expression patterns were observed in prokaryotic and eukaryotic-based systems. The wild-type and mutant enzyme showed high activity for both genes (up to 10 U/ml) in eukaryotic hosts P. pastoris and L. tarentolae, while those of E. coli exhibited about 1 and 15 U/ml, respectively. The different activity level in prokaryotic systems but the same level among the eukaryotic hosts indicate the phenomenon is specific to the E. coli system. Both the wild-type and mutant enzymes were functionally expressed in eukaryotic systems, probably using the folding assistants such as chaperones. Properties of expression systems used in this study were precisely compared, too.

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

    Science.gov (United States)

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

    2015-08-28

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

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. Gene

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene integrates information from a wide range of species. A record may include nomenclature, Reference Sequences (RefSeqs), maps, pathways, variations, phenotypes,...

  13. Both decrease in ACL1 gene expression and increase in ICL1 gene expression in marine-derived yeast Yarrowia lipolytica expressing INU1 gene enhance citric acid production from inulin.

    Science.gov (United States)

    Liu, Xiao-Yan; Chi, Zhe; Liu, Guang-Lei; Madzak, Catherine; Chi, Zhen-Ming

    2013-02-01

    In this study, some of the ATP-citrate lyase genes (ACL1) were deleted and the copy number of the iso-citrate lyase gene (ICL1) was increased in the marine-derived yeast Yarrowia lipolytica SWJ-1b displaying the recombinant inulinase. It was found that lipid content and iso-citric acid in the transformant 30 obtained were greatly reduced and citric acid production was greatly enhanced. It was also found that the ACL1 gene expression and ATP-citrate lyase activity in the transformant 30 were declined and the ICL1 gene expression and iso-citrate lyase activity were promoted. During the 2-l fermentation, 84.0 g/l of citric acid and 1.8 g/l of iso-citric acid in the fermented medium were attained from 10.0 % of inulin by the transformant 30 within 214 h. The results showed that only 0.36 % of the residual reducing sugar and 1.0 % of the residual total sugar were left in the fermented medium, suggesting that 89.6 % of the total sugar was used for citric acid production and cell growth by the transformant 30.

  14. 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...

  15. Prevention of Diet-Induced Metabolic Dysregulation, Inflammation, and Atherosclerosis in Ldlr(-/-) Mice by Treatment With the ATP-Citrate Lyase Inhibitor Bempedoic Acid.

    Science.gov (United States)

    Samsoondar, Joshua P; Burke, Amy C; Sutherland, Brian G; Telford, Dawn E; Sawyez, Cynthia G; Edwards, Jane Y; Pinkosky, Stephen L; Newton, Roger S; Huff, Murray W

    2017-04-01

    Bempedoic acid (ETC-1002, 8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel low-density lipoprotein cholesterol-lowering compound. In animals, bempedoic acid targets the liver where it inhibits cholesterol and fatty acid synthesis through inhibition of ATP-citrate lyase and through activation of AMP-activated protein kinase. In this study, we tested the hypothesis that bempedoic acid would prevent diet-induced metabolic dysregulation, inflammation, and atherosclerosis. APPROACH AND RESULTS: Ldlr(-/-) mice were fed a high-fat, high-cholesterol diet (42% kcal fat, 0.2% cholesterol) supplemented with bempedoic acid at 0, 3, 10 and 30 mg/kg body weight/day. Treatment for 12 weeks dose-dependently attenuated diet-induced hypercholesterolemia, hypertriglyceridemia, hyperglycemia, hyperinsulinemia, fatty liver and obesity. Compared to high-fat, high-cholesterol alone, the addition of bempedoic acid decreased plasma triglyceride (up to 64%) and cholesterol (up to 50%) concentrations, and improved glucose tolerance. Adiposity was significantly reduced with treatment. In liver, bempedoic acid prevented cholesterol and triglyceride accumulation, which was associated with increased fatty acid oxidation and reduced fatty acid synthesis. Hepatic gene expression analysis revealed that treatment significantly increased expression of genes involved in fatty acid oxidation while suppressing inflammatory gene expression. In full-length aorta, bempedoic acid markedly suppressed cholesteryl ester accumulation, attenuated the expression of proinflammatory M1 genes and attenuated the iNos/Arg1 ratio. Treatment robustly attenuated atherosclerotic lesion development in the aortic sinus by 44%, with beneficial changes in morphology, characteristic of earlier-stage lesions. Bempedoic acid effectively prevents plasma and tissue lipid elevations and attenuates the onset of inflammation, leading to the prevention of atherosclerotic lesion development in a mouse model of metabolic

  16. Phenylpropanoids, Phenylalanine Ammonia Lyase and Peroxidases in Elicitor‐challenged Cassava (Manihot esculenta) Suspension Cells and Leaves

    Science.gov (United States)

    GÓMEZ‐VÁSQUEZ, ROCÍO; DAY, ROBERT; BUSCHMANN, HOLGER; RANDLES, SOPHIE; BEECHING, JOHN R.; COOPER, RICHARD M.

    2004-01-01

    • Background and aims Control of diseases in the key tropical staple, cassava, is dependent on resistant genotypes, but the innate mechanisms are unknown. The aim was to study phenylpropanoids and associated enzymes as possible defence components. • Methods Phenylalanine ammonia‐lyase (PAL), phenylpropanoids and peroxidases (POD) were investigated in elicited cassava suspension cells and leaves. Yeast elicitor was the most effective of several microbial and endogenous elicitors. Fungitoxicity was determined against the cassava pathogens Fusarium solani, F. oxysporum and the saprotroph Trichoderma harzianum. • Key results A single and rapid (≥2–3 min) oxidative burst, measured as hydrogen peroxide, occurred in elicited cells. PAL activity was induced maximally at 15 h and was preceded by PAL mRNA accumulation, which peaked at 9 h. Symplasmic POD activity increased four‐fold in cells, 48 h post‐elicitation. POD isoforms (2–7 isoforms, pI 3·1–8·8) were detected in elicited and unelicited cells, extracellular medium and leaves but two extracellular isoforms were enhanced post‐elicitation. Also expression of a cassava peroxidase gene MecPOD1 increased in elicited cells. Only anionic forms oxidized scopoletin, with highest activity by isoform pI 3·6, present in all samples. Unidentified phenolics and possibly scopolin increased post‐elicitation, but there was no enhancement of scopoletin, rutin or kaempferol‐3‐O‐rutinoside concentration. Fungal germ tube elongation was inhibited more than germination by esculetin, ferulic acid, quercetin and scopoletin. T. harzianum was generally more sensitive than the pathogens and was inhibited by ≥50 µg mL–1 of ferulic acid and quercetin and ≥10 µg mL–1 of scopoletin. • Conclusions Phenolic levels in cells were not enhanced and were, theoretically, too low to be inhibitory. However, in combination and when oxidized they may contribute to defence, because oxidation of esculetin and

  17. Bioproduction of L-Aspartic Acid and Cinnamic Acid by L-Aspartate Ammonia Lyase from Pseudomonas aeruginosa PAO1.

    Science.gov (United States)

    Patel, Arti T; Akhani, Rekha C; Patel, Manisha J; Dedania, Samir R; Patel, Darshan H

    2017-06-01

    Aspartase (L-aspartate ammonia lyase, EC 4.3.1.1) catalyses the reversible amination and deamination of L-aspartic acid to fumaric acid which can be used to produce important biochemical. In this study, we have explored the characteristics of aspartase from Pseudomonas aeruginosa PAO1 (PA-AspA). To overproduce PA-AspA, the 1425-bp gene was introduced in Escherichia coli BL21 and purified. A 51.0-kDa protein was observed as a homogenous purified protein on SDS-PAGE. The enzyme was optimally active at pH 8.0 and 35 °C. PA-AspA has retained 56% activity after 7 days of incubation at 35 °C, which displays the hyperthermostablility characteristics of the enzyme. PA-AspA is activated in the presence of metal ions and Mg2+ is found to be most effective. Among the substrates tested for specificity of PA-AspA, L-phenylalanine (38.35 ± 2.68) showed the highest specific activity followed by L-aspartic acid (31.21 ± 3.31) and fumarate (5.42 ± 2.94). K m values for L-phenylalanine, L-aspartic acid and fumarate were 1.71 mM, 0.346 μM and 2 M, respectively. The catalytic efficiency (k cat/K m) for L-aspartic acid (14.18 s(-1) mM(-1)) was higher than that for L-phenylalanine (4.65 s(-1) mM(-1)). For bioconversion, from an initial concentration of 1000 mM of fumarate and 30 mM of L-phenylalanine, PA-AspA was found to convert 395.31 μM L-aspartic acid and 3.47 mM cinnamic acid, respectively.

  18. 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.

    Directory of Open Access Journals (Sweden)

    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.

  19. In Vivo Multienzyme Complex Coconstruction of N-Acetylneuraminic Acid Lyase and N-Acetylglucosamine-2-epimerase for Biosynthesis of N-Acetylneuraminic Acid.

    Science.gov (United States)

    Wang, Zhenfu; Zhuang, Wei; Cheng, Jian; Sun, Wujin; Wu, Jinglan; Chen, Yong; Ying, Hanjie

    2017-08-30

    Metabolic channeling enables efficient transfer of the intermediates by forming a multienzyme complex. To leverage the metabolic channeling for improved biosynthesis, we coexpressed N-acetylneuraminic acid lyase from C. glutamicum ATCC 13032 (CgNal) and N-acetylglucosamine-2-epimerase from Anabaena sp. CH1 (anAGE) in Escherichia coli and used the whole cell to synthesize N-acetylneuraminic acid (Neu5Ac) from N-acetylglucosamine (GlcNAc) and pyruvate. To get the multienzyme complex, polycistronic plasmid with high levels of CgNal and anAGE expression was constructed by tuning the orders of the genes. The Shine-Dalgarno (SD) sequence and aligned spacing (AS) distance were optimized. The E. coli Rosetta harboring the polycistronic plasmid pET-28a-SD2-AS1-CgNal-SD-AS-anAGE increased the production of Neu5Ac by 58.7% to 92.5 g/L in 36 h by whole-cell catalysis and by 21.9% up to 112.8 g/L in 24 h with the addition of Triton X-100.

  20. Identificação de variáveis cataclísmicas eruptivas na direção do bojo galáctico e Nuvens de Magalhães usando dados do OGLE

    Science.gov (United States)

    Cieslinski, D.; Diaz, M. P.; Mennickent, R.; Pietrzyski, G.

    2003-08-01

    Na década de 90 iniciaram-se vários programas para a pesquisa de matéria escura na Galáxia usando o efeito de microlentes gravitacionais. Entre os projetos mais bem conhecidos podemos mencionar o OGLE (Optical Gravitational Lensing Experiment) e o MACHO (MAssive Compact Halo Objects). A estratégia usada por eles consiste em fazer fotometria de banda larga (normalmente B, R e I) de um grande número de estrelas (dezenas de milhões) tão freqüentemente quanto possí vel e por longos perí odos de tempo (anos). Uma tal sistemática de observação, além de descobrir inúmeras lentes gravitacionais, é também muito apropriada para a descoberta de estrelas variáveis. De fato, inúmeras novas variáveis de vários tipos foram descobertas como subproduto. Exemplos podem ser encontrados nos endereços http://bulge.princeton.edu/~ogle/ e http://wwwmacho.mcmaster.ca/. As variáveis cataclí smicas eruptivas (novas clássicas, novas recorrentes e novas anãs) são objetos que apresentam variabilidade de grande amplitude com escalas de tempo de dias a centenas de dias e, por esta razão, devem ter sido detectadas em grande número nestes "surveys". Para testar esta possibilidade nós procuramos nos dados do OGLE por tais sistemas e o presente trabalho mostra os resultados desta pesquisa. Os objetos foram selecionados entre as variáveis detectadas usando a amplitude de variação de brilho como critério principal. Este critério forneceu 13756 objetos, sendo 2169 na direção da Grande Nuvem de Magalhães, 1162 na direção da Pequena Nuvem de Magalhães e o restante na direção do Bojo Galáctico. A análise foi feita inspecionando-se visualmente cada curva de luz por erupções com as características acima mencionadas. Os resultados obtidos podem ser sumarizados como: descoberta de duas novas clássicas e 33 novas anãs. Além disso, pode-se mencionar a identificação de candidatas a outros tipos de variáveis como: estrelas simbióticas, RV Tauri, R Coronae

  1. 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...

  2. 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

    The base excision repair (BER) process requires removal of an abasic deoxyribose-5-phosphate group, a catalytic activity that has been demonstrated for the N-terminal 8 kDa domain of DNA polymerase beta (Pol beta), and for the homologous domain of DNA polymerase lambda (Pol lambda). Previous studies have demonstrated that this activity results from formation of a Schiff base adduct of the abasic deoxyribose C-1' with a lysine residue (K312 in the case of Pol lambda), followed by a beta-elimination reaction. To better understand the underlying chemistry, we have determined pKa values for the lysine residues in the Pol lambda lyase domain labeled with [epsilon-13C]lysine. At neutral pH, the H(epsilon) protons on 3 of the 10 lysine residues in this domain, K287, K291, and K312, exhibit chemical shift inequivalence that results from immobilization of the lysyl side chains. For K287 and K291, this results from the K287-E261 and K291-E298 salt bridge interactions, while for K312, immobilization apparently results from steric and hydrogen-bonding interactions that constrain the position of the lysine side chain. The pKa value of K312 is depressed to 9.58, a value indicating that at physiological pH K312 will exist predominantly in the protonated form. Titration of the domain with hairpin DNA containing a 5'-tetrahydrofuran terminus to model the abasic site produced shifts of the labeled lysine resonances that were in fast exchange but appeared to be complete at a stoichiometry of approximately 1:1.3, consistent with a dissociation constant of approximately 1 microM. The epsilon-proton shifts of K273 were the most sensitive to the addition of the DNA, apparently due to changes in the relative orientation between K273 and W274 in the DNA complex. The average pKa values increased by 0.55, consistent with the formation of some DNA-lysine salt bridges and with the general pH increase expected to result from a reduction in the net positive charge of the complex. A general

  3. 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.

  4. 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.

  5. An ancient relative of cyclooxygenase in cyanobacteria is a linoleate 10S-dioxygenase that works in tandem with a catalase-related protein with specific 10S-hydroperoxide lyase activity.

    Science.gov (United States)

    Brash, Alan R; Niraula, Narayan P; Boeglin, William E; Mashhadi, Zahra

    2014-05-09

    In the course of exploring the scope of catalase-related hemoprotein reactivity toward fatty acid hydroperoxides, we detected a novel candidate in the cyanobacterium Nostoc punctiforme PCC 73102. The immediate neighboring upstream gene, annotated as "cyclooxygenase-2," appeared to be a potential fatty acid heme dioxygenase. We cloned both genes and expressed the cDNAs in Escherichia coli, confirming their hemoprotein character. Oxygen electrode recordings demonstrated a rapid (>100 turnovers/s) reaction of the heme dioxygenase with oleic and linoleic acids. HPLC, including chiral column analysis, UV, and GC-MS of the oxygenated products, identified a novel 10S-dioxygenase activity. The catalase-related hemoprotein reacted rapidly and specifically with linoleate 10S-hydroperoxide (>2,500 turnovers/s) with a hydroperoxide lyase activity specific for the 10S-hydroperoxy enantiomer. The products were identified by NMR as (8E)10-oxo-decenoic acid and the C8 fragments, 1-octen-3-ol and 2Z-octen-1-ol, in ∼3:1 ratio. Chiral HPLC analysis established strict enzymatic control in formation of the 3R alcohol configuration (99% enantiomeric excess) and contrasted with racemic 1-octen-3-ol formed in reaction of linoleate 10S-hydroperoxide with hematin or ferrous ions. The Nostoc linoleate 10S-dioxygenase, the sequence of which contains the signature catalytic sequence of cyclooxygenases and fungal linoleate dioxygenases (YRWH), appears to be a heme dioxygenase ancestor. The novel activity of the lyase expands the known reactions of catalase-related proteins and functions in Nostoc in specific transformation of the 10S-hydroperoxylinoleate.

  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. 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.

  8. 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.

  9. 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.

  10. Inhibition of Escherichia coli tryptophan indole-lyase by tryptophan homologues.

    Science.gov (United States)

    Do, Quang T; Nguyen, Giang T; Celis, Victor; Phillips, Robert S

    2014-10-15

    We have designed, synthesized and evaluated homotryptophan analogues as possible mechanism-based inhibitors for Escherichia coli tryptophan indole-lyase (tryptophanase, TIL, E.C. 4.1.99.1). As a quinonoid structure is an intermediate in the reaction mechanism of TIL, we anticipated that homologation of the physiological substrate, L-Trp would provide analogues resembling the transition state for β-elimination, and potentially inhibit TIL. Our results demonstrate that L-homotryptophan (1a) is a moderate competitive inhibitor of TIL, with Ki=67 μM, whereas L-bishomotryptophan (1b) displays more potent inhibition, with Ki=4.7 μM. Pre-steady-state kinetics indicated the formation of an external aldimine and quinonoid with 1a, but only the formation of an external aldimine for 1b, suggesting differences in the inhibition mechanism. These results demonstrate that formation of a quinonoid complex is not required for strong inhibition. In addition, the Trp analogues were evaluated as inhibitors of Salmonella typhimurium Trp synthase. Our results indicate that compound 1b is at least 25-fold more selective toward TIL than Trp synthase. We report that compound 1b is comparable to the most potent inhibitor previously reported, while displaying high selectivity for TIL. Thus, 1b is a potential lead for the development of novel antibacterials.

  11. Sphingosine-1-phosphate lyase mutations cause primary adrenal insufficiency and steroid-resistant nephrotic syndrome

    Science.gov (United States)

    Prasad, Rathi; Hadjidemetriou, Irene; Meimaridou, Eirini; Buonocore, Federica; Saleem, Moin; Hurcombe, Jenny; Bierzynska, Agnieszka; Barbagelata, Eliana; Bergadá, Ignacio; Cassinelli, Hamilton; Das, Urmi; Krone, Ruth; Hacihamdioglu, Bulent; Sari, Erkan; Yesilkaya, Ediz; Storr, Helen L.; Clemente, Maria; Fernandez-Cancio, Monica; Camats, Nuria; Ram, Nanik; Achermann, John C.; Van Veldhoven, Paul P.; Guasti, Leonardo; Braslavsky, Debora; Guran, Tulay; Metherell, Louise A.

    2017-01-01

    Primary adrenal insufficiency is life threatening and can present alone or in combination with other comorbidities. Here, we have described a primary adrenal insufficiency syndrome and steroid-resistant nephrotic syndrome caused by loss-of-function mutations in sphingosine-1-phosphate lyase (SGPL1). SGPL1 executes the final decisive step of the sphingolipid breakdown pathway, mediating the irreversible cleavage of the lipid-signaling molecule sphingosine-1-phosphate (S1P). Mutations in other upstream components of the pathway lead to harmful accumulation of lysosomal sphingolipid species, which are associated with a series of conditions known as the sphingolipidoses. In this work, we have identified 4 different homozygous mutations, c.665G>A (p.R222Q), c.1633_1635delTTC (p.F545del), c.261+1G>A (p.S65Rfs*6), and c.7dupA (p.S3Kfs*11), in 5 families with the condition. In total, 8 patients were investigated, some of whom also manifested other features, including ichthyosis, primary hypothyroidism, neurological symptoms, and cryptorchidism. Sgpl1–/– mice recapitulated the main characteristics of the human disease with abnormal adrenal and renal morphology. Sgpl1–/– mice displayed disrupted adrenocortical zonation and defective expression of steroidogenic enzymes as well as renal histology in keeping with a glomerular phenotype. In summary, we have identified SGPL1 mutations in humans that perhaps represent a distinct multisystemic disorder of sphingolipid metabolism. PMID:28165343

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

    Science.gov (United States)

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

    2016-01-01

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

  13. 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.

  14. Design of benzimidazole- and benzoxazole-2-thione derivatives as inhibitors of bacterial hyaluronan lyase.

    Science.gov (United States)

    Braun, Stephan; Botzki, Alexander; Salmen, Sunnhild; Textor, Christian; Bernhardt, Günther; Dove, Stefan; Buschauer, Armin

    2011-09-01

    Bacterial hyaluronan lyases (Hyal) degrade hyaluronan, an important component of the extracellular matrix, and are involved in microbial spread. Hyal inhibitors may serve as tools to study the role of the enzyme, its substrates and products in the course of bacterial infections. Moreover, such enzyme inhibitors are potential candidates for antibacterial combination therapy. Based on crystal structures of Streptococcus pneumoniae Hyal in complex with a hexasaccharide substrate and with different inhibitors, 1-acylated benzimidazole-2-thiones and benzoxazole-2-thiones were derived as new leads for the inhibition of Streptococcus agalactiae strain 4755 Hyal. Structure-based optimization led to N-(3-phenylpropionyl)benzoxazole-2-thione, one of the most potent compounds known to date (IC(50) values: 24 μM at pH 7.4, 15 μM at pH 5). Among the 27 new derivatives, other N-acylated benzimidazoles and benzoxazoles are just as active at pH 7.4, but not at pH 5. The results support a binding mode characterized by interactions with residues in the catalytic site and with a hydrophobic patch.

  15. A high-throughput scintillation proximity assay for sphingosine-1-phosphate lyase.

    Science.gov (United States)

    Kashem, Mohammed A; Wa, Chunling; Wolak, John P; Grafos, Nicholas S; Ryan, Kelli R; Sanville-Ross, Mary L; Fogarty, Kylie E; Rybina, Irina V; Shoultz, Alycia; Molinaro, Teresa; Desai, Sudha N; Rajan, Anusha; Huber, John D; Nelson, Richard M

    2014-06-01

    The emergence of sphingosine-1-phosphate lyase (SPL) as a promising therapeutic target for inflammatory diseases has heightened interest in the identification of small molecules that modulate its activity. The enzymatic activity of SPL is typically measured using radiometric or fluorescence-based assays that require a lipid extraction step, or by direct quantitation of reaction products using mass spectrometry (MS). To facilitate testing large numbers of compounds to identify SPL modulators, we developed a robust scintillation proximity assay (SPA) that is compatible with high-throughput screening (HTS). This assay employs recombinant human full-length SPL in insect cell membrane preparations to catalyze the conversion of biotinylated aminosphingosine-1-[(33)P]phosphate (S1(33)P-biotin) to trans-2-hexadecenal-biotin and ethanolamine [(33)P]phosphate. To validate the SPA and confirm the fidelity of its measurement of SPL enzyme activity, we developed a Rapid-Fire MS method that quantitates nonradiolabeled S1P-biotin. In addition, we developed a simple, scalable method to produce S1(33)P-biotin in quantities sufficient for HTS. The optimized SPA screen in 384-well microplates produced a mean plate-wise Z'-statistic of 0.58 across approximately 3,000 plates and identified several distinct structural classes of SPL inhibitor. Among the inhibitors that the screen identified was one compound with an IC50 of 1.6 μM in the SPA that induced dose-dependent lymphopenia in mice.

  16. Revised domain structure of ulvan lyase and characterization of the first ulvan binding domain

    Science.gov (United States)

    Melcher, Rebecca L. J.; Neumann, Marten; Fuenzalida Werner, Juan Pablo; Gröhn, Franziska; Moerschbacher, Bruno M.

    2017-01-01

    Biomass waste products from green algae have recently been given new life, as these polysaccharides have potential applications in industry, agriculture, and medicine. One such polysaccharide group called ulvans displays many different, potentially useful properties that arise from their structural versatility. Hence, performing structural analyses on ulvan is crucial for future applications. However, chemical reaction–based analysis methods cannot fully characterize ulvan and tend to alter its structure. Thus, better methods require well-characterized ulvan-degrading enzymes. Therefore, we analysed a previously sequenced ulvan lyase (GenebankTM reference number JN104480) and characterized its domains. We suggest that the enzyme consists of a shorter than previously described catalytic domain, a newly identified substrate binding domain, and a C-terminal type 9 secretion system signal peptide. By separately expressing the two domains in E. coli, we confirmed that the binding domain is ulvan specific, having higher affinity for ulvan than most lectins for their ligands (affinity constant: 105 M−1). To our knowledge, this is the first description of an ulvan-binding domain. Overall, identifying this new binding domain is one step towards engineering ulvan enzymes that can be used to characterize ulvan, e.g. through enzymatic/mass spectrometric fingerprinting analyses, and help unlock its full potential. PMID:28327560

  17. Engineered Citrobacter freundii methionine γ-lyase effectively produces antimicrobial thiosulfinates.

    Science.gov (United States)

    Morozova, Elena A; Kulikova, Vitalia V; Rodionov, Alexei N; Revtovich, Svetlana V; Anufrieva, Natalya V; Demidkina, Tatyana V

    2016-01-01

    Antimicrobial activity of thiosulfinates in situ produced by mixtures of Citrobacter freundii methionine γ-lyase (MGL) with new substrates, l-methionine and S-(alkyl/allyl)-l-cysteine sulfoxides has been recently demonstrated (Anufrieva et al., 2015). This opens a way to the rational design of a new biotechnologically relevant antimicrobial drug producer. To increase the efficiency of the enzyme toward sulfoxides, the mutant forms of MGL, with the replacements of active site cysteine 115 with alanine (C115A MGL) and histidine (C115H MGL) were obtained. The replacement of cysteine 115 by histidine results in the loss of activity of the mutant enzyme in the γ-elimination reaction of physiological substrate, whereas the activity in the β-elimination reaction of characteristic substrates persists. However, the catalytic efficiency of C115H MGL in the β-elimination reaction of S-substituted l-cysteine sulfoxides is increased by about an order of magnitude compared to the wild type MGL. The antibacterial activity of C115H MGL mixtures with a number of sulfoxides was assessed against Gram-positive and Gram-negative bacteria. The bacteriostatic effect was more pronounced against Gram-positive than against Gram-negative bacteria, while antibacterial potential proved to be quite similar. Thus, the mutant enzyme C115H MGL is an effective catalyst, in particular, for decomposition of sulfoxides and the pharmacological couples of the mutant form with sulfoxides might be new antimicrobial agents.

  18. 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.

  19. Immunolocalization of phenylalanine ammonia-lyase and cinnamate-4-hydroxylase in differentiating xylem of poplar.

    Science.gov (United States)

    Sato, Takahiko; Takabe, Keiji; Fujita, Minoru

    2004-01-01

    Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) and cinnamate-4-hydroxylase (C4H; EC 1.14.13.11) are pivotal enzymes involved in lignification. We synthesized peptides as the epitopes according to the amino acid sequences of these enzymes, coupled them with hemocyanin, and injected them into mice. The antiserums against peptides of PAL and C4H specifically detected PAL and C4H in the crude enzymes extracted from differentiating xylem of poplar, respectively. PAL and C4H were localized in differentiating xylem of poplar. PAL labeling was mainly localized in the cytosol, and somewhat localized on the rough-endoplasmic reticulum (r-ER) and the Golgi apparatus. In contrast, C4H was mainly observed on r-ER and the Golgi apparatus. These findings suggest that conversion of phenylalanine to cinnamic acid occurs in the cytosol and the following reaction occurs near the membrane of r-ER and the Golgi apparatus. The possibility of coordinated localization of PAL and C4H is discussed.

  20. Mutational analysis of phenylalanine ammonia lyase to improve reactions rates for various substrates.

    Science.gov (United States)

    Bartsch, Sebastian; Bornscheuer, Uwe T

    2010-12-01

    Phenylalanine ammonia lyases (PAL) catalyze the reversible, non-reductive amination of trans-cinnamic acid to l-phenylalanine in the presence of high ammonia concentrations. Since neither cofactor recycling nor other additives are needed and by this asymmetric synthesis theoretical yields of 100% can be reached, it is an interesting reaction for industrial processes. In this study we demonstrate the superior properties of p-nitro-cinnamic acid (p-n-CA) in the amination reaction using the PAL from Petroselinum crispum (pcPAL). By focused-directed evolution, three mutants were identified showing increased reaction rates and decreased substrate inhibition. Together, the F137V mutant with p-n-CA showed a 15-fold increased reaction rate compared with the pcPAL WT with the natural cinnamic acid. The high reaction rates were also proven in preparative scale experiments. Activities towards other p-substituted cinnamic acids showing different electronic effects of the substituent were analyzed. Focused-directed evolution around the carboxylic acid- and amine-binding site always decreased PAL activity, due to a sensitive H-bond network.

  1. Arsenic Demethylation by a C·As Lyase in Cyanobacterium Nostoc sp. PCC 7120.

    Science.gov (United States)

    Yan, Yu; Ye, Jun; Xue, Xi-Mei; Zhu, Yong-Guan

    2015-12-15

    Arsenic, a ubiquitous toxic substance, exists mainly as inorganic forms in the environment. It is perceived that organoarsenicals can be demethylated and degraded into inorganic arsenic by microorganisms. Few studies have focused on the mechanism of arsenic demethylation in bacteria. Here, we investigated arsenic demethylation in a typical freshwater cyanobacterium Nostoc sp. PCC 7120. This bacterium was able to demethylate monomethylarsenite [MAs(III)] rapidly to arsenite [As(III)] and also had the ability to demethylate monomethylarsenate [MAs(V)] to As(III). The NsarsI encoding a C·As lyase responsible for MAs(III) demethylation was cloned from Nostoc sp. PCC 7120 and heterologously expressed in an As-hypersensitive strain Escherichia coli AW3110 (ΔarsRBC). Expression of NsarsI was shown to confer MAs(III) resistance through arsenic demethylation. The purified NsArsI was further identified and functionally characterized in vitro. NsArsI existed mainly as the trimeric state, and the kinetic data were well-fit to the Hill equation with K0.5 = 7.55 ± 0.33 μM for MAs(III), Vmax = 0.79 ± 0.02 μM min(-1), and h = 2.7. Both of the NsArsI truncated derivatives lacking the C-terminal 10 residues (ArsI10) or 23 residues (ArsI23) had a reduced ability of MAs(III) demethylation. These results provide new insights for understanding the important role of cyanobacteria in arsenic biogeochemical cycling in the environment.

  2. Phycoerythrin-specific bilin lyase-isomerase controls blue-green chromatic acclimation in marine Synechococcus.

    Science.gov (United States)

    Shukla, Animesh; Biswas, Avijit; Blot, Nicolas; Partensky, Frédéric; Karty, Jonathan A; Hammad, Loubna A; Garczarek, Laurence; Gutu, Andrian; Schluchter, Wendy M; Kehoe, David M

    2012-12-04

    The marine cyanobacterium Synechococcus is the second most abundant phytoplanktonic organism in the world's oceans. The ubiquity of this genus is in large part due to its use of a diverse set of photosynthetic light-harvesting pigments called phycobiliproteins, which allow it to efficiently exploit a wide range of light colors. Here we uncover a pivotal molecular mechanism underpinning a widespread response among marine Synechococcus cells known as "type IV chromatic acclimation" (CA4). During this process, the pigmentation of the two main phycobiliproteins of this organism, phycoerythrins I and II, is reversibly modified to match changes in the ambient light color so as to maximize photon capture for photosynthesis. CA4 involves the replacement of three molecules of the green light-absorbing chromophore phycoerythrobilin with an equivalent number of the blue light-absorbing chromophore phycourobilin when cells are shifted from green to blue light, and the reverse after a shift from blue to green light. We have identified and characterized MpeZ, an enzyme critical for CA4 in marine Synechococcus. MpeZ attaches phycoerythrobilin to cysteine-83 of the α-subunit of phycoerythrin II and isomerizes it to phycourobilin. mpeZ RNA is six times more abundant in blue light, suggesting that its proper regulation is critical for CA4. Furthermore, mpeZ mutants fail to normally acclimate in blue light. These findings provide insights into the molecular mechanisms controlling an ecologically important photosynthetic process and identify a unique class of phycoerythrin lyase/isomerases, which will further expand the already widespread use of phycoerythrin in biotechnology and cell biology applications.

  3. Olive Recombinant Hydroperoxide Lyase, an Efficient Biocatalyst for Synthesis of Green Leaf Volatiles.

    Science.gov (United States)

    Jacopini, Sabrina; Mariani, Magali; de Caraffa, Virginie Brunini-Bronzini; Gambotti, Claude; Vincenti, Sophie; Desjobert, Jean-Marie; Muselli, Alain; Costa, Jean; Berti, Liliane; Maury, Jacques

    2016-06-01

    Volatile C6-aldehydes are the main contributors to the characteristic odor of plants known as "green note" and are widely used by the flavor industry. Biotechnological processes were developed to fulfill the high demand in C6-aldehydes in natural flavorants and odorants. Recombinant hydroperoxide lyases (HPLs) constitute an interesting alternative to overcome drawbacks arising from the use of HPL from plant extracts. Thus, olive recombinant 13-HPL was assayed as biocatalysts to produce C6-aldehydes. Firstly, a cDNA encoding for olive HPL of Leccino variety was isolated and cloned in pQE-30 expression vector. In order to improve the enzyme solubility, its chloroplast transit peptide was deleted. Both enzymes (HPL wild type and HPL deleted) were expressed into Escherichia coli strain M15, purified, characterized, and then used for bioconversion of 13-hydroperoxides of linoleic and linolenic acids. Aldehydes produced were extracted, then identified and quantified using gas chromatography and mass spectrometry. Recombinant HPL wild type (HPLwt) allowed producing 5.61 mM of hexanal and 4.39 mM of 3Z-hexenal, corresponding to high conversion yields of 93.5 and 73 %, respectively. Using HPL deleted (HPLdel) instead of HPLwt failed to obtain greater quantities of hexanal or 3Z-hexenal. No undesirable products were formed, and no isomerization of 3Z-hexenal in 2E-hexenal occurred. The olive recombinant HPLwt appears to be a promising efficient biocatalyst for the production of C6-aldehydes.

  4. Inhibition of Arabidopsis O-acetylserine(thiol)lyase A1 by tyrosine nitration.

    Science.gov (United States)

    Alvarez, Consolación; Lozano-Juste, Jorge; Romero, Luís C; García, Irene; Gotor, Cecilia; León, José

    2011-01-07

    The last step of sulfur assimilation is catalyzed by O-acetylserine(thiol)lyase (OASTL) enzymes. OASTLs are encoded by a multigene family in the model plant Arabidopsis thaliana. Cytosolic OASA1 enzyme is the main source of OASTL activity and thus crucial for cysteine homeostasis. We found that nitrating conditions after exposure to peroxynitrite strongly inhibited OASTL activity. Among OASTLs, OASA1 was markedly sensitive to nitration as demonstrated by the comparative analysis of OASTL activity in nitrated crude protein extracts from wild type and different oastl mutants. Furthermore, nitration assays on purified recombinant OASA1 protein led to 90% reduction of the activity due to inhibition of the enzyme, as no degradation of the protein occurred under these conditions. The reduced activity was due to nitration of the protein because selective scavenging of peroxynitrite with epicatechin impaired OASA1 nitration and the concomitant inhibition of OASTL activity. Inhibition of OASA1 activity upon nitration correlated with the identification of a modified OASA1 protein containing 3-nitroTyr(302) residue. The essential role of the Tyr(302) residue for the catalytic activity was further demonstrated by the loss of OASTL activity of a Y302A-mutated version of OASA1. Inhibition caused by Tyr(302) nitration on OASA1 activity seems to be due to a drastically reduced O-acetylserine substrate binding to the nitrated protein, and also to reduced stabilization of the pyridoxal-5'-phosphate cofactor through hydrogen bonds. This is the first report identifying a Tyr nitration site of a plant protein with functional effect and the first post-translational modification identified in OASA1 enzyme.

  5. Selectivity of commonly used pharmacological inhibitors for cystathionine β synthase (CBS) and cystathionine γ lyase (CSE).

    Science.gov (United States)

    Asimakopoulou, Antonia; Panopoulos, Panagiotis; Chasapis, Christos T; Coletta, Ciro; Zhou, Zongmin; Cirino, Giuseppe; Giannis, Athanassios; Szabo, Csaba; Spyroulias, Georgios A; Papapetropoulos, Andreas

    2013-06-01

    Hydrogen sulfide (H₂S) is a signalling molecule that belongs to the gasotransmitter family. Two major sources for endogenous enzymatic production of H₂S are cystathionine β synthase (CBS) and cystathionine γ lyase (CSE). In the present study, we examined the selectivity of commonly used pharmacological inhibitors of H₂S biosynthesis towards CSE and CBS. To address this question, human CSE or CBS enzymes were expressed and purified from Escherichia coli as fusion proteins with GSH-S-transferase. After purification, the activity of the recombinant enzymes was tested using the methylene blue method. β-Cyanoalanine (BCA) was more potent in inhibiting CSE than propargylglycine (PAG) (IC₅₀ 14 ± 0.2 μM vs. 40 ± 8 μM respectively). Similar to PAG, L-aminoethoxyvinylglycine (AVG) only inhibited CSE, but did so at much lower concentrations. On the other hand, aminooxyacetic acid (AOAA), a frequently used CBS inhibitor, was more potent in inhibiting CSE compared with BCA and PAG (IC₅₀ 1.1 ± 0.1 μM); the IC₅₀ for AOAA for inhibiting CBS was 8.5 ± 0.7 μM. In line with our biochemical observations, relaxation to L-cysteine was blocked by AOAA in aortic rings that lacked CBS expression. Trifluoroalanine and hydroxylamine, two compounds that have also been used to block H₂S biosynthesis, blocked the activity of CBS and CSE. Trifluoroalanine had a fourfold lower IC₅₀ for CBS versus CSE, while hydroxylamine was 60-fold more selective against CSE. In conclusion, although PAG, AVG and BCA exhibit selectivity in inhibiting CSE versus CBS, no selective pharmacological CBS inhibitor is currently available. © 2013 The Authors. British Journal of Pharmacology © 2013 The British Pharmacological Society.

  6. 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.

  7. 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.

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

    Science.gov (United States)

    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)-specific base catalyst and abstracts the 3S-proton from l-threo-3-methylaspartate, resulting in an enolate anion intermediate. This enolic intermediate is stabilized by coordination to the essential active site Mg(2+) ion and hydrogen bonding to the Gln-329 residue. Collapse of this intermediate results in the release of ammonia and the formation of mesaconate. His-194 likely acts as the (R)-specific base catalyst and abstracts the 3R-proton from the l-erythro isomer of 3-methylaspartate, yielding the enolic intermediate. In the present study, we have investigated the importance of the residues Gln-73, Phe-170, Gln-172, Tyr-356, Thr-360, Cys-361 and Leu-384 for the catalytic activity of C. tetanomorphum MAL. These residues, which are part of the enzyme surface lining the substrate binding pocket, were subjected to site-directed mutagenesis and the mutant enzymes were characterized for their structural integrity, ability to catalyze the amination of mesaconate, and regio- and diastereoselectivity. Based on the observed properties of the mutant enzymes, combined with previous structural studies and protein engineering work, we propose a detailed catalytic mechanism for the MAL-catalyzed reaction, in which the side chains of Gln-73, Gln-172, Tyr-356, Thr-360, and Leu-384 provide favorable interactions with the substrate, which are important for substrate binding and activation. This detailed knowledge of the catalytic mechanism of MAL can serve as a guide for future protein engineering experiments.

  9. The role of active site tyrosine 58 in Citrobacter freundii methionine γ-lyase.

    Science.gov (United States)

    Anufrieva, Natalya V; Faleev, Nicolai G; Morozova, Elena A; Bazhulina, Natalia P; Revtovich, Svetlana V; Timofeev, Vladimir P; Tkachev, Yaroslav V; Nikulin, Alexei D; Demidkina, Tatyana V

    2015-09-01

    In the spatial structure of methionine γ-lyase (MGL, EC 4.4.1.11) from Citrobacter freundii, Tyr58 is located at H-bonding distance to the oxygen atom of the phosphate "handle" of pyridoxal 5'-phosphate (PLP). It was replaced for phenylalanine by site-directed mutagenesis. The X-ray structure of the mutant enzyme was determined at 1.96Å resolution. Comparison of spatial structures and absorption spectra of wild-type and mutant holoenzymes demonstrated that the replacement did not result in essential changes of the conformation of the active site Tyr58Phe MGL. The Kd value of PLP for Tyr58Phe MGL proved to be comparable to the Kd value for the wild-type enzyme. The replacement led to a decrease of catalytic efficiencies in both γ- and β-elimination reactions of about two orders of magnitude as compared to those for the wild-type enzyme. The rates of exchange of C-α- and C-β- protons of inhibitors in D2O catalyzed by the mutant form are comparable with those for the wild-type enzyme. Spectral data on the complexes of the mutant form with the substrates and inhibitors showed that the replacement led to a change of rate the limiting step of the physiological reaction. The results allowed us to conclude that Tyr58 is involved in an optimal positioning of the active site Lys210 at some stages of γ- and β-elimination reactions. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

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

    Directory of Open Access Journals (Sweden)

    Salas, Joaquín J.

    2000-06-01

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

  11. ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae.

    Science.gov (United States)

    Rodriguez, Sarah; Denby, Charles M; Van Vu, T; Baidoo, Edward E K; Wang, George; Keasling, Jay D

    2016-03-03

    With increasing concern about the environmental impact of a petroleum based economy, focus has shifted towards greener production strategies including metabolic engineering of microbes for the conversion of plant-based feedstocks to second generation biofuels and industrial chemicals. Saccharomyces cerevisiae is an attractive host for this purpose as it has been extensively engineered for production of various fuels and chemicals. Many of the target molecules are derived from the central metabolite and molecular building block, acetyl-CoA. To date, it has been difficult to engineer S. cerevisiae to continuously convert sugars present in biomass-based feedstocks to acetyl-CoA derived products due to intrinsic physiological constraints-in respiring cells, the precursor pyruvate is directed away from the endogenous cytosolic acetyl-CoA biosynthesis pathway towards the mitochondria, and in fermenting cells pyruvate is directed towards the byproduct ethanol. In this study we incorporated an alternative mode of acetyl-CoA biosynthesis mediated by ATP citrate lyase (ACL) that may obviate such constraints. We characterized the activity of several heterologously expressed ACLs in crude cell lysates, and found that ACL from Aspergillus nidulans demonstrated the highest activity. We employed a push/pull strategy to shunt citrate towards ACL by deletion of the mitochondrial NAD(+)-dependent isocitrate dehydrogenase (IDH1) and engineering higher flux through the upper mevalonate pathway. We demonstrated that combining the two modifications increases accumulation of mevalonate pathway intermediates, and that both modifications are required to substantially increase production. Finally, we incorporated a block strategy by replacing the native ERG12 (mevalonate kinase) promoter with the copper-repressible CTR3 promoter to maximize accumulation of the commercially important molecule mevalonate. By combining the push/pull/block strategies, we significantly improved mevalonate

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

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    Andreas Billich

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

  13. SGPL1 (sphingosine phosphate lyase 1) modulates neuronal autophagy via phosphatidylethanolamine production.

    Science.gov (United States)

    Mitroi, Daniel N; Karunakaran, Indulekha; Gräler, Markus; Saba, Julie D; Ehninger, Dan; Ledesma, María Dolores; van Echten-Deckert, Gerhild

    2017-05-04

    Macroautophagy/autophagy defects have been identified as critical factors underlying the pathogenesis of neurodegenerative diseases. The roles of the bioactive signaling lipid sphingosine-1-phosphate (S1P) and its catabolic enzyme SGPL1/SPL (sphingosine phosphate lyase 1) in autophagy are increasingly recognized. Here we provide in vitro and in vivo evidence for a previously unidentified route through which SGPL1 modulates autophagy in neurons. SGPL1 cleaves S1P into ethanolamine phosphate, which is directed toward the synthesis of phosphatidylethanolamine (PE) that anchors LC3-I to phagophore membranes in the form of LC3-II. In the brains of SGPL1(fl/fl/Nes) mice with developmental neural specific SGPL1 ablation, we observed significantly reduced PE levels. Accordingly, alterations in basal and stimulated autophagy involving decreased conversion of LC3-I to LC3-II and increased BECN1/Beclin-1 and SQSTM1/p62 levels were apparent. Alterations were also noticed in downstream events of the autophagic-lysosomal pathway such as increased levels of lysosomal markers and aggregate-prone proteins such as APP (amyloid β [A4] precursor protein) and SNCA/α-synuclein. In vivo profound deficits in cognitive skills were observed. Genetic and pharmacological inhibition of SGPL1 in cultured neurons promoted these alterations, whereas addition of PE was sufficient to restore LC3-I to LC3-II conversion, and control levels of SQSTM1, APP and SNCA. Electron and immunofluorescence microscopy showed accumulation of unclosed phagophore-like structures, reduction of autolysosomes and altered distribution of LC3 in SGPL1(fl/fl/Nes) brains. Experiments using EGFP-mRFP-LC3 provided further support for blockage of the autophagic flux at initiation stages upon SGPL1 deficiency due to PE paucity. These results emphasize a formerly overlooked direct role of SGPL1 in neuronal autophagy and assume significance in the context that autophagy modulators hold an enormous therapeutic potential in the

  14. PhnJ – A novel radical SAM enzyme from the C–P lyase complex

    Directory of Open Access Journals (Sweden)

    Siddhesh S. Kamat

    2015-03-01

    Full Text Available PhnJ from the C–P lyase complex catalyzes the cleavage of the carbon–phosphorus bond in ribose-1-phosphonate-5-phosphate (PRPn to produce methane and ribose-1,2-cyclic-phosphate-5-phosphate (PRcP. This protein is a novel radical SAM enzyme that uses glycyl and thiyl radicals as reactive intermediates in the proposed reaction mechanism. The overall reaction is initiated with the reductive cleavage of S-adenosylmethionine (SAM by a reduced [4Fe–4S]1+-cluster to form an Ado-CH2∙ radical intermediate. This intermediate abstracts the proR hydrogen from Gly-32 of PhnJ to form Ado-CH3 and a glycyl radical. In the next step, there is hydrogen atom transfer from Cys-272 to the Gly-32 radical to generate a thiyl radical. The thiyl radical attacks the phosphorus center of the substrate, PRPn, to form a transient thiophosphonate radical intermediate. This intermediate collapses via homolytic C–P bond cleavage and hydrogen atom transfer from the proS hydrogen of Gly-32 to produce a thiophosphate intermediate, methane, and a radical intermediate at Gly-32. The final product, PRcP, is formed by nucleophilic attack of the C2-hydroxyl on the transient thiophosphate intermediate. This reaction regenerates the free thiol group of Cys-272. After hydrogen atom transfer from Cys-272 to the Gly-32 radical, the entire process is repeated with another substrate molecule without the use of another molecule of SAM or involvement from the [4Fe–4S]-cluster again.

  15. Formulation and PEGylation optimization of the therapeutic PEGylated phenylalanine ammonia lyase for the treatment of phenylketonuria

    Science.gov (United States)

    Bell, Sean M.; Wendt, Dan J.; Zhang, Yanhong; Long, Shinong; Tsuruda, Laurie; Zhao, Bin; Laipis, Phillip; Fitzpatrick, Paul A.

    2017-01-01

    Phenylketonuria (PKU) is a genetic metabolic disease in which the decrease or loss of phenylalanine hydroxylase (PAH) activity results in elevated, neurotoxic levels of phenylalanine (Phe). Due to many obstacles, PAH enzyme replacement therapy is not currently an option. Treatment of PKU with an alternative enzyme, phenylalanine ammonia lyase (PAL), was first proposed in the 1970s. However, issues regarding immunogenicity, enzyme production and mode of delivery needed to be overcome. Through the evaluation of PAL enzymes from multiple species, three potential PAL enzymes from yeast and cyanobacteria were chosen for evaluation of their therapeutic potential. The addition of polyethylene glycol (PEG, MW = 20,000), at a particular ratio to modify the protein surface, attenuated immunogenicity in an animal model of PKU. All three PEGylated PAL candidates showed efficacy in a mouse model of PKU (BTBR Pahenu2) upon subcutaneous injection. However, only PEGylated Anabaena variabilis (Av) PAL-treated mice demonstrated sustained low Phe levels with weekly injection and was the only PAL evaluated that maintained full enzymatic activity upon PEGylation. A PEGylated recombinant double mutant version of AvPAL (Cys503Ser/Cys565Ser), rAvPAL-PEG, was selected for drug development based on its positive pharmacodynamic profile and favorable expression titers. PEGylation was shown to be critical for rAvPAL-PEG efficacy as under PEGylated rAvPAL had a lower pharmacodynamic effect. rAvPAL and rAvPAL-PEG had poor stability at 4°C. L-Phe and trans-cinnamate were identified as activity stabilizing excipients. rAvPAL-PEG is currently in Phase 3 clinical trials to assess efficacy in PKU patients. PMID:28282402

  16. A carbon-nitrogen lyase from Leucaena leucocephala catalyzes the first step of mimosine degradation.

    Science.gov (United States)

    Negi, Vishal Singh; Bingham, Jon-Paul; Li, Qing X; Borthakur, Dulal

    2014-02-01

    The tree legume Leucaena leucocephala contains a large amount of a toxic nonprotein aromatic amino acid, mimosine, and also an enzyme, mimosinase, for mimosine degradation. In this study, we isolated a 1,520-bp complementary DNA (cDNA) for mimosinase from L. leucocephala and characterized the encoded enzyme for mimosine-degrading activity. The deduced amino acid sequence of the coding region of the cDNA was predicted to have a chloroplast transit peptide. The nucleotide sequence, excluding the sequence for the chloroplast transit peptide, was codon optimized and expressed in Escherichia coli. The purified recombinant enzyme was used in mimosine degradation assays, and the chromatogram of the major product was found to be identical to that of 3-hydroxy-4-pyridone (3H4P), which was further verified by electrospray ionization-tandem mass spectrometry. The enzyme activity requires pyridoxal 5'-phosphate but not α-keto acid; therefore, the enzyme is not an aminotransferase. In addition to 3H4P, we also identified pyruvate and ammonia as other degradation products. The dependence of the enzyme on pyridoxal 5'-phosphate and the production of 3H4P with the release of ammonia indicate that it is a carbon-nitrogen lyase. It was found to be highly efficient and specific in catalyzing mimosine degradation, with apparent Km and Vmax values of 1.16×10(-4) m and 5.05×10(-5) mol s(-1) mg(-1), respectively. The presence of other aromatic amino acids, including l-tyrosine, l-phenylalanine, and l-tryptophan, in the reaction did not show any competitive inhibition. The isolation of the mimosinase cDNA and the biochemical characterization of the recombinant enzyme will be useful in developing transgenic L. leucocephala with reduced mimosine content in the future.

  17. Transcriptional Regulation of Cystathionine-γ-Lyase in Endothelial Cells by NADPH Oxidase 4-Dependent Signaling*

    Science.gov (United States)

    Mistry, Rajesh K.; Murray, Thomas V. A.; Prysyazhna, Oleksandra; Martin, Daniel; Burgoyne, Joseph R.; Santos, Celio; Eaton, Philip; Shah, Ajay M.; Brewer, Alison C.

    2016-01-01

    The gasotransmitter, hydrogen sulfide (H2S) is recognized as an important mediator of endothelial cell homeostasis and function that impacts upon vascular tone and blood pressure. Cystathionine-γ-lyase (CSE) is the predominant endothelial generator of H2S, and recent evidence suggests that its transcriptional expression is regulated by the reactive oxygen species, H2O2. However, the cellular source of H2O2 and the redox-dependent molecular signaling pathway that modulates this is not known. We aimed to investigate the role of Nox4, an endothelial generator of H2O2, in the regulation of CSE in endothelial cells. Both gain- and loss-of-function experiments in human endothelial cells in vitro demonstrated Nox4 to be a positive regulator of CSE transcription and protein expression. We demonstrate that this is dependent upon a heme-regulated inhibitor kinase/eIF2α/activating transcription factor 4 (ATF4) signaling module. ATF4 was further demonstrated to bind directly to cis-regulatory sequences within the first intron of CSE to activate transcription. Furthermore, CSE expression was also increased in cardiac microvascular endothelial cells, isolated from endothelial-specific Nox4 transgenic mice, compared with wild-type littermate controls. Using wire myography we demonstrate that endothelial-specific Nox4 transgenic mice exhibit a hypo-contractile phenotype in response to phenylephrine that was abolished when vessels were incubated with a CSE inhibitor, propargylglycine. We, therefore, conclude that Nox4 is a positive transcriptional regulator of CSE in endothelial cells and propose that it may in turn contribute to the regulation of vascular tone via the modulation of H2S production. PMID:26620565

  18. 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.

  19. INFLUENCE OF COBALT IONS ON ENZYME ACTIVTY OF ISOCITRIATE LYASE AND ITS REGULATION IN CONDITION OF SEED GERMINATION OF GLYCINE MAX L.

    Directory of Open Access Journals (Sweden)

    Chechui O. F.

    2012-12-01

    Full Text Available 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. We fixed the increase in the activity of isocitrate lyase under influence of cobalt ions occurs by means of enzyme induction on third day of experiment while maintaining performance of enzyme activity on the fifth day; one of the reasons caused the increased activity of the key enzyme of the glyoxylate cycle under the influence of cobalt ions can be increasing of the concentration of lipid peroxidation. In addition, during experiments with usage of actinomycin D we determined the increasing of activity ofisocitrate lyase under the influence of cobalt ions by enzyme induction.

  20. 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.

  1. Effects of pectin lyase-modified red ginseng extracts in high-fat diet-fed obese mice

    OpenAIRE

    Lee, Hak-Yong; Park, Kwang-Hyun; PARK, Young-mi; Moon, Dae-In; Oh, Hong-Geun; Kwon, Dae-Young; Yang, Hye-Jeong; Kim, Okjin; Kim, Dong-Woo; Yoo, Ji-Hyun; Hong, Se-Chul; Lee, Kun-Hee; Seol, Su-Yeon; Park, Yong-Sik; Park, Jong-Dae

    2014-01-01

    Red ginseng and its extracts have been used as traditional medicines and functional foods in countries worldwide. The aim of this study was to examine the bioavailability of pectin lyase-modified red ginseng extracts (GS-E3D), and the effects of GS-E3D on adipogenesis of 3T3-L1 adipocytes, as well as on metabolic disorders such as hyperglycemia, dyslipidemia, and fatty liver in high-fat diet fed obese C57BL/6 mice. Mice were divided into 5 groups: normal diet group, high fat diet-vehicle grou...

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    similar to that of the tRNase Z endonucleases but lacks the long exosite module used by these enzymes to bind their tRNA substrates. The active site of PhnP contains what are probably two Mn2+ ions surrounded by an array of active site residues that are identical to those observed in the tRNase Z enzymes......Carbon-phosphorus lyase is a multienzyme system encoded by the phn operon that enables bacteria to metabolize organophosphonates when the preferred nutrient, inorganic phosphate, is scarce. One of the enzymes encoded by this operon, PhnP, is predicted by sequence homology to be a metal...

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

    Directory of Open Access Journals (Sweden)

    Kobkiat Saengnil

    2011-11-01

    Full Text Available 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 unexposed side. It is concluded that sunlight increases red colour development of the mango exocarp by inducing PAL activity. Exposure to sunlight also enhances endogenous sugar accumulation in mango fruit.

  4. Elicitor modulation of the turnover of L-phenylalanine ammonia-lyase in French bean cell suspension cultures.

    Science.gov (United States)

    Lawton, M A; Dixon, R A; Lamb, C J

    1980-12-01

    (1) The mechanisms underlying the transient increase in phenylalanine ammonia-lyase activity during phaseollin accumulation in cell suspension cultures of Dwarf French bean (Phaseolus volgaris) have been investigated using density labelling with 3H from 2H2O coupled with residual analysis of the equilibrium distribution of enzyme activity in high-resolution KBr density gradients. (2) The resolution achieved in this system is sufficient to allow quantitative analysis of the relative proportions of light, unlabelled, pre-existing enzyme and heavy, labelled, newly synthesised enzyme. (3) Elicitor released by heat treatment of cell walls of Colletotrichum lindemuthianum, the causal agent of anthracnose disease of French bean, caused a marked but transient increase in phenylalanine ammonia-lyase activity concomitant with the onset of phaseollin accumulation in the bean cultures. The induction of enzyme activity was highly dependent on elicitor concentration, with maximum induction occurring in two discrete concentration ranges; at an intermediate elicitor concentration, or at supra-optimal elicitor concentrations, no enzyme induction was observed. (4) At low concentrations of elicitor the induction of enzyme was entirely a result of elicitor stimulation of the rate of de novo enzyme production. In contrast, at higher elicitor concentrations the increase in enzyme activity was accompanied by a marked apparent stabilization of the enzyme in vivo, and the rapid but transient increase in enzyme activity was achieved by a programme of reciprocal changes in the rate constant for de novo enzyme production and the rate constant for removal of enzyme activity. Such reciprocal control of the rates of enzyme production and removal may be crucial in determining the magnitude and duration of the phytoalexin defense response. (5) Information on the specific activity of 2H label in the amino acid pools was obtained from analysis of the equilibrium distribution of residual, labelled

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

    Directory of Open Access Journals (Sweden)

    Nyalwidhe Julius

    2008-05-01

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

  6. Regulation of phosphoenolpyruvate carboxykinase gene transcription by insulin and cAMP: reciprocal actions on initiation and elongation.

    OpenAIRE

    1988-01-01

    Nuclei isolated from H4IIE rat hepatoma cells were used in an in vitro run-on assay, with probes directed against various regions of the phosphoenolpyruvate carboxykinase [GTP: oxaloacetate carboxy-lyase (transphosphorylating); EC 4.1.1.32] gene, to analyze whether transcription proceeds uniformly across this gene in response to insulin and cAMP treatment. Fewer polymerase II complexes were associated with the phosphoenolpyruvate carboxykinase gene after insulin treatment, as compared with cA...

  7. A mutation in the cytosolic O-acetylserine (thiol lyase induces a genome-dependent early leaf death phenotype in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Schippers Jos HM

    2010-04-01

    Full Text Available Abstract Background Cysteine is a component in organic compounds including glutathione that have been implicated in the adaptation of plants to stresses. O-acetylserine (thiol lyase (OAS-TL catalyses the final step of cysteine biosynthesis. OAS-TL enzyme isoforms are localised in the cytoplasm, the plastids and mitochondria but the contribution of individual OAS-TL isoforms to plant sulphur metabolism has not yet been fully clarified. Results The seedling lethal phenotype of the Arabidopsis onset of leaf death3-1 (old3-1 mutant is due to a point mutation in the OAS-A1 gene, encoding the cytosolic OAS-TL. The mutation causes a single amino acid substitution from Gly162 to Glu162, abolishing old3-1 OAS-TL activity in vitro. The old3-1 mutation segregates as a monogenic semi-dominant trait when backcrossed to its wild type accession Landsberg erecta (Ler-0 and the Di-2 accession. Consistent with its semi-dominant behaviour, wild type Ler-0 plants transformed with the mutated old3-1 gene, displayed the early leaf death phenotype. However, the old3-1 mutation segregates in an 11:4:1 (wild type: semi-dominant: mutant ratio when backcrossed to the Colombia-0 and Wassilewskija accessions. Thus, the early leaf death phenotype depends on two semi-dominant loci. The second locus that determines the old3-1 early leaf death phenotype is referred to as odd-ler (for old3 determinant in the Ler accession and is located on chromosome 3. The early leaf death phenotype is temperature dependent and is associated with increased expression of defence-response and oxidative-stress marker genes. Independent of the presence of the odd-ler gene, OAS-A1 is involved in maintaining sulphur and thiol levels and is required for resistance against cadmium stress. Conclusions The cytosolic OAS-TL is involved in maintaining organic sulphur levels. The old3-1 mutation causes genome-dependent and independent phenotypes and uncovers a novel function for the mutated OAS-TL in cell

  8. RegA Plays a Key Role in Oxygen-Dependent Establishment of Persistence and in Isocitrate Lyase Activity, a Critical Determinant of In vivo Brucella suis Pathogenicity

    Directory of Open Access Journals (Sweden)

    Elias Abdou

    2017-05-01

    Full Text Available For aerobic human pathogens, adaptation to hypoxia is a critical factor for the establishment of persistent infections, as oxygen availability is low inside the host. The two-component system RegB/A of Brucella suis plays a central role in the control of respiratory systems adapted to oxygen deficiency, and in persistence in vivo. Using an original “in vitro model of persistence” consisting in gradual oxygen depletion, we compared transcriptomes and proteomes of wild-type and ΔregA strains to identify the RegA-regulon potentially involved in the set-up of persistence. Consecutive to oxygen consumption resulting in growth arrest, 12% of the genes in B. suis were potentially controlled directly or indirectly by RegA, among which numerous transcriptional regulators were up-regulated. In contrast, genes or proteins involved in envelope biogenesis and in cellular division were repressed, suggesting a possible role for RegA in the set-up of a non-proliferative persistence state. Importantly, the greatest number of the RegA-repressed genes and proteins, including aceA encoding the functional IsoCitrate Lyase (ICL, were involved in energy production. A potential consequence of this RegA impact may be the slowing-down of the central metabolism as B. suis progressively enters into persistence. Moreover, ICL is an essential determinant of pathogenesis and long-term interactions with the host, as demonstrated by the strict dependence of B. suis on ICL activity for multiplication and persistence during in vivo infection. RegA regulates gene or protein expression of all functional groups, which is why RegA is a key regulator of B. suis in adaptation to oxygen depletion. This function may contribute to the constraint of bacterial growth, typical of chronic infection. Oxygen-dependent activation of two-component systems that control persistence regulons, shared by several aerobic human pathogens, has not been studied in Brucella sp. before. This work

  9. Salmonella type III effector SpvC, a phosphothreonine lyase, contributes to reduction in inflammatory response during intestinal phase of infection.

    Science.gov (United States)

    Haneda, Takeshi; Ishii, Yuta; Shimizu, Hiromichi; Ohshima, Keiko; Iida, Naoyuki; Danbara, Hirofumi; Okada, Nobuhiko

    2012-04-01

    Salmonella phosphothreonine lyase SpvC inactivates the dual-phosphorylated host mitogen-activated protein kinases (MAPK) through β-elimination. While SpvC can be secreted in vitro by both Salmonella pathogenicity island (SPI)-1 and SPI-2 type III secretion systems (T3SSs), translocation of this protein into the host cell cytosol has only been demonstrated by SPI-2 T3SS. In this study, we show that SpvC can be delivered into the host cell cytoplasm by both SPI-1 and SPI-2 T3SSs. Dephosphorylation of the extracellular signal-regulated protein kinases (ERK) was detected in an SPI-1 T3SS-dependent manner 2 h post infection. Using a mouse model for Salmonella enterocolitis, which was treated with streptomycin prior to infection, we observed that mice infected with Salmonella enterica serovar Typhimurium strains lacking the spvC gene showed pronounced colitis when compared with mice infected with the wild-type strain 1 day after infection. The effect of SpvC on the development of colitis was characterized by reduced mRNA levels of the pro-inflammatory cytokines and chemokines, and reduced inflammation with less infiltration of neutrophils. Furthermore, the reduction in inflammation by SpvC resulted in increased bacterial dissemination in spleen of mice infected with Salmonella. Collectively, our findings suggest that SpvC exerts as an anti-inflammatory effector and the attenuation of intestinal inflammatory response by SpvC is involved in systemic infection of Salmonella.

  10. Successful fertilization requires the presence of at least one major O-acetylserine(thiol)lyase for cysteine synthesis in pollen of Arabidopsis.

    Science.gov (United States)

    Birke, Hannah; Heeg, Corinna; Wirtz, Markus; Hell, Rüdiger

    2013-10-01

    The synthesis of cysteine (Cys) is a master control switch of plant primary metabolism that coordinates the flux of sulfur with carbon and nitrogen metabolism. In Arabidopsis (Arabidopsis thaliana), nine genes encode for O-acetylserine(thiol)lyase (OAS-TL)-like proteins, of which the major isoforms, OAS-TL A, OAS-TL B, and OAS-TL C, catalyze the formation of Cys by combining O-acetylserine and sulfide in the cytosol, the plastids, and the mitochondria, respectively. So far, the significance of individual OAS-TL-like enzymes is unresolved. Generation of all major OAS-TL double loss-of-function mutants in combination with radiolabeled tracer studies revealed that subcellular localization of OAS-TL proteins is more important for efficient Cys synthesis than total cellular OAS-TL activity in leaves. The absence of oastl triple embryos after targeted crosses indicated the exclusiveness of Cys synthesis by the three major OAS-TLs and ruled out alternative sulfur fixation by other OAS-TL-like proteins. Analyses of oastlABC pollen demonstrated that the presence of at least one functional OAS-TL isoform is essential for the proper function of the male gametophyte, although the synthesis of histidine, lysine, and tryptophan is dispensable in pollen. Comparisons of oastlABC pollen derived from genetically different parent plant combinations allowed us to separate distinct functions of Cys and glutathione in pollen and revealed an additional role of glutathione for pollen germination. In contrast, female gametogenesis was not affected by the absence of major OAS-TLs, indicating significant transport of Cys into the developing ovule from the mother plant.

  11. Identification of an L-methionine γ-lyase involved in the production of hydrogen sulfide from L-cysteine in Fusobacterium nucleatum subsp. nucleatum ATCC 25586.

    Science.gov (United States)

    Suwabe, Kyosuke; Yoshida, Yasuo; Nagano, Keiji; Yoshimura, Fuminobu

    2011-10-01

    Fusobacterium nucleatum produces an abundance of hydrogen sulfide (H(2)S) in the oral cavity that is mediated by several enzymes. The identification and characterization of three distinct enzymes (Fn0625, Fn1055 and Fn1220) in F. nucleatum that catalyse the production of H(2)S from l-cysteine have been reported. In the current study, a novel enzyme involved in the production of H(2)S in F. nucleatum ATCC 25586, whose molecular mass had been estimated to be approximately 130 kDa, was identified by two-dimensional electrophoresis combined with MALDI-TOF MS. The enzyme, Fn1419, has previously been characterized as an l-methionine γ-lyase. SDS-PAGE and gel-filtration chromatography indicated that Fn1419 has a molecular mass of 43 kDa and forms tetramers in solution. Unlike other enzymes associated with H(2)S production in F. nucleatum, the quaternary structure of Fn1419 was not completely disrupted by exposure to SDS. The purified recombinant enzyme exhibited a K(m) of 0.32±0.02 mM and a k(cat) of 0.69±0.01 s(-1). Based on current and published data, the enzymic activity for H(2)S production from l-cysteine in F. nucleatum is ranked as follows: Fn1220>Fn1055>Fn1419>Fn0625. Based on kinetic values and relative mRNA levels of the respective genes, as determined by real-time quantitative PCR, the amount of H(2)S produced by Fn1419 was estimated to be 1.9 % of the total H(2)S produced from l-cysteine in F. nucleatum ATCC 25586. In comparison, Fn1220 appeared to contribute significantly to H(2)S production (87.6 %).

  12. Exploration of structure-function relationships in Escherichia coli cystathionine γ-synthase and cystathionine β-lyase via chimeric constructs and site-specific substitutions.

    Science.gov (United States)

    Manders, Adrienne L; Jaworski, Allison F; Ahmed, Mohammed; Aitken, Susan M

    2013-06-01

    Cystathionine γ-synthase (CGS) and cystathionine β-lyase (CBL) share a common structure and several active-site residues, but catalyze distinct side-chain rearrangements in the two-step transsulfuration pathway that converts cysteine to homocysteine, the precursor of methionine. A series of 12 chimeric variants of Escherichia coli CGS (eCGS) and CBL (eCBL) was constructed to probe the roles of two structurally distinct, ~25-residue segments situated in proximity to the amino and carboxy termini and located at the entrance of the active-site. In vivo complementation of methionine-auxotrophic E. coli strains, lacking the genes encoding eCGS and eCBL, demonstrated that exchange of the targeted regions impairs the activity of the resulting enzymes, but does not produce a corresponding interchange of reaction specificity. In keeping with the in vivo results, the catalytic efficiency of the native reactions is reduced by at least 95-fold, and α,β versus α,γ-elimination specificity is not modified. The midpoint of thermal denaturation monitored by circular dichroism, ranges between 59 and 80°C, compared to 66°C for the two wild-type enzymes, indicating that the chimeric enzymes adopt a stable folded structure and that the observed reductions in catalytic efficiency are due to reorganization of the active site. Alanine-substitution variants of residues S32 and S33, as well as K42 of eCBL, situated in proximity to and within, respectively, the targeted amino-terminal region were also investigated to explore their role as determinants of reaction specificity via positioning of key active-site residues. The catalytic efficiency of the S32A, S33A and the K42A site-directed variants of eCBL is reduced by less than 10-fold, demonstrating that, while these residues may participate in positioning S339, which tethers the catalytic base, their role is minor.

  13. Regulation of a phenylalanine ammonia lyase (BbPAL) by calmodulin in response to environmental changes in the entomopathogenic fungus Beauveria bassiana.

    Science.gov (United States)

    Kim, Jiyoung; Park, Hyesung; Han, Jae-Gu; Oh, Junsang; Choi, Hyung-Kyoon; Kim, Seong Hwan; Sung, Gi-Ho

    2015-11-01

    Phenylalanine ammonia lyase (PAL, E.C. 4.3.1.5) catalyses the deamination of L -phenylalanine to trans-cinnamic acid and ammonia, facilitating a critical step in the phenylpropanoid pathway that produces a variety of secondary metabolites. In this study, we isolated BbPAL gene in the entomopathogenic fungus Beauveria bassiana. According to multiple sequence alignment, homology modelling and in vitro PAL activity, we demonstrated that BbPAL acts as a typical PAL enzyme in B. bassiana. BbPAL interacted with calmodulin (CaM) in vitro and in vivo, indicating that BbPAL is a novel CaM-binding protein. The functional role of CaM in BbPAL action was to negatively regulate the BbPAL activity in B. bassiana. High-performance liquid chromatography analysis revealed that L -phenylalanine was reduced and trans-cinnamic acid was increased in response to the CaM inhibitor W-7. Dark conditions suppressed BbPAL activity in B. bassiana, compared with light. In addition, heat and cold stresses inhibited BbPAL activity in B. bassiana. Interestingly, these negative effects of BbPAL activity by dark, heat and cold conditions were recovered by W-7 treatment, suggesting that the inhibitory mechanism is mediated through stimulation of CaM activity. Therefore, this work suggests that BbPAL plays a role in the phenylpropanoid pathway mediated by environmental stimuli via the CaM signalling pathway.

  14. A phenylalanine ammonia-lyase ortholog (PkPAL1) from Picrorhiza kurrooa Royle ex. Benth: molecular cloning, promoter analysis and response to biotic and abiotic elicitors.

    Science.gov (United States)

    Bhat, Wajid Waheed; Razdan, Sumeer; Rana, Satiander; Dhar, Niha; Wani, Tariq Ahmad; Qazi, Parvaiz; Vishwakarma, Ram; Lattoo, Surrinder K

    2014-09-01

    Picrorhiza kurrooa Royle ex Benth. is a highly reputed medicinal herb utilised in the preparation of a number of herbal drug formulations, principally due to the presence of novel monoterpene iridoid glycosides kenned as picrosides. Phenylalanine ammonia-lyase catalyses an important rate-limiting step in phenylpropanoid pathway and supplies precursors like cinnamic acid, vanillic acid, ferulic acid, etc., to a variety of secondary metabolites including picrosides. The imperilled status of P. ku