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Sample records for coli phosphoribosyl diphosphate

  1. Binding of divalent magnesium by Escherichia coli phosphoribosyl diphosphate synthetase

    DEFF Research Database (Denmark)

    Willemoës, Martin; Hove-Jensen, Bjarne

    1997-01-01

    The mechanism of binding of the substrates Mg x ATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-D-ribosyl (alpha-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, alpha,beta-methylene ATP ...

  2. Binding of Divalent Magnesium by Escherichia coli Phosphoribosyl Diphosphate Synthetase

    DEFF Research Database (Denmark)

    Willemoës, Martin; Hove-Jensen, Bjarne

    1997-01-01

    The mechanism of binding of the substrates MgATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-d-ribosyl a-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, a,ß-methylene ATP and (+)-1-a,2-a,3...... of substrates and products indicated a role of Mg2+ in preparing the active site of phosphoribosyl diphosphate synthetase for binding of the highly phosphorylated ligands MgATP and phosphoribosyl diphosphate, as evaluated by analysis of the effects of the inhibitors adenosine and ribose 1,5-bisphosphate....... Calcium ions, which inhibit the enzyme even in the presence of high concentrations of Mg2+, appeared to compete with free Mg2+ for binding to its activator site on the enzyme. Analysis of the inhibition of Mg2+ binding by MgADP indicated that MgADP binding to the allosteric site may occur in competition...

  3. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    2004-01-01

    The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS...

  4. Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichia coli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose-5-phosphate binding

    DEFF Research Database (Denmark)

    Willemoës, Martin; Nilsson, Dan; Hove-Jensen, Bjarne

    1996-01-01

    The three conserved aspartic acid residues of the 5-phospho-d-ribosyl a-1-diphosphate binding site (213-GRDCVLVDDMIDTGGT-228) of Escherichia coli phosphoribosyl diphosphate synthetase were studied by analysis of the mutant enzymes D220E, D220F, D221A, D224A, and D224S. The mutant enzymes showed...... an increase in KM for ribose 5-phosphate in the presence of at least one of the divalent metal ions Mg2+, Mn2+, Co2+, or Cd2+, with the most dramatic changes revealed by the D220E and D220F enzymes in the presence of Co2+ and the D221A enzyme in the presence of Mn2+ or Co2+. The D220F and D221A enzymes both...... showed large decreases in Vapp in the presence of the various divalent metal ions, except for the D221A enzyme in the presence of Co2+. Vapp of the D220E enzyme was similar to that of the wild-type enzyme in the presence of Mg2+, Mn2+, or Cd2+, whereas the Vapp was increased in the presence of Co2+. Vapp...

  5. Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichia coli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose 5-phosphate binding

    DEFF Research Database (Denmark)

    Willemoës, Martin; Nilsson, Dan; Hove-Jensen, Bjarne

    1996-01-01

    The three conserved aspartic acid residues of the 5-phospho-D-ribosyl α-1-diphosphate binding site (213-GRDCVLVDDMIDTGGT-228) of Escherichia coli phosphoribosyl diphosphate synthetase were studied by analysis of the mutant enzymes D220E, D220F, D221A, D224A, and D224S. The mutant enzymes showed...... an increase in KM for ribose 5-phosphate in the presence of at least one of the divalent metal ions Mg2+, Mn2+, Co2+, or Cd2+, with the most dramatic changes revealed by the D220E and D220F enzymes in the presence of Co2+ and the D221A enzyme in the presence of Mn2+ or Co2+. The D220F and D221A enzymes both...... showed large decreases in Vapp in the presence of the various divalent metal ions, except for the D221A enzyme in the presence of Co2+. Vapp of the D220E enzyme was similar to that of the wild-type enzyme in the presence of Mg2+, Mn2+, or Cd2+, whereas the Vapp was increased in the presence of Co2+. Vapp...

  6. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae: combinatorial expression of the five PRS genes in Escherichia coli.

    Science.gov (United States)

    Hove-Jensen, Bjarne

    2004-09-24

    The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS gene product had no PRPP synthase activity. In contrast, expression of five pairwise combinations of PRS genes resulted in the formation of active PRPP synthase. These combinations were PRS1 PRS2, PRS1 PRS3, and PRS1 PRS4, as well as PRS5 PRS2 and PRS5 PRS4. None of the remaining five possible pairwise combinations of PRS genes appeared to produce active enzyme. Extract of an E. coli strain containing a plasmid-borne PRS1 gene and a chromosome-borne PRS3 gene contained detectable PRPP synthase activity, whereas extracts of strains containing PRS1 PRS2, PRS1 PRS4, PRS5 PRS2, or PRS5 PRS4 contained no detectable PRPP synthase activity. In contrast PRPP could be detected in growing cells containing PRS1 PRS2, PRS1 PRS3, PRS5 PRS2, or PRS5 PRS4. These apparent conflicting results indicate that, apart from the PRS1 PRS3-specified enzyme, PRS-specified enzyme is functional in vivo but unstable when released from the cell. Certain combinations of three PRS genes appeared to produce an enzyme that is stable in vitro. Thus, extracts of strains harboring PRS1 PRS2 PRS5, PRS1 PRS4 PRS5, or PRS2 PRS4 PRS5 as well as extracts of strains harboring combinations with PRS1 PRS3 contained readily assayable PRPP synthase activity. The data indicate that although certain pairwise combinations of subunits produce an active enzyme, yeast PRPP synthase requires at least three different subunits to be stable in vitro. The activity of PRPP synthases containing subunits 1 and 3 or subunits 1, 2, and 5 was found to be dependent on Pi, to be temperature-sensitive, and inhibited by ADP.

  7. Bacillus caldolyticus prs gene encoding phosphoribosyl-diphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-1-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  8. Purification and properties of phosphoribosyl-diphosphate synthetase from Bacillus subtilis

    DEFF Research Database (Denmark)

    Arnvig, Kirsten; Hove-Jensen, Bjarne; Switzer, Robert L.

    1990-01-01

    Phosphoribosyl-diphosphate (PPRibP) synthetase from Bacillus subtiliis has been purified to near homogeneity from an Escherichia coli Δprs strain bearing the cloned B. subtilis prs gene, encoding PPRibP synthentase, on a plasmid. The Mr of the subunit (34,000) and its amino-terminal amino acid...

  9. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase

    DEFF Research Database (Denmark)

    Andersen, Rune W.; Lo Leggio, Leila; Hove-Jensen, Bjarne

    2015-01-01

    The enzyme 5-phosphoribosyl-1-α-diphosphate (PRPP) synthase (EC 2.7.6.1) catalyses the Mg2+-dependent transfer of a diphosphoryl group from ATP to the C1 hydroxyl group of ribose 5-phosphate resulting in the production of PRPP and AMP. A nucleotide sequence specifying Sulfolobus solfataricus PRPP...... PRPP synthase as a search model. The two amino acid sequences share 35 % identity. The resulting asymmetric unit consists of three separated dimers. The protein was co-crystallised in the presence of AMP and ribose 5-phosphate, but in the electron density map of the active site only AMP and a sulphate...

  10. Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Andersen, Kasper R; Kilstrup, Mogens;

    2017-01-01

    is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP......Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds...... synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of the five diphosphoryltransferases are compared, as are those of selected enzymes of diphosphoryl transfer, phosphoryl transfer, and nucleotidyl transfer reactions. PRPP is used as a substrate by a large number...

  11. Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance.

    Science.gov (United States)

    Hove-Jensen, Bjarne; Andersen, Kasper R; Kilstrup, Mogens; Martinussen, Jan; Switzer, Robert L; Willemoës, Martin

    2017-03-01

    Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds. PRPP is utilized in the biosynthesis of purine and pyrimidine nucleotides, the amino acids histidine and tryptophan, the cofactors NAD and tetrahydromethanopterin, arabinosyl monophosphodecaprenol, and certain aminoglycoside antibiotics. The participation of PRPP in each of these metabolic pathways is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of the five diphosphoryltransferases are compared, as are those of selected enzymes of diphosphoryl transfer, phosphoryl transfer, and nucleotidyl transfer reactions. PRPP is used as a substrate by a large number phosphoribosyltransferases. The protein structures and reaction mechanisms of these phosphoribosyltransferases vary and demonstrate the versatility of PRPP as an intermediate in cellular physiology. PRPP synthases appear to have originated from a phosphoribosyltransferase during evolution, as demonstrated by phylogenetic analysis. PRPP, furthermore, is an effector molecule of purine and pyrimidine nucleotide biosynthesis, either by binding to PurR or PyrR regulatory proteins or as an allosteric activator of carbamoylphosphate synthetase. Genetic analyses have disclosed a number of mutants altered in the PRPP synthase-specifying genes in humans as well as bacterial species.

  12. Novel class III phosphoribosyl diphosphate synthase: structure and properties of the tetrameric, phosphate-activated, non-allosterically inhibited enzyme from Methanocaldococcus jannaschii

    DEFF Research Database (Denmark)

    Kadziola, Anders; Jepsen, Clemens H; Johansson, Eva;

    2005-01-01

    The prs gene encoding phosphoribosyl diphosphate (PRPP) synthase of the hyperthermophilic autotrophic methanogenic archaeon Methanocaldococcus jannaschii has been cloned and expressed in Escherichia coli. Subsequently, M.jannaschii PRPP synthase has been purified, characterised, crystallised, and....... The properties of M.jannaschii PRPP synthase differ widely from previously characterised PRPP synthases by its tetrameric quaternary structure and the simultaneous phosphate ion-activation and lack of allosteric inhibition, and, thus, constitute a novel class of PRPP synthases....

  13. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase: a bent dimer defining the adenine specificity of the substrate ATP.

    Science.gov (United States)

    Andersen, Rune W; Leggio, Leila Lo; Hove-Jensen, Bjarne; Kadziola, Anders

    2015-03-01

    The enzyme 5-phosphoribosyl-1-α-diphosphate (PRPP) synthase (EC 2.7.6.1) catalyses the Mg(2+)-dependent transfer of a diphosphoryl group from ATP to the C1 hydroxyl group of ribose 5-phosphate resulting in the production of PRPP and AMP. A nucleotide sequence specifying Sulfolobus solfataricus PRPP synthase was synthesised in vitro with optimised codon usage for expression in Escherichia coli. Following expression of the gene in E. coli PRPP synthase was purified by heat treatment and ammonium sulphate precipitation and the structure of S. solfataricus PRPP synthase was determined at 2.8 Å resolution. A bent dimer oligomerisation was revealed, which seems to be an abundant feature among PRPP synthases for defining the adenine specificity of the substrate ATP. Molecular replacement was used to determine the S. solfataricus PRPP synthase structure with a monomer subunit of Methanocaldococcus jannaschii PRPP synthase as a search model. The two amino acid sequences share 35 % identity. The resulting asymmetric unit consists of three separated dimers. The protein was co-crystallised in the presence of AMP and ribose 5-phosphate, but in the electron density map of the active site only AMP and a sulphate ion were observed. Sulphate ion, reminiscent of the ammonium sulphate precipitation step of the purification, seems to bind tightly and, therefore, presumably occupies and blocks the ribose 5-phosphate binding site. The activity of S. solfataricus PRPP synthase is independent of phosphate ion.

  14. Surface exposed amino acid differences between mesophilic and thermophilic phosphoribosyl diphosphate synthase

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; McGuire, James N

    2004-01-01

    The amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the thermophile Bacillus caldolyticus is 81% identical to the amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the mesophile Bacillus subtilis. Nevertheless the enzyme from the two organisms...... competitive with respect to ATP. A predicted structure of the B. caldolyticus enzyme based on homology modelling with the structure of B. subtilis 5-phospho-alpha-D-ribosyl 1-diphosphate synthase shows 92% of the amino acid differences to be on solvent exposed surfaces in the hexameric structure....

  15. Crystallization and preliminary X-ray diffraction study of phosphoribosyl pyrophosphate synthetase from E. Coli

    Energy Technology Data Exchange (ETDEWEB)

    Timofeev, V. I., E-mail: inna@ns.crys.ras.ru; Abramchik, Yu. A., E-mail: tostars@mail.ru; Zhukhlistova, N. E., E-mail: ugama@yandex.ru; Kuranova, I. P. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

    2015-09-15

    Enzymes of the phosphoribosyl pyrophosphate synthetase family (PRPPS, EC 2.7.6.1) catalyze the formation of 5-phosphoribosyl pyrophosphate (5-PRPP) from adenosine triphosphate and ribose 5-phosphate. 5-Phosphoribosyl pyrophosphate is an important intermediate in the synthesis of purine, pyrimidine, and pyridine nucleotides, as well as of the amino acids histidine and tryptophan. The crystallization conditions for E. coli PRPPS were found by the vapor-diffusion technique and were optimized to apply the capillary counter-diffusion technique. The X-ray diffraction data set was collected from the crystals grown by the counter-diffusion technique using a synchrotron radiation source to 3.1-Å resolution. The crystals of PRPPS belong to sp. gr. P6{sub 3}22 and have the following unit-cell parameters: a = b = 104.44 Å, c = 124.98 Å, α = β = 90°, γ = 120°. The collected X-ray diffraction data set is suitable for the solution of the three-dimensional structure of PRPPS at 3.1-Å resolution.

  16. Crystallization and preliminary X-ray diffraction study of phosphoribosyl pyrophosphate synthetase from E. Coli

    Science.gov (United States)

    Timofeev, V. I.; Abramchik, Yu. A.; Zhukhlistova, N. E.; Kuranova, I. P.

    2015-09-01

    Enzymes of the phosphoribosyl pyrophosphate synthetase family (PRPPS, EC 2.7.6.1) catalyze the formation of 5-phosphoribosyl pyrophosphate (5-PRPP) from adenosine triphosphate and ribose 5-phosphate. 5-Phosphoribosyl pyrophosphate is an important intermediate in the synthesis of purine, pyrimidine, and pyridine nucleotides, as well as of the amino acids histidine and tryptophan. The crystallization conditions for E. coli PRPPS were found by the vapor-diffusion technique and were optimized to apply the capillary counter-diffusion technique. The X-ray diffraction data set was collected from the crystals grown by the counter-diffusion technique using a synchrotron radiation source to 3.1-Å resolution. The crystals of PRPPS belong to sp. gr. P6322 and have the following unit-cell parameters: a = b = 104.44 Å, c = 124.98 Å, α = β = 90°, γ = 120°. The collected X-ray diffraction data set is suitable for the solution of the three-dimensional structure of PRPPS at 3.1-Å resolution.

  17. Tricistronic operon expression of the genes gcaD (tms), which encodes N-acetylglucosamine 1-phosphate uridyltransferase, prs, which encodes phosphoribosyl diphosphate synthetase, and ctc in vegetative cells of Bacillus subtilis

    DEFF Research Database (Denmark)

    Hilden, Ida; Krath, Britta N.; Hove-Jensen, Bjarne

    1995-01-01

    The gcaD, prs, and ctc genes were shown to be organized as a tricistronic operon. The transcription of the prs gene, measured as phosphoribosyl diphosphate synthetase activity, and of the ctc gene, measured as β-galactosidase activity specified by a ctc-lacZ protein fusion, were dependent...

  18. Implications of secondary structure prediction and amino acid sequence comparison of class I and class II phosphoribosyl diphosphate synthases on catalysis, regulation, and quaternary structure

    DEFF Research Database (Denmark)

    Krath, B N; Hove-Jensen, B

    2001-01-01

    Spinach 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) synthase isozyme 4 was synthesized in Escherichia coli and purified to near homogeneity. The activity of the enzyme is independent of P(i); it is inhibited by ADP in a competitive manner, indicating a lack of an allosteric site; and it accepts...

  19. Three-dimensional structure of phosphoribosyl pyrophosphate synthetase from E. coli at 2.71 Å resolution

    Energy Technology Data Exchange (ETDEWEB)

    Timofeev, V. I., E-mail: inna@ns.crys.ras.ru, E-mail: tostars@mail.ru, E-mail: ugama@yandex.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Abramchik, Yu. A. [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Zhukhlistova, N. E. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Muravieva, T. I.; Esipov, R. S. [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Kuranova, I. P. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

    2016-01-15

    Phosphoribosyl pyrophosphate synthetase from Escherichia coli was cloned, purified, and crystallized. Single crystals of the enzyme were grown under microgravity. The X-ray diffraction data set was collected at the Spring-8 synchrotron facility and used to determine the three-dimensional structure of the enzyme by the molecular-replacement method at 2.71 Å resolution. The active and regulatory sites in the molecule of E. coli phosphoribosyl pyrophosphate synthetase were revealed by comparison with the homologous protein from Bacillus subtilis, the structure of which was determined in a complex with functional ligands. The conformations of polypeptide-chain fragments surrounding and composing the active and regulatory sites were shown to be identical in both proteins.

  20. Three-dimensional structure of phosphoribosyl pyrophosphate synthetase from E. coli at 2.71 Å resolution

    Science.gov (United States)

    Timofeev, V. I.; Abramchik, Yu. A.; Zhukhlistova, N. E.; Muravieva, T. I.; Esipov, R. S.; Kuranova, I. P.

    2016-01-01

    Phosphoribosyl pyrophosphate synthetase from Escherichia coli was cloned, purified, and crystallized. Single crystals of the enzyme were grown under microgravity. The X-ray diffraction data set was collected at the Spring-8 synchrotron facility and used to determine the three-dimensional structure of the enzyme by the molecular-replacement method at 2.71 Å resolution. The active and regulatory sites in the molecule of E. coli phosphoribosyl pyrophosphate synthetase were revealed by comparison with the homologous protein from Bacillus subtilis, the structure of which was determined in a complex with functional ligands. The conformations of polypeptide-chain fragments surrounding and composing the active and regulatory sites were shown to be identical in both proteins.

  1. Adenosine diphosphate sugar pyrophosphatase prevents glycogen biosynthesis in Escherichia coli

    Science.gov (United States)

    Moreno-Bruna, Beatriz; Baroja-Fernández, Edurne; Muñoz, Francisco José; Bastarrica-Berasategui, Ainara; Zandueta-Criado, Aitor; Rodríguez-López, Milagros; Lasa, Iñigo; Akazawa, Takashi; Pozueta-Romero, Javier

    2001-01-01

    An adenosine diphosphate sugar pyrophosphatase (ASPPase, EC 3.6.1.21) has been characterized by using Escherichia coli. This enzyme, whose activities in the cell are inversely correlated with the intracellular glycogen content and the glucose concentration in the culture medium, hydrolyzes ADP-glucose, the precursor molecule of glycogen biosynthesis. ASPPase was purified to apparent homogeneity (over 3,000-fold), and sequence analyses revealed that it is a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated as “nudix” hydrolases. Insertional mutagenesis experiments leading to the inactivation of the ASPPase encoding gene, aspP, produced cells with marginally low enzymatic activities and higher glycogen content than wild-type bacteria. aspP was cloned into an expression vector and introduced into E. coli. Transformed cells were shown to contain a dramatically reduced amount of glycogen, as compared with the untransformed bacteria. No pleiotropic changes in the bacterial growth occurred in both the aspP-overexpressing and aspP-deficient strains. The overall results pinpoint the reaction catalyzed by ASPPase as a potential step of regulating glycogen biosynthesis in E. coli. PMID:11416161

  2. Steady state kinetic model for the binding of substrates and allosteric effectors to Escherichia coli phosphoribosyl-diphosphate synthase

    DEFF Research Database (Denmark)

    Willemoës, Martin; Hove-Jensen, Bjarne; Larsen, Sine

    2000-01-01

    saturation with ribose 5-phosphate leads to the binding of Mg2+ and substrates via a slow pathway where Pi binds to the enzyme last. The random mechanism for Pi binding was further supported by studies with competitive inhibitors of Mg2+, MgATP, and ribose 5-phosphate that all appeared noncompetitive when...... varying Pi at either saturating or unsaturating ribose 5-phosphate concentrations. Furthermore, none of the inhibitors induced inhibition at increasing Pi concentrations. Results from ADP inhibition of Pi activation suggest that these effectors compete for binding to a common regulatory site....

  3. Tricistronic operon expression of the genes gcaD (tms), which encodes N-acetylglucosamine 1-phosphate uridyltransferase, prs, which encodes phosphoribosyl diphosphate synthetase, and ctc in vegetative cells of Bacillus subtilis

    DEFF Research Database (Denmark)

    Hilden, Ida; Krath, Britta N.; Hove-Jensen, Bjarne

    1995-01-01

    The gcaD, prs, and ctc genes were shown to be organized as a tricistronic operon. The transcription of the prs gene, measured as phosphoribosyl diphosphate synthetase activity, and of the ctc gene, measured as β-galactosidase activity specified by a ctc-lacZ protein fusion, were dependent...... on the promoter in front of the gcaD gene. Analysis of cDNA molecules prepared with gcaD-prs-ctc-specified mRNA as the template revealed an RNA transcript that encompassed all three cistrons....

  4. Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichia coli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose 5-phosphate binding

    DEFF Research Database (Denmark)

    Willemoës, Martin; Nilsson, Dan; Hove-Jensen, Bjarne

    1996-01-01

    an increase in KM for ribose 5-phosphate in the presence of at least one of the divalent metal ions Mg2+, Mn2+, Co2+, or Cd2+, with the most dramatic changes revealed by the D220E and D220F enzymes in the presence of Co2+ and the D221A enzyme in the presence of Mn2+ or Co2+. The D220F and D221A enzymes both...... showed large decreases in Vapp in the presence of the various divalent metal ions, except for the D221A enzyme in the presence of Co2+. Vapp of the D220E enzyme was similar to that of the wild-type enzyme in the presence of Mg2+, Mn2+, or Cd2+, whereas the Vapp was increased in the presence of Co2+. Vapp...... enzymes were dependent on the metal ion present, suggesting a function of the investigated aspartic acid residues both in the binding of ribose 5-phosphate, possibly via a divalent metal ion, and in the interaction with a divalent metal ion during catalysis...

  5. Products of the inactivation of ribonucleoside diphosphate reductase from Escherichia coli with 2'-azido-2'-deoxyuridine 5'-diphosphate

    Energy Technology Data Exchange (ETDEWEB)

    Salowe, S.P.; Ator, M.A.; Stubbe, J.A.

    1987-06-16

    Ribonucleoside diphosphate reductase (RDPR) from Escherichia coli was completely inactivated by 1 equiv of the mechanism-based inhibitor 2'-azido-2'-azido-2'-deoxyuridine 5'-diphosphate (N/sub 3/UDP). Incubation of RDPR with (3'-/sup 3/H)N/sub 3/UDP resulted in 0.2 mol of /sup 3/H released to solvent per mole of enzyme inactivated, indicating that cleavage of the 3' carbon-hydrogen bond occurred in the reaction. Incubation of RDPR with (..beta..-/sup 32/P)N/sub 3/UDP resulted in stoichiometric production of inorganic pyrophosphate. One equivalent of uracil was eliminated from N/sub 3/UDP, but no azide release was detected. Analysis of the reaction of RDPR with (/sup 15/N/sub 3/)N/sub 3/UDP by mass spectrometry revealed that the azide moiety was converted to 0.9 mol of nitrogen gas per mole of enzyme inactivated. The tyrosyl radical of the B2 subunit was destroyed during the inactivation by N/sub 3/UDP as reported previously, while the specific activity of the B1 subunit was reduced by half. Incubation of (5'-/sup 3/H)N/sub 3/UDP with RDPR resulted in stoichiometric covalent radiolabeling of the enzyme. Separation of the enzyme's subunits by chromatofocusing revealed that the modification was specific for the B1 subunit.

  6. Thiamine diphosphate binds to intermediates in the assembly of adenovirus fiber knob trimers in Escherichia coli.

    Science.gov (United States)

    Schulz, Ryan; Zhang, Yian-Biao; Liu, Chang-Jun; Freimuth, Paul

    2007-12-01

    Assembly of the adenovirus (Ad) homotrimeric fiber protein is nucleated by its C-terminal knob domain, which itself can trimerize when expressed as a recombinant protein fragment. The non-interlocked, globular structure of subunits in the knob trimer implies that trimers assemble from prefolded monomers through a dimer intermediate, but these intermediates have not been observed and the mechanism of assembly therefore remains uncharacterized. Here we report that expression of the Ad serotype 2 (Ad2) knob was toxic for thi- strains of Escherichia coli, which are defective in de novo synthesis of thiamine (vitamin B1). Ad2 knob trimers isolated from a thi+ strain copurified through multiple chromatography steps with a small molecule of mass equivalent to that of thiamine diphosphate (ThDP). Mutant analysis did not implicate any specific site for ThDP binding. Our results suggest that ThDP may associate with assembly intermediates and become trapped in assembled trimers, possibly within one of several large cavities that are partially solvent-accessible or buried completely within the trimer interior.

  7. Thiamine diphosphate adenylyl transferase from E. coli: functional characterization of the enzyme synthesizing adenosine thiamine triphosphate

    Directory of Open Access Journals (Sweden)

    Brans Alain

    2007-08-01

    Full Text Available Abstract Background We have recently identified a new thiamine derivative, adenosine thiamine triphosphate (AThTP, in E. coli. In intact bacteria, this nucleotide is synthesized only in the absence of a metabolizable carbon source and quickly disappears as soon as the cells receive a carbon source such as glucose. Thus, we hypothesized that AThTP may be a signal produced in response to carbon starvation. Results Here we show that, in bacterial extracts, the biosynthesis of AThTP is carried out from thiamine diphosphate (ThDP and ADP or ATP by a soluble high molecular mass nucleotidyl transferase. We partially purified this enzyme and characterized some of its functional properties. The enzyme activity had an absolute requirement for divalent metal ions, such as Mn2+ or Mg2+, as well as for a heat-stable soluble activator present in bacterial extracts. The enzyme has a pH optimum of 6.5–7.0 and a high Km for ThDP (5 mM, suggesting that, in vivo, the rate of AThTP synthesis is proportional to the free ThDP concentration. When ADP was used as the variable substrate at a fixed ThDP concentration, a sigmoid curve was obtained, with a Hill coefficient of 2.1 and an S0.5 value of 0.08 mM. The specificity of the AThTP synthesizing enzyme with respect to nucleotide substrate is restricted to ATP/ADP, and only ThDP can serve as the second substrate of the reaction. We tentatively named this enzyme ThDP adenylyl transferase (EC 2.7.7.65. Conclusion This is the first demonstration of an enzyme activity transferring a nucleotidyl group on thiamine diphosphate to produce AThTP. The existence of a mechanism for the enzymatic synthesis of this compound is in agreement with the hypothesis of a non-cofactor role for thiamine derivatives in living cells.

  8. Effect of guanosine 5'-diphosphate 3'-diphosphate and related nucleoside polyphosphates on induction of tryptophanase and beta-galactosidase in permeabilized cells of Escherichia coli.

    Science.gov (United States)

    Yoshimoto, A; Oki, T; Inui, T

    1978-10-04

    Exogenous addition of guanosine and adenosine 5'-(mono, di and tri) phosphate 3'-diphosphates (pppGpp, ppGpp, pGpp, pppApp, ppApp and pApp) stimulated the synthesis of tryptophanase and beta-galactosidase in permeabilized cells of Escherichia coli. From the results obtained with ppGpp and pppApp, this effect appeared to be at a transcriptional level and depended greatly on the growth condition; the largest effect was observed in cells under shiftdown or grown on poor enrgy source. ppGpp and pppApp, unlike cyclic AMP, did not act to overcome the inhibition of enzyme induction by glucose, but in combination with cyclic AMP caused a synergistic stimulation effect. In the shiftdown cells, ppGpp and pppApp gave 30% or more stimulation effect on tryptophanase induction while cyclic AMP did not stimulate induction. There was therefore a pronounced difference between cyclic AMP and ppGpp or pppApp in stimulatory function.

  9. The defective phosphoribosyl diphosphate synthase in a temperature-sensitive prs-2 mutant of Escherichia coli is compensated by increased enzyme synthesis

    DEFF Research Database (Denmark)

    Post, David A.; Switzer, Robert L.; Hove-Jensen, Bjarne

    1996-01-01

    at 25 degrees C. The mutant enzyme had nearly normal heat stability, as long as it was synthesized at 25 degrees C. In contrast, there was hardly any PRPP synthase activity or anti-PRPP synthase antibody cross-reactive material present in cells harbouring the glycine to serine alteration following...... synthase activity as a strain harbouring a plasmid-borne wild-type prs allele. In cells harbouring both mutations, the C -> T mutation appeared to compensate for the G -> A mutation by increasing the amount of a partially defective enzyme at the permissive temperature....

  10. New Stetter reactions catalyzed by thiamine diphosphate dependent MenD from E. coli.

    Science.gov (United States)

    Beigi, Maryam; Waltzer, Simon; Zarei, Mostafa; Müller, Michael

    2014-12-10

    The intermolecular asymmetric Stetter reaction is a rarely found biocatalysts transformation. MenD, the second enzyme of the menaquinone biosynthetic pathway, catalyzes as a physiological reaction a Stetter-like addition of α-ketoglutarate to isochorismate. The substrate range of MenD for similar 1,4-additions is highly restricted. All other thiamine diphosphate dependent enzymes known to act as stetterases are members of the PigD enzyme subfamily, which accept aliphatic and aromatic α,β-unsaturated ketones and thioesters as Michael acceptor substrates. Here, we describe the unexpected activity of MenD with short-chain α,β-unsaturated acids and derivatives as substrates in Stetter reactions. MenD possesses a characteristic substrate range with respect to Michael acceptor substrates which is distinctly different from the classical stetterases. This provides biocatalytic access to new types of products which are not related to the products currently accessible by thiamine diphosphate dependent enzyme catalysis.

  11. Metabolic engineering of Escherichia coli to produce 2'-fucosyllactose via salvage pathway of guanosine 5'-diphosphate (GDP)-l-fucose.

    Science.gov (United States)

    Chin, Young-Wook; Seo, Nari; Kim, Jae-Han; Seo, Jin-Ho

    2016-11-01

    2'-Fucosyllactose (2-FL) is one of the key oligosaccharides in human milk. In the present study, the salvage guanosine 5'-diphosphate (GDP)-l-fucose biosynthetic pathway from fucose was employed in engineered Escherichia coli BL21star(DE3) for efficient production of 2-FL. Introduction of the fkp gene coding for fucokinase/GDP-l-fucose pyrophosphorylase (Fkp) from Bacteroides fragilis and the fucT2 gene encoding α-1,2-fucosyltransferase from Helicobacter pylori allows the engineered E. coli to produce 2-FL from fucose, lactose and glycerol. To enhance the lactose flux to 2-FL production, the attenuated, and deleted mutants of β-galactosidase were employed. Moreover, the 2-FL yield and productivity were further improved by deletion of the fucI-fucK gene cluster coding for fucose isomerase (FucI) and fuculose kinase (FucK). Finally, fed-batch fermentation of engineered E. coli BL21star(DE3) deleting lacZ and fucI-fucK, and expressing fkp and fucT2 resulted in 23.1 g/L of extracellular concentration of 2-FL and 0.39 g/L/h productivity. Biotechnol. Bioeng. 2016;113: 2443-2452. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  12. Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Andersen, Kasper R; Kilstrup, Mogens

    2017-01-01

    is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP...... synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of the five diphosphoryltransferases are compared, as are those of selected enzymes of diphosphoryl transfer, phosphoryl transfer, and nucleotidyl transfer reactions. PRPP is used as a substrate by a large number...... phosphoribosyltransferases. The protein structures and reaction mechanisms of these phosphoribosyltransferases vary and demonstrate the versatility of PRPP as an intermediate in cellular physiology. PRPP synthases appear to have originated from a phosphoribosyltransferase during evolution, as demonstrated by phylogenetic...

  13. Thiamine diphosphate adenylyl transferase from E. coli: functional characterization of the enzyme synthesizing adenosine thiamine triphosphate

    OpenAIRE

    Brans Alain; Makarchikov Alexander F; Bettendorff Lucien

    2007-01-01

    Abstract Background We have recently identified a new thiamine derivative, adenosine thiamine triphosphate (AThTP), in E. coli. In intact bacteria, this nucleotide is synthesized only in the absence of a metabolizable carbon source and quickly disappears as soon as the cells receive a carbon source such as glucose. Thus, we hypothesized that AThTP may be a signal produced in response to carbon starvation. Results Here we show that, in bacterial extracts, the biosynthesis of AThTP is carried o...

  14. Different oligomeric states are involved in the allosteric behavior of uracil phosphoribosyltransferase from Escherichia coli

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank; Mygind, Bente

    1996-01-01

    Uracil phosphoribosyltransferase, catalyzing the formation of UMP and pyrophosphate from uracil and 5-phosphoribosyl-a-1-diphosphate (PP RibP), was purified from an overproducing strain of Escherichia coli. GTP was shown to activate the enzyme by reducing Km for PP RibP by about fivefold without...... affecting Vmax. When started by addition of enzyme, the reactions accelerated over an extended period of time, while enzyme solutions incubated first with GTP and PP RibP displayed constant velocities. This indicated that PP RibP and GTP influenced the structure of the enzyme. Gel....... It appears that the smaller form predominates in the absence of substrates, while the larger form predominates in the presence of GTP and PP RibP. Guanosine-3',5'-bis(diphosphate) was found to activate the enzyme much like GTP....

  15. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

    1999-03-02

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

  16. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wildung, Mark Raymond (Colfax, WA); Burke, Charles Cullen (Moscow, ID); Gershenzon, Jonathan (Jena, DE)

    1999-01-01

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

  17. Determination of Activity of the Enzymes Hypoxanthine Phosphoribosyl Transferase (HPRT) and Adenine Phosphoribosyl Transferase (APRT) in Blood Spots on Filter Paper.

    Science.gov (United States)

    Auler, Kasie; Broock, Robyn; Nyhan, William L

    2015-07-01

    Hypoxanthine-guanine phosphoribosyl-transferase (HPRT) deficiency is the cause of Lesch-Nyhan disease. Adenine phosphoribosyl-transferase (APRT) deficiency causes renal calculi. The activity of each enzyme is readily determined on spots of whole blood on filter paper. This unit describes a method for detecting deficiencies of HPRT and APRT. Copyright © 2015 John Wiley & Sons, Inc.

  18. The design, synthesis and biological evaluation of novel thiamin diphosphate analog inhibitors against the pyruvate dehydrogenase multienzyme complex E1 from Escherichia coli.

    Science.gov (United States)

    Feng, Lingling; He, Junbo; He, Haifeng; Zhao, Lulu; Deng, Lingfu; Zhang, Li; Zhang, Lin; Ren, Yanliang; Wan, Jian; He, Hongwu

    2014-11-28

    Pyruvate dehydrogenase multienzyme complex E1 (PDHc E1) is a potential target enzyme when looking for inhibitors to combat microbial disease. In this study, we designed and synthesized a series of novel thiamin diphosphate (ThDP) analogs with triazole ring and oxime ether moieties as potential inhibitors of PDHc E1. Their inhibitory activities against PDHc E1 were examined both in vitro and in vivo. Most of the tested compounds exhibited moderate inhibitory activities against PDHc E1 (IC50 = 6.1-75.5 μM). The potent inhibitors 4g, 4h and 4j, had strong inhibitory activities with IC50 values of 6.7, 6.9 and 6.1 μM against PDHc E1 in vitro and with inhibition rates of 35%, 50% and 33% at 100 μg mL(-1) against Gibberella zeae in vivo, respectively. The binding mode of 4j to PDHc E1 was analyzed by a molecular docking method. Furthermore, the possible interactions of the important residues of PDHc E1 with compound 4j were examined by site-directed mutagenesis, enzymatic assays and spectral fluorescence studies. The theoretical and experimental results are in good agreement and suggest that compound 4j could be used as a lead compound for further optimization, and may have potential as a new microbicide.

  19. Manganese dipyridoxyl diphosphate:

    DEFF Research Database (Denmark)

    H, Brurok; Ardenkjær-Larsen, Jan Henrik; G, Hansson

    1999-01-01

    Manganese dipyridoxyl diphosphate (MnDPDP) is a contrast agent for magnetic resonance imaging (MRI) of the liver. Aims of the study were to examine if MnDPDP possesses superoxide dismutase (SOD) mimetic activity in vitro, and if antioxidant protection can be demonstrated in an ex vivo rat heart...

  20. A Versatile Photoactivatable Probe Designed to Label the Diphosphate Binding Site of Farnesyl Diphosphate Utilizing Enzymes

    Science.gov (United States)

    Henry, Olivier; Lopez-Gallego, Fernando; Agger, Sean A.; Schmidt-Dannert, Claudia; Sen, Stephanie; Shintani, David; Cornish, Katrina; Distefano, Mark D.

    2009-01-01

    Farnesyl diphosphate (FPP) is a substrate for a diverse number of enzymes found in nature. Photoactive analogues of isoprenoid diphosphates containing either benzophenone, diazotrifluropropionate or azide groups have been useful for studying both the enzymes that synthesize FPP as well as those that employ FPP as a substrate. Here we describe the synthesis and properties of a new class of FPP analogues that links an unmodified farnesyl group to a diphosphate mimic containing a photoactive benzophenone moiety; thus, importantly, these compounds are photoactive FPP analogues that contain no modifications of the isoprenoid portion of the molecule that may interfere with substrate binding in the active site of an FPP utilizing enzyme. Two isomeric compounds containing meta- and para-substituted benzophenones were prepared. These two analogues inhibit S. cerevisiae protein farnesyltransferase (ScPFTase) with IC50 values of 5.8 (meta isomer) and 3.0 µM (para isomer); the more potent analogue, the para isomer, was shown to be a competitive inhibitor of ScPFTase with respect to FPP with a KI of 0.46 µM. Radiolabeled forms of both analogues selectively labelled the β-subunit of ScPFTase. The para isomer was also shown to label E. coli farnesyl diphosphate synthase and Drosophila melanogaster farnesyl diphosphate synthase. Finally, the para isomer was shown to be an alternative substrate for a sesquiterpene synthase from Nostoc sp. strain PCC7120, a cyanobacterial source; the compound also labeled the purified enzyme upon photolysis. Taken together, these results using a number of enzymes demonstrate that this new class of probes should be useful for a plethora of studies of FPP-utilizing enzymes. PMID:19447628

  1. Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines.

    Science.gov (United States)

    López-Cruz, Roberto I; Crocker, Daniel E; Gaxiola-Robles, Ramón; Bernal, Jaime A; Real-Valle, Roberto A; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

    2016-01-01

    Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements.

  2. Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines

    Science.gov (United States)

    López-Cruz, Roberto I.; Crocker, Daniel E.; Gaxiola-Robles, Ramón; Bernal, Jaime A.; Real-Valle, Roberto A.; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

    2016-01-01

    Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements. PMID:27375492

  3. A unique deubiquitinase that deconjugates phosphoribosyl-linked protein ubiquitination

    Energy Technology Data Exchange (ETDEWEB)

    Qiu, Jiazhang; Yu, Kaiwen; Fei, Xiaowen; Liu, Yao; Nakayasu, Ernesto S.; Piehowski, Paul D.; Shaw, Jared B.; Puvar, Kedar; Das, Chittaranjan; Liu, Xiaoyun; Luo, Zhao-Qing

    2017-05-12

    Ubiquitination regulates many aspects of host immunity and thus is a common target for infectious agents. Recent studies revealed that members of the SidE effector family of the bacterial pathogen Legionella pneumophila attacked several small GTPases associated with the endoplasmic reticulum by a novel ubiquitination mechanism that does not require the E1 and E2 enzymes of the host ubiquitination machinery. Following ubiquitin activation by ADP- ribosylation via a mono-ADP-ribosylation motif, ADP-ribosylated ubiquitin is cleaved by a phosphodiesterasedomainwithinSdeA,whichisconcomitantwiththelinkof phosphoribosylated ubiquitin to serine residues in the substrate. Here we demonstrate that the activity of SidEs is regulated by SidJ, another effector encoded by a gene situated in the locus coding for three members of the SidE family (SdeC, SdeB and SdeA). SidJ functions to remove ubiquitin from SidEs-modified substrates by cleaving the phosphodiester bond that links phosphoribosylated ubiquitin to protein substrates. Further, the deubiquitinase activity of SidJ is essential for its role in L. pneumophila infection. Finally, the activity of SidJ is required for efficiently reducing the abundance of ubiquitinated Rab33b in infected cells within a few hours after bacterial uptake. Our results establish SidJ as a deubiquitinase that functions to impose temporal regulation of the activity of the SidE effectors. The identification of SidJ may shed light on future study of signaling cascades mediated by this unique ubiquitination that also potentially regulates cellular processes in eukaryotic cells.

  4. Bis(4-methoxybenzylammonium dihydrogen diphosphate

    Directory of Open Access Journals (Sweden)

    Adel Elboulali

    2013-02-01

    Full Text Available In the title compound, 2C8H12NO+·H2P2O72−, the linked PO4 groups of the diphosphate anion are almost eclipsed and the P—O—P angle is 134.45 (7°. In the crystal, infinite ribbons of H2P2O72− anions propagate in [100], being linked by strong O—H...O hydrogen bonds. The 4-methoxybenzylammonium cations bond to the diphosphate chains by N—H...O and C—H...O links, and are themselves linked by C—H...π interactions.

  5. Partial hypoxanthine-guanine phosphoribosyl transferase deficiency without elevated urinary hypoxanthine excretion

    NARCIS (Netherlands)

    van Dael, C. M. L.; Pierik, L. J. W. M.; Reijngoud, D. J.; Niezen-Koning, K. E.; van Diggelen, O. P.; van Spronsen, F. J.

    Partial hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficiency, also known as the Kelley-Seegmiller syndrome, can give rise to a wide range of neurological symptoms, and renal insufficiency. Biochemically, it is characterized by high uric acid concentrations in blood, high uric acid and

  6. Partial hypoxanthine-guanine phosphoribosyl transferase deficiency without elevated urinary hypoxanthine excretion

    NARCIS (Netherlands)

    van Dael, C. M. L.; Pierik, L. J. W. M.; Reijngoud, D. J.; Niezen-Koning, K. E.; van Diggelen, O. P.; van Spronsen, F. J.

    2007-01-01

    Partial hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficiency, also known as the Kelley-Seegmiller syndrome, can give rise to a wide range of neurological symptoms, and renal insufficiency. Biochemically, it is characterized by high uric acid concentrations in blood, high uric acid and

  7. The 1.25 Å resolution structure of phosphoribosyl-ATP pyrophosphohydrolase from Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Javid-Majd, Farah; Yang, Dong [Department of Biochemistry and Biophysics, Texas A& M University, College Station, Texas 77843-2128 (United States); Ioerger, Thomas R. [Department of Computer Science, Texas A& M University, College Station, Texas 77843-2128 (United States); Sacchettini, James C., E-mail: sacchett@tamu.edu [Department of Biochemistry and Biophysics, Texas A& M University, College Station, Texas 77843-2128 (United States)

    2008-06-01

    The crystal structure of M. tuberculosis phosphoribosyl-ATP pyrophosphohydrolase, the second enzyme in the histidine-biosynthetic pathway, is presented. The structural and inferred functional relationships between M. tuberculosis phosphoribosyl-ATP pyrophosphohydrolase and other members of the nucleoside-triphosphate pyrophosphatase-fold family are described. Phosphoribosyl-ATP pyrophosphohydrolase is the second enzyme in the histidine-biosynthetic pathway, irreversibly hydrolyzing phosphoribosyl-ATP to phosphoribosyl-AMP and pyrophosphate. It is encoded by the hisE gene, which is present as a separate gene in many bacteria and archaea but is fused to hisI in other bacteria, fungi and plants. Because of its essentiality for growth in vitro, HisE is a potential drug target for tuberculosis. The crystal structures of two native (uncomplexed) forms of HisE from Mycobacterium tuberculosis have been determined to resolutions of 1.25 and 1.79 Å. The structure of the apoenzyme reveals that the protein is composed of five α-helices with connecting loops and is a member of the α-helical nucleoside-triphosphate pyrophosphatase superfamily. The biological unit of the protein is a homodimer, with an active site on each subunit composed of residues exclusively from that subunit. A comparison with the Campylobacter jejuni dUTPase active site allowed the identification of putative metal- and substrate-binding sites in HisE, including four conserved glutamate and glutamine residues in the sequence that are consistent with a motif for pyrophosphohydrolase activity. However, significant differences between family members are observed in the loop region between α-helices H1 and H3. The crystal structure of M. tuberculosis HisE provides insights into possible mechanisms of substrate binding and the diversity of the nucleoside-triphosphate pyrophosphatase superfamily.

  8. Bioconversion of lactose/whey to fructose diphosphate with recombinant Saccharomyces cerevisiae cells

    Energy Technology Data Exchange (ETDEWEB)

    Compagno, C.; Tura, A.; Ranzi, B.M.; Martegani, E. (Univ. di Milano (Italy))

    1993-07-01

    Genetically engineered Saccharomyces cerevisiae strains that express Escherichia coli [beta]-galactosidase gene are able to bioconvert lactose or whey into fructose-1,6-diphosphate (FDP). High FDP yields from whey were obtained with an appropriate ratio between cell concentration and inorganic phosphate. The biomass of transformed cells can be obtained from different carbon sources, according to the expression vector bearing the lacZ gene. The authors showed that whey can be used as the carbon source for S. cerevisiae growth and as the substrate for bioconversion to fructose diphosphate.

  9. The 1.25 Å resolution structure of phosphoribosyl-ATP pyrophosphohydrolase from Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Javid-Majd, Farah; Yang, Dong; Ioerger, Thomas R.; Sacchettini, James C. (TAM)

    2008-06-23

    Phosphoribosyl-ATP pyrophosphohydrolase is the second enzyme in the histidine-biosynthetic pathway, irreversibly hydrolyzing phosphoribosyl-ATP to phosphoribosyl-AMP and pyrophosphate. It is encoded by the hisE gene, which is present as a separate gene in many bacteria and archaea but is fused to hisI in other bacteria, fungi and plants. Because of its essentiality for growth in vitro, HisE is a potential drug target for tuberculosis. The crystal structures of two native (uncomplexed) forms of HisE from Mycobacterium tuberculosis have been determined to resolutions of 1.25 and 1.79 {angstrom}. The structure of the apoenzyme reveals that the protein is composed of five -helices with connecting loops and is a member of the {alpha}-helical nucleoside-triphosphate pyrophosphatase superfamily. The biological unit of the protein is a homodimer, with an active site on each subunit composed of residues exclusively from that subunit. A comparison with the Campylobacter jejuni dUTPase active site allowed the identification of putative metal- and substrate-binding sites in HisE, including four conserved glutamate and glutamine residues in the sequence that are consistent with a motif for pyrophosphohydrolase activity. However, significant differences between family members are observed in the loop region between {alpha}-helices H1 and H3. The crystal structure of M. tuberculosis HisE provides insights into possible mechanisms of substrate binding and the diversity of the nucleoside-triphosphate pyrophosphatase superfamily.

  10. Activation of thiamin diphosphate in enzymes.

    Science.gov (United States)

    Hübner, G; Tittmann, K; Killenberg-Jabs, M; Schäffner, J; Spinka, M; Neef, H; Kern, D; Kern, G; Schneider, G; Wikner, C; Ghisla, S

    1998-06-29

    Activation of the coenzyme ThDP was studied by measuring the kinetics of deprotonation at the C2 carbon of thiamin diphosphate in the enzymes pyruvate decarboxylase, transketolase, pyruvate dehydrogenase complex, pyruvate oxidase, in site-specific mutant enzymes and in enzyme complexes containing coenzyme analogues by proton/deuterium exchange detected by 1H-NMR spectroscopy. The respective deprotonation rate constant is above the catalytic constant in all enzymes investigated. The fast deprotonation requires the presence of an activator in pyruvate decarboxylase from yeast, showing the allosteric regulation of this enzyme to be accomplished by an increase in the C2-H dissociation rate of the enzyme-bound thiamin diphosphate. The data of the thiamin diphosphate analogues and of the mutant enzymes show the N1' atom and the 4'-NH2 group to be essential for the activation of the coenzyme and a conserved glutamate involved in the proton abstraction mechanism of the enzyme-bound thiamin diphosphate.

  11. Micronuclei rate and hypoxanthine phosphoribosyl transferase mutation in radon-exposed rats

    Institute of Scientific and Technical Information of China (English)

    Fengmei Cui; Saijun Fan; Mingjiang Hu; Jihua Nie; Hongmei Li; Jian Tong

    2008-01-01

    The genetic changes in rats with radon exposure were studied by the micronucleus technology and detection of hypoxanthine phosphoribosyl transferase (hprt) mutations.The rate of the micronuclei in peripheral blood lymphocytes and tracheal-bronchial epithelial cells in the radon-inhaled rats was higher than that of the controls (P < 0.05).A similar result was obtained from the hprt assay,which showed a higher mutation frequency in radon-exposed rats.Our results suggested that micronuclei rate and hprt deficiency could be used as biomarkers for the genetic changes with radon exposure.

  12. Ammonium ytterbium(III diphosphate(V

    Directory of Open Access Journals (Sweden)

    Michal Dušek

    2008-12-01

    Full Text Available The title compound, NH4YbP2O7, crystallizes in the KAlP2O7 structure type and consists of distorted YbO6 octahedra and bent P2O74− diphosphate units forming together a three-dimensional network. There are channels in the structure running along the c axis, where the NH4+ cations are located. They are connected via N—H...O hydrogen bonds to the terminal O atoms of the diphosphate anions.

  13. Motesanib diphosphate in progressive differentiated thyroid cancer

    DEFF Research Database (Denmark)

    Sherman, Steven I; Wirth, Lori J; Droz, Jean-Pierre

    2008-01-01

    BACKGROUND: The expression of vascular endothelial growth factor (VEGF) is characteristic of differentiated thyroid cancer and is associated with aggressive tumor behavior and a poor clinical outcome. Motesanib diphosphate (AMG 706) is a novel oral inhibitor of VEGF receptors, platelet......-derived growth-factor receptor, and KIT. METHODS: In an open-label, single-group, phase 2 study, we treated 93 patients who had progressive, locally advanced or metastatic, radioiodine-resistant differentiated thyroid cancer with 125 mg of motesanib diphosphate, administered orally once daily. The primary end...... point was an objective response as assessed by an independent radiographic review. Additional end points included the duration of the response, progression-free survival, safety, and changes in serum thyroglobulin concentration. RESULTS: Of the 93 patients, 57 (61%) had papillary thyroid carcinoma...

  14. How thiamine diphosphate is activated in enzymes.

    Science.gov (United States)

    Kern, D; Kern, G; Neef, H; Tittmann, K; Killenberg-Jabs, M; Wikner, C; Schneider, G; Hübner, G

    1997-01-03

    The controversial question of how thiamine diphosphate, the biologically active form of vitamin B1, is activated in different enzymes has been addressed. Activation of the coenzyme was studied by measuring thermodynamics and kinetics of deprotonation at the carbon in the 2-position (C2) of thiamine diphosphate in the enzymes pyruvate decarboxylase and transketolase by use of nuclear magnetic resonance spectroscopy, proton/deuterium exchange, coenzyme analogs, and site-specific mutant enzymes. Interaction of a glutamate with the nitrogen in the 1'-position in the pyrimidine ring activated the 4'-amino group to act as an efficient proton acceptor for the C2 proton. The protein component accelerated the deprotonation of the C2 atom by several orders of magnitude, beyond the rate of the overall enzyme reaction. Therefore, the earlier proposed concerted mechanism or stabilization of a C2 carbanion can be excluded.

  15. Geranyl diphosphate synthase molecules, and nucleic acid molecules encoding same

    Science.gov (United States)

    Croteau, Rodney Bruce; Burke, Charles Cullen

    2008-06-24

    In one aspect, the present invention provides isolated nucleic acid molecules that each encode a geranyl diphosphate synthase protein, wherein each isolated nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:1 under conditions of 5.times.SSC at 45.degree. C. for one hour. The present invention also provides isolated geranyl diphosphate synthase proteins, and methods for altering the level of expression of geranyl diphosphate synthase protein in a host cell.

  16. Asymmetric Stetter reactions catalyzed by thiamine diphosphate-dependent enzymes.

    Science.gov (United States)

    Kasparyan, Elena; Richter, Michael; Dresen, Carola; Walter, Lydia S; Fuchs, Georg; Leeper, Finian J; Wacker, Tobias; Andrade, Susana L A; Kolter, Geraldine; Pohl, Martina; Müller, Michael

    2014-12-01

    The intermolecular asymmetric Stetter reaction is an almost unexplored transformation for biocatalysts. Previously reported thiamine diphosphate (ThDP)-dependent PigD from Serratia marcescens is the first enzyme identified to catalyze the Stetter reaction of α,β-unsaturated ketones (Michael acceptor substrates) and α-keto acids. PigD is involved in the biosynthesis of the potent cytotoxic agent prodigiosin. Here, we describe the investigation of two new ThDP-dependent enzymes, SeAAS from Saccharopolyspora erythraea and HapD from Hahella chejuensis. Both show a high degree of homology to the amino acid sequence of PigD (39 and 51 %, respectively). The new enzymes were heterologously overproduced in Escherichia coli, and the yield of soluble protein was enhanced by co-expression of the chaperone genes groEL/ES. SeAAS and HapD catalyze intermolecular Stetter reactions in vitro with high enantioselectivity. The enzymes possess a characteristic substrate range with respect to Michael acceptor substrates. This provides support for a new type of ThDP-dependent enzymatic activity, which is abundant in various species and not restricted to prodigiosin biosynthesis in different strains. Moreover, PigD, SeAAS, and HapD are also able to catalyze asymmetric carbon-carbon bond formation reactions of aldehydes and α-keto acids, resulting in 2-hydroxy ketones.

  17. Activation of Variants of Hypoxanthine-Guanine Phosphoribosyl Transferase by the Normal Enzyme

    Science.gov (United States)

    Bakay, Bohdan; Nyhan, William L.

    1972-01-01

    Deficient hypoxanthine-guanine phosphoribosyl transferase (HGPRT; EC 2.4.2.8) enzymes from erythrocytes of patients with hyperuricemia and with the Lesch-Nyhan syndrome migrate 15% faster in polyacrylamide gel disc electrophoresis than the normal enzyme. A half-sister of two males with partial deficiency, who had 34% of normal HGPRT activity in her erythrocytes, yielded profiles containing two distinct zones of activity; one corresponded to the enzyme found in normal individuals and one to the variant of her half-brothers. However, in her profile her variant enzyme showed notably greater activity than that observed in her half-brothers. This increase was due to an activation of the variant by normal enzyme. Electrophoresis of mixtures of normal enzyme with partially deficient enzymes from patients with hyperuricemia and with the Lesch-Nyhan syndrome also led to activation of deficient HGPRT variants by normal enzymes. Deficient variants were also activated by normal enzyme on filtration through Sephadex G-25. Experiments in which deficient variant enzymes were activated with purified normal enzyme labeled with 125I indicated that deficient enzymes incorporate components of the normal enzyme. No such activation of deficient enzymes was ever obtained when mixtures of deficient and normal enzymes were put together in a test tube. Images PMID:4341698

  18. Functional Analysis of the Isopentenyl Diphosphate Isomerase of Salvia miltiorrhiza via Color Complementation and RNA Interference

    Directory of Open Access Journals (Sweden)

    Xianan Zhang

    2015-11-01

    Full Text Available Isopentenyl diphosphate isomerase (IPI catalyzes the isomerization between the common terpene precursor substances isopentenyl diphosphate (IPP and dimethylallyl diphosphate (DMAPP during the terpenoid biosynthesis process. In this study, tissue expression analysis revealed that the expression level of the Salvia miltiorrhiza IPI1 gene (SmIPI1 was higher in the leaves than in the roots and stems. Furthermore, color complementation and RNA interference methods were used to verify the function of the SmIPI1 gene from two aspects. A recombinant SmIPI1 plasmid was successfully constructed and transferred into engineered E. coli for validating the function of SmIPI1 through the color difference in comparison to the control group; the observed color difference indicated that SmIPI1 served in promoting the accumulation of lycopene. Transformant hairy root lines with RNA interference of SmIPI1 were successfully constructed mediated by Agrobacterium rhizogenes ACCC 10060. RNA interference hairy roots had a severe phenotype characterized by withering, deformity or even death. The mRNA expression level of SmIPI1 in the RSi3 root line was only 8.4% of that of the wild type. Furthermore the tanshinone content was too low to be detected in the RNA interference lines. These results suggest that SmIPI1 plays a critical role in terpenoid metabolic pathways. Addition of an exogenous SmIPI1 gene promoted metabolic flow toward the biosynthesis of carotenoids in E. coli, and SmIPI1 interference in S. miltiorrhiza hairy roots may cause interruption of the 2-C-methyl-D-erythritol-4-phosphate metabolic pathway.

  19. Functional Analysis of the Isopentenyl Diphosphate Isomerase of Salvia miltiorrhiza via Color Complementation and RNA Interference.

    Science.gov (United States)

    Zhang, Xianan; Guan, Hongyu; Dai, Zhubo; Guo, Juan; Shen, Ye; Cui, Guanghong; Gao, Wei; Huang, Luqi

    2015-11-10

    Isopentenyl diphosphate isomerase (IPI) catalyzes the isomerization between the common terpene precursor substances isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) during the terpenoid biosynthesis process. In this study, tissue expression analysis revealed that the expression level of the Salvia miltiorrhiza IPI1 gene (SmIPI1) was higher in the leaves than in the roots and stems. Furthermore, color complementation and RNA interference methods were used to verify the function of the SmIPI1 gene from two aspects. A recombinant SmIPI1 plasmid was successfully constructed and transferred into engineered E. coli for validating the function of SmIPI1 through the color difference in comparison to the control group; the observed color difference indicated that SmIPI1 served in promoting the accumulation of lycopene. Transformant hairy root lines with RNA interference of SmIPI1 were successfully constructed mediated by Agrobacterium rhizogenes ACCC 10060. RNA interference hairy roots had a severe phenotype characterized by withering, deformity or even death. The mRNA expression level of SmIPI1 in the RSi3 root line was only 8.4% of that of the wild type. Furthermore the tanshinone content was too low to be detected in the RNA interference lines. These results suggest that SmIPI1 plays a critical role in terpenoid metabolic pathways. Addition of an exogenous SmIPI1 gene promoted metabolic flow toward the biosynthesis of carotenoids in E. coli, and SmIPI1 interference in S. miltiorrhiza hairy roots may cause interruption of the 2-C-methyl-D-erythritol-4-phosphate metabolic pathway.

  20. Rv0989c encodes a novel (E)-geranyl diphosphate synthase facilitating decaprenyl diphosphate biosynthesis in Mycobacterium tuberculosis.

    Science.gov (United States)

    Mann, Francis M; Thomas, Jill A; Peters, Reuben J

    2011-02-04

    Mycobacterium tuberculosis (Mtb) has a highly complex cell wall, which is required for both bacterial survival and infection. Cell wall biosynthesis is dependent on decaprenyl diphosphate as a glyco-carrier, which is hence an essential metabolite in this pathogen. Previous biochemical studies indicated (E)-geranyl diphosphate (GPP) is required for the synthesis of decaprenyl diphosphate. Here we demonstrate that Rv0989c encodes the "missing" GPP synthase, representing the first such enzyme to be characterized from bacteria, and which presumably is involved in decaprenyl diphosphate biosynthesis in Mtb. Our investigation also has revealed previously unrecognized substrate plasticity of the farnesyl diphosphate synthases from Mtb, resolving previous discrepancies between biochemical and genetic studies of cell wall biosynthesis.

  1. Labeling embryonic stem cells with enhanced green fluorescent protein on the hypoxanthineguanine phosphoribosyl transferase locus

    Institute of Scientific and Technical Information of China (English)

    滕路; 孟国良; 刑阳; 尚克刚; 王小珂; 顾军

    2003-01-01

    Objective To label embryonic stem (ES) cells with enhanced green fluorescent protein (EGF P) on the hypoxanthineguanine phosphoribosyl transferase (HPRT) gene locus for t he first time to provide a convenient and efficient way for cell tracking and ma nipulation in the studies of transplantation and stem cell therapy.Methods Homologous fragments were obtained by polymerase chain reaction (PCR), from whic h the gene targeting vector pHPRT-EGFP was constructed. The linearized vector was introduced into ES cells by electroporation. The G418r6TGr cell clones were obtained after selection with G418 and 6TG media. The integration patterns of these resistant cell clones were identified with Southern blotting.Results EGFP expressing ES cells on the locus of HPRT were successfu lly generated. They have normal properties, such as karyotype, viability and di fferentiation ability. The green fluorescence of EGFP expressing cells was main tained in propagation of the ES cells for more than 30 passages and in different iated cells. Cultured in suspension, the "green" ES cells aggregated and forme d embryoid bodies, retaining the green fluorescence at varying developmental sta ges. The "green" embryoid bodies could expand and differentiate into various t ypes of cells, exhibiting ubiquitous green fluorescence. Conclusions This generation of "green" targeted ES cells is described in an efficient proto col for obtaining the homologous fragments by PCR. Introducing the marker gene in the genome of ES cells, we should be able to manipulate them in vitro and use them as vehicles in cell-replacement therapy as well as for other biomedical a nd research purposes.

  2. Additional diterpenes from Physcomitrella patens synthesized by copalyl diphosphate/kaurene synthase (PpCPS/KS).

    Science.gov (United States)

    Zhan, Xin; Bach, Søren Spanner; Hansen, Nikolaj Lervad; Lunde, Christina; Simonsen, Henrik Toft

    2015-11-01

    The bifunctional diterpene synthase, copalyl diphosphate/kaurene synthase from the moss Physcomitrella patens (PpCPS/KS), catalyses the formation of at least four diterpenes, including ent-beyerene, ent-sandaracopimaradiene, ent-kaur-16-ene, and 16-hydroxy-ent-kaurene. The enzymatic activity has been confirmed through generation of a targeted PpCPS/KS knock-out mutant in P. patens via homologous recombination, through transient expression of PpCPS/KS in Nicotiana benthamiana, and expression of PpCPS/KS in E. coli. GC-MS analysis of the knock-out mutant shows that it lacks the diterpenoids, supporting that all are products of PpCPS/KS as observed in N. benthamiana and E. coli. These results provide additional knowledge of the mechanism of this bifunctional diterpene synthase, and are in line with proposed reaction mechanisms in kaurene biosynthesis.

  3. Studies on thiamine diphosphate-dependent enzymes.

    Science.gov (United States)

    Leeper, F J; Hawksley, D; Mann, S; Perez Melero, C; Wood, M D H

    2005-08-01

    The 3-deaza analogue of TPP (thiamine diphosphate), a close mimic of the ylid intermediate, has been synthesized and is an extremely potent inhibitor of a variety of TPP-dependent enzymes, binding much more tightly than TPP itself. Results using deazaTPP complexed with the E1 subunit of PDH (pyruvate dehydrogenase) have led to a novel proposal about the mechanism of this enzyme. The 2-substituted forms of deazaTPP, which mimic other intermediates in the catalytic mechanism, can also be synthesized and 2-(1-hydroxyethyl)deazaTPP is also an extremely potent inhibitor of PDC (pyruvate decarboxylase). Attachment of such 2-substituents is expected to be a way to introduce selectivity in the inhibition of various TPP-dependent enzymes.

  4. Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase.

    Science.gov (United States)

    Bergstrom, J D; Bostedor, R G; Masarachia, P J; Reszka, A A; Rodan, G

    2000-01-01

    Alendronate, a nitrogen-containing bisphosphonate, is a potent inhibitor of bone resorption used for the treatment and prevention of osteoporosis. Recent findings suggest that alendronate and other N-containing bisphosphonates inhibit the isoprenoid biosynthesis pathway and interfere with protein prenylation, as a result of reduced geranylgeranyl diphosphate levels. This study identified farnesyl disphosphate synthase as the mevalonate pathway enzyme inhibited by bisphosphonates. HPLC analysis of products from a liver cytosolic extract narrowed the potential targets for alendronate inhibition (IC(50) = 1700 nM) to isopentenyl diphosphate isomerase and farnesyl diphosphate synthase. Recombinant human farnesyl diphosphate synthase was inhibited by alendronate with an IC(50) of 460 nM (following 15 min preincubation). Alendronate did not inhibit isopentenyl diphosphate isomerase or GGPP synthase, partially purified from liver cytosol. Recombinant farnesyl diphosphate synthase was also inhibited by pamidronate (IC(50) = 500 nM) and risedronate (IC(50) = 3.9 nM), negligibly by etidronate (IC50 = 80 microM), and not at all by clodronate. In osteoclasts, alendronate inhibited the incorporation of [(3)H]mevalonolactone into proteins of 18-25 kDa and into nonsaponifiable lipids, including sterols. These findings (i) identify farnesyl diphosphate synthase as the selective target of alendronate in the mevalonate pathway, (ii) show that this enzyme is inhibited by other N-containing bisphosphonates, such as risendronate, but not by clodronate, supporting a different mechanism of action for different bisphosphonates, and (iii) document in purified osteoclasts alendronate inhibition of prenylation and sterol biosynthesis.

  5. Monoterpenes in the glandular trichomes of tomato are synthesized from a neryl diphosphate precursor rather than geranyl diphosphate.

    Science.gov (United States)

    Schilmiller, Anthony L; Schauvinhold, Ines; Larson, Matthew; Xu, Richard; Charbonneau, Amanda L; Schmidt, Adam; Wilkerson, Curtis; Last, Robert L; Pichersky, Eran

    2009-06-30

    We identified a cis-prenyltransferase gene, neryl diphosphate synthase 1 (NDPS1), that is expressed in cultivated tomato (Solanum lycopersicum) cultivar M82 type VI glandular trichomes and encodes an enzyme that catalyzes the formation of neryl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. mRNA for a terpene synthase gene, phellandrene synthase 1 (PHS1), was also identified in these glands. It encodes an enzyme that uses neryl diphosphate to produce beta-phellandrene as the major product as well as a variety of other monoterpenes. The profile of monoterpenes produced by PHS1 is identical with the monoterpenes found in type VI glands. PHS1 and NDPS1 map to chromosome 8, and the presence of a segment of chromosome 8 derived from Solanum pennellii LA0716 causes conversion from the M82 gland monoterpene pattern to that characteristic of LA0716 plants. The data indicate that, contrary to the textbook view of geranyl diphosphate as the "universal" substrate of monoterpene synthases, in tomato glands neryl diphosphate serves as a precursor for the synthesis of monoterpenes.

  6. Orotate phosphoribosyl transferase mRNA expression and the response of cholangiocarcinoma to 5-fluorouracil

    Institute of Scientific and Technical Information of China (English)

    Chariya Hahnvajanawong; Jariya Chaiyagool; Wunchana Seubwai; Vajarabhongsa Bhudhisawasdi; Nisana Namwat; Narong Khuntikeo; Banchob Sripa

    2012-01-01

    AIM:To determine whether expression of certain enzymes related to 5-fluorouracil (5-FU) metabolism predicts 5-FU chemosensitivity in cholangiocarcinoma (CCA).METHODS:The histoculture drug response assay (HDRA) was performed using surgically resected CCA tissues.Tumor cell viability was determined morphologically with hematoxylin and eosin-and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling-stained tissues.The mRNA expression of thymidine phosphorylase (TP),orotate phosphoribosyl transferase (OPRT),thymidylate synthase (TS),and dihydropyrimidine dehydrogenase (DPD) was determined with realtime reverse transcriptase-polymerase chain reaction.The levels of gene expression and the sensitivity to 5-FU were evaluated.RESULTS:Twenty-three CCA tissues were obtained from patients who had been diagnosed with intrahepatic CCA and who underwent surgical resection at Srinagarind Hospital,Khon Kaen University from 2007 to 2009.HDRA was used to determine the response of these CCA tissues to 5-FU.Based on the dose-response curve,200 μg/mL 5-FU was selected as the test concentration.The percentage of inhibition index at the median point was selected as the cut-off point to differentiate the responding and non-responding tumors to 5-FU.When the relationship between TP,OPRT,TS and DPD mRNA expression levels and the sensitivity of CCA tissues to 5-FU was examined,only OPRT mRNA expression was significantly correlated with the response to 5-FU.The mean expression level of OPRT was significantly higher in the responder group compared to the non-responder group (0.41 ± 0.25 vs 0.22 ± 0.12,P < 0.05).CONCLUSION:OPRT mRNA expression may be a useful predictor of 5-FU chemosensitivity of CCA.Whether OPRT mRNA could be used to predict the success of 5-FU chemotherapy in CCA patients requires confirmation in patients.

  7. The function of the three phosphoribosyl pyrophosphate synthetase (Prs) genes in hyphal growth and conidiation in Aspergillus nidulans.

    Science.gov (United States)

    Jiang, Ping; Wei, Wen-Fan; Zhong, Guo-Wei; Zhou, Xiao-Gang; Qiao, Wei-Ran; Fisher, Reinhard; Lu, Ling

    2017-02-01

    Phosphoribosyl pyrophosphate synthetase, which is encoded by the Prs gene, catalyses the reaction of ribose-5-phosphate and adenine ribonucleotide triphosphate (ATP) and has central importance in cellular metabolism. However, knowledge about how Prs family members function and contribute to total 5-phosphoribosyl-α-1-pyrophosphate (PRPP) synthetase activity is limited. In this study, we identified that the filamentous fungus Aspergillus nidulans genome contains three PRPP synthase-homologous genes (AnprsA, AnprsB and AnprsC), among which AnprsB and AnprsC but not AnprsA are auxotrophic genes. Transcriptional expression profiles revealed that the mRNA levels of AnprsA, AnprsB and AnprsC are dynamic during germination, hyphal growth and sporulation and that they all showed abundant expression during the vigorous hyphal growth time point. Inhibiting the expression of AnprsB or AnprsC in conditional strains produced more effects on the total PRPP synthetase activity than did inhibiting AnprsA, thus indicating that different AnPrs proteins are unequal in their contributions to Prs enzyme activity. In addition, the constitutive overexpression of AnprsA or AnprsC could significantly rescue the defective phenotype of the AnprsB-absent strain, suggesting that the function of AnprsB is not a specific consequence of this auxotrophic gene but instead comes from the contribution of Prs proteins to PRPP synthetase activity.

  8. The function of the three phosphoribosyl-pyrophosphate synthetase (Prs) genes in hyphal growth and conidiation in Aspergillus nidulans.

    Science.gov (United States)

    Jiang, Ping; Wei, Wen-Fan; Zhong, Guo-Wei; Zhou, Xiao-Gang; Qiao, Wei-Ran; Lu, Ling

    2017-01-12

    Phosphoribosyl pyrophosphate synthetase, which is encoded by the Prs gene, catalyzes the reaction of ribose-5-phosphate and adenine ribonucleotide triphosphate (ATP) and has central importance in cellular metabolism. However, knowledge about how Prs family members function and contribute to total PRPP synthetase activity is limited. In this study, we identified that the filamentous fungus Aspergillus nidulans genome contains three 5-phosphoribosyl-α-1-pyrophosphate (PRPP) synthase-homologous genes (AnprsA, B, and C), among which AnprsB and AnprsC but not AnprsA are auxotrophic genes. Transcriptional expression profiles revealed that the mRNA levels of AnprsA, B and C are dynamic during germination, hyphal growth and sporulation and that they all showed abundant expression during the vigorous hyphal growth time-point. Inhibiting the expression of AnprsB or AnprsC in conditional strains produced more effects on the total PRPP synthetase activity than did inhibiting AnprsA, thus indicating that different AnPrs proteins are unequal in their contributions to Prs enzyme activity. In addition, the constitutive overexpression of AnprsA or AnprsC could significantly rescue the defective phenotype of the AnprsB-absent strain, suggesting that the function of AnprsB is not a specific consequence of this auxotrophic gene but instead comes from the contribution of Prs proteins to PRPP synthetase activity.

  9. Genetic analysis and enzyme activity suggest the existence of more than one minimal functional unit capable of synthesizing phosphoribosyl pyrophosphate in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hernando, Yolanda; Carter, Andrew T.; Parr, Adrian;

    1999-01-01

    The PRS gene family in Saccharomyces cerevisiae consists of five genes each capable of encoding a 5-phosphoribosyl-1(α)-pyrophosphate synthetase polypeptide. To gain insight into the functional organization of this gene family we have constructed a collection of strains containing all possible co...

  10. Radioimmunoassay for guanosin-5'-diphosphate-3'-diphosphate and adenosine-5'-triphosphate-3'-diphosphate

    Energy Technology Data Exchange (ETDEWEB)

    Hamagishi, Y.; Oki, T.; Tone, H.; Inui, T. (Sanraku-Ocean Co. Ltd., Fujisawa, Kanagawa (Japan). Central Research Lab.)

    1980-12-01

    A radioimmunoassay for guanosine-5'-diphosphate-3'-diphosphate (ppGpp) and adenosine-5'-triphosphate-3'-diphosphate (pppApp) has been developed. The assay method is based on competition of an unlabeled highly phosphorylated nucleotide with /sup 3/H-labeled highly phosphorylated nucleotide for binding sites on a specific antibody. Antibodies to ppGpp and pppApp were obtained by immunizing rabbits with the antigen prepared by conjugating ppGpp with human serum albumin using 1-ethyl-3-(3-dimethylaminoprophyl)carbodiimide, and with the antigen prepared by conjugating 8-(6-aminohexyl)amino-adenosine-5'-triphosphate-3'-diphosphate with human serum albumin using glutaraldehyde, respectively. Antibody-bound /sup 3/H-labeled highly phosphorylated nucleotides were separated from the free /sup 3/H-labeled highly phosphorylated nucleotides by selective adsorption on dextran-coated charcoal. Displacement plots were linear over a concentration range of 5 - 1,000 pmol/assay tube in a log-probit percentage plot. Application of this method to biological systems offers improved accuracy and convenience compared with the previous /sup 32/PO/sub 4/-labeling technique.

  11. C2-alpha-lactylthiamin diphosphate is an intermediate on the pathway of thiamin diphosphate-dependent pyruvate decarboxylation. Evidence on enzymes and models.

    Science.gov (United States)

    Zhang, Sheng; Liu, Min; Yan, Yan; Zhang, Zhen; Jordan, Frank

    2004-12-24

    Thiamin diphosphate (ThDP)-dependent decarboxylations are usually assumed to proceed by a series of covalent intermediates, the first one being the C2-trimethylthiazolium adduct with pyruvate, C2-alpha-lactylthiamin diphosphate (LThDP). Herein is addressed whether such an intermediate is kinetically competent with the enzymatic turnover numbers. In model studies it is shown that the first-order rate constant for decarboxylation can indeed exceed 50 s(-1) in tetrahydrofuran as solvent, approximately 10(3) times faster than achieved in previous model systems. When racemic LThDP was exposed to the E91D yeast pyruvate decarboxylase variant, or to the E1 subunit of the pyruvate dehydrogenase complex (PDHc-E1) from Escherichia coli, it was partitioned between reversion to pyruvate and decarboxylation. Under steady-state conditions, the rate of these reactions is severely limited by the release of ThDP from the enzyme. Under pre-steady-state conditions, the rate constant for decarboxylation on exposure of LThDP to the E1 subunit of the pyruvate dehydrogenase complex was 0.4 s(-1), still more than a 100-fold slower than the turnover number. Because these experiments include binding, decarboxylation, and oxidation (for detection purposes), this is a lower limit on the rate constant for decarboxylation. The reasons for this slow reaction most likely include a slow conformational change of the free LThDP to the V conformation enforced by the enzyme. Between the results from model studies and those from the two enzymes, it is proposed that LThDP is indeed on the decarboxylation pathway of the two enzymes studied, and once LThDP is bound the protein needs to provide little assistance other than a low polarity environment.

  12. A new motif for inhibitors of geranylgeranyl diphosphate synthase.

    Science.gov (United States)

    Foust, Benjamin J; Allen, Cheryl; Holstein, Sarah A; Wiemer, David F

    2016-08-15

    The enzyme geranylgeranyl diphosphate synthase (GGDPS) is believed to receive the substrate farnesyl diphosphate through one lipophilic channel and release the product geranylgeranyl diphosphate through another. Bisphosphonates with two isoprenoid chains positioned on the α-carbon have proven to be effective inhibitors of this enzyme. Now a new motif has been prepared with one isoprenoid chain on the α-carbon, a second included as a phosphonate ester, and the potential for a third at the α-carbon. The pivaloyloxymethyl prodrugs of several compounds based on this motif have been prepared and the resulting compounds have been tested for their ability to disrupt protein geranylgeranylation and induce cytotoxicity in myeloma cells. The initial biological studies reveal activity consistent with GGDPS inhibition, and demonstrate a structure-function relationship which is dependent on the nature of the alkyl group at the α-carbon.

  13. A Geranylfarnesyl Diphosphate Synthase Provides the Precursor for Sesterterpenoid (C25) Formation in the Glandular Trichomes of the Mint Species Leucosceptrum canum.

    Science.gov (United States)

    Liu, Yan; Luo, Shi-Hong; Schmidt, Axel; Wang, Guo-Dong; Sun, Gui-Ling; Grant, Marcus; Kuang, Ce; Yang, Min-Jie; Jing, Shu-Xi; Li, Chun-Huan; Schneider, Bernd; Gershenzon, Jonathan; Li, Sheng-Hong

    2016-03-01

    Plant sesterterpenoids, an important class of terpenoids, are widely distributed in various plants, including food crops. However, little is known about their biosynthesis. Here, we cloned and functionally characterized a plant geranylfarnesyl diphosphate synthase (Lc-GFDPS), the enzyme producing the C25 prenyl diphosphate precursor to all sesterterpenoids, from the glandular trichomes of the woody plant Leucosceptrum canum. GFDPS catalyzed the formation of GFDP after expression in Escherichia coli. Overexpressing GFDPS in Arabidopsis thaliana also gave an extract catalyzing GFDP formation. GFDPS was strongly expressed in glandular trichomes, and its transcript profile was completely in accordance with the sesterterpenoid accumulation pattern. GFDPS is localized to the plastids, and inhibitor studies indicated its use of isoprenyl diphosphate substrates supplied by the 2-C-methyl-D-erythritol 4-phosphate pathway. Application of a jasmonate defense hormone induced GFDPS transcript and sesterterpenoid accumulation, while reducing feeding and growth of the generalist insect Spodoptera exigua, suggesting that these C25 terpenoids play a defensive role. Phylogenetic analysis suggested that GFDPS probably evolved from plant geranylgeranyl diphosphate synthase under the influence of positive selection. The isolation of GFDPS provides a model for investigating sesterterpenoid formation in other species and a tool for manipulating the formation of this group in plants and other organisms.

  14. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemolsynthase activity

    NARCIS (Netherlands)

    Yang, T.; Gao, L.; Hu, H.; Stoopen, G.M.; Wang, C.; Jongsma, M.A.

    2014-01-01

    Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme inthe biosynthesis of pyrethrins, the most widely used plant-derivedpesticide.CDScatalyzes c1’-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP).

  15. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemolsynthase activity

    NARCIS (Netherlands)

    Yang, T.; Gao, L.; Hu, H.; Stoopen, G.M.; Wang, C.; Jongsma, M.A.

    2014-01-01

    Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme inthe biosynthesis of pyrethrins, the most widely used plant-derivedpesticide.CDScatalyzes c1’-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP).

  16. Structure of active IspH enzyme from escherichia coli provides mechanistic insights into substrate reduction

    KAUST Repository

    Gräwert, Tobias

    2009-07-20

    The terminal step of the non-mevalonate pathway of terpene biosynthesis is catalyzed by IspH (see scheme). In the crystal structure of IspH from E. coli, a bound inorganic diphosphate ligand occupies the position of the diphosphate residue of the substrate. Together with mutation studies and theoretical calculations, these data support a mechanism which is analogous to the Birch reduction of allylic alcohols. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.

  17. HbIDI, SlIDI and EcIDI: A comparative study of isopentenyl diphosphate isomerase activity and structure.

    Science.gov (United States)

    Berthelot, Karine; Estevez, Yannick; Quiliano, Miguel; Baldera-Aguayo, Pedro A; Zimic, Mirko; Pribat, Anne; Bakleh, Marc-Elias; Teyssier, Emeline; Gallusci, Philippe; Gardrat, Christian; Lecomte, Sophie; Peruch, Frédéric

    2016-08-01

    In this study, we cloned, expressed and purified the isopentenyl diphosphate isomerases (IDIs) from two plants, Hevea brasiliensis and Solanum lycopersicum, and compared them to the already well characterized Escherichia coli IDI. Phylogenetic analysis showed high homology between the three enzymes. Their catalytic activity was investigated in vitro with recombinant purified enzymes and in vivo by complementation colorimetric tests. The three enzymes displayed consistent activities both in vitro and in vivo. In term of structure, studied by ATR-FTIR and molecular modeling, it is clear that both plant enzymes are more related to their human homologue than to E. coli IDI. But it is assumed that EcIDI represent the minimalistic part of the catalytic core, as both plant enzymes present a supplementary sequence forming an extra α-helice surrounding the catalytic site that could facilitate the biocatalysis. New potential biotechnological applications may be envisaged.

  18. Nicotinamide phosphoribosyl transferase (Nampt is a target of microRNA-26b in colorectal cancer cells.

    Directory of Open Access Journals (Sweden)

    Chenpeng Zhang

    Full Text Available A number of cancers show increased expression of Nicotinamide phosphoribosyl transferase (Nampt. However, the mechanism through which Nampt is upregulated is unclear. In our study, we found that the Nampt-specific chemical inhibitor FK866 significantly inhibited cell survival and reduced nicotinamide adenine dinucleotide (NAD levels in LoVo and SW480 cell lines. Bioinformatics analyses suggested that miR-26b targets Nampt mRNA. We identified Nampt as a new target of miR-26b and demonstrated that miR-26b inhibits Nampt expression at the protein and mRNA levels by binding to the Nampt 3'-UTR. Moreover, we found that miR-26b was down regulated in cancer tissues relative to that in adjacent normal tissues in 18 colorectal cancer patients. A statistically significant inverse correlation between miR-26b and Nampt expression was observed in samples from colorectal cancer patients and in 5 colorectal cell lines (HT-29, SW480, SW1116, LoVo, and HCT116. In addition, over expression of miR-26b strongly inhibited LoVo cell survival and invasion, an effect partially abrogated by the addition of NAD. In conclusion, this study demonstrated that the NAD-salvaging biosynthesis pathway involving Nampt might play a role in colorectal cancer cell survival. MiR-26b may serve as a tumor suppressor by targeting Nampt.

  19. Human T cell recognition of the blood stage antigen Plasmodium hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT in acute malaria

    Directory of Open Access Journals (Sweden)

    Woodberry Tonia

    2009-06-01

    Full Text Available Abstract Background The Plasmodium purine salvage enzyme, hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT can protect mice against Plasmodium yoelii pRBC challenge in a T cell-dependent manner and has, therefore, been proposed as a novel vaccine candidate. It is not known whether natural exposure to Plasmodium falciparum stimulates HGXPRT T cell reactivity in humans. Methods PBMC and plasma collected from malaria-exposed Indonesians during infection and 7–28 days after anti-malarial therapy, were assessed for HGXPRT recognition using CFSE proliferation, IFNγ ELISPOT assay and ELISA. Results HGXPRT-specific T cell proliferation was found in 44% of patients during acute infection; in 80% of responders both CD4+ and CD8+ T cell subsets proliferated. Antigen-specific T cell proliferation was largely lost within 28 days of parasite clearance. HGXPRT-specific IFN-γ production was more frequent 28 days after treatment than during acute infection. HGXPRT-specific plasma IgG was undetectable even in individuals exposed to malaria for at least two years. Conclusion The prevalence of acute proliferative and convalescent IFNγ responses to HGXPRT demonstrates cellular immunogenicity in humans. Further studies to determine minimal HGXPRT epitopes, the specificity of responses for Plasmodia and associations with protection are required. Frequent and robust T cell proliferation, high sequence conservation among Plasmodium species and absent IgG responses distinguish HGXPRT from other malaria antigens.

  20. An intersubunit disulfide bridge stabilizes the tetrameric nucleoside diphosphate kinase of Aquifex aeolicus.

    Science.gov (United States)

    Boissier, Fanny; Georgescauld, Florian; Moynié, Lucile; Dupuy, Jean-William; Sarger, Claude; Podar, Mircea; Lascu, Ioan; Giraud, Marie-France; Dautant, Alain

    2012-06-01

    The nucleoside diphosphate kinase (Ndk) catalyzes the reversible transfer of the γ-phosphate from nucleoside triphosphate to nucleoside diphosphate. Ndks form hexamers or two types of tetramers made of the same building block, namely, the common dimer. The secondary interfaces of the Type I tetramer found in Myxococcus xanthus Ndk and of the Type II found in Escherichia coli Ndk involve the opposite sides of subunits. Up to now, the few available structures of Ndk from thermophiles were hexameric. Here, we determined the X-ray structures of four crystal forms of the Ndk from the hyperthermophilic bacterium Aquifex aeolicus (Aa-Ndk). Aa-Ndk displays numerous features of thermostable proteins and is made of the common dimer but it is a tetramer of Type I. Indeed, the insertion of three residues in a surface-exposed spiral loop, named the Kpn-loop, leads to the formation of a two-turn α-helix that prevents both hexamer and Type II tetramer assembly. Moreover, the side chain of the cysteine at position 133, which is not present in other Ndk sequences, adopts two alternate conformations. Through the secondary interface, each one forms a disulfide bridge with the equivalent Cys133 from the neighboring subunit. This disulfide bridge was progressively broken during X-ray data collection by radiation damage. Such crosslinks counterbalance the weakness of the common-dimer interface. A 40% decrease of the kinase activity at 60°C after reduction and alkylation of the protein corroborates the structural relevance of the disulfide bridge on the tetramer assembly and enzymatic function.

  1. The Metabolism of 2-Caroboxy-4-Ketopentitol Diphosphate

    Energy Technology Data Exchange (ETDEWEB)

    Moses, V.; Calvin, M.

    1958-07-15

    2-Carboxy-4-ketopentitol is converted enzymatically by a cell-free preparation from spinach leaves into a substance undergoing acid-lactone interconversion. This substance has no phosphate or letone group and is probably a dicarboxylic, six-carbon sugar acid or the saccharic or saccharinic acid type. The significance of these findings with regard to the metabolic role of 2-carboxy-4-ketopentitol diphosphate is discussed.

  2. Biosynthesis of the diterpenoid lycosantalonol via nerylneryl diphosphate in Solanum lycopersicum.

    Science.gov (United States)

    Matsuba, Yuki; Zi, Jiachen; Jones, A Daniel; Peters, Reuben J; Pichersky, Eran

    2015-01-01

    We recently reported that three genes involved in the biosynthesis of monoterpenes in trichomes, a cis-prenyltransferase named neryl diphosphate synthase 1 (NDPS1) and two terpene synthases (TPS19 and TPS20), are present in close proximity to each other at the tip of chromosome 8 in the genome of the cultivated tomato (Solanum lycopersicum). This terpene gene "cluster" also contains a second cis-prenyltransferase gene (CPT2), three other TPS genes, including TPS21, and the cytochrome P450-oxidoreductase gene CYP71BN1. CPT2 encodes a neryneryl diphosphate synthase. Co-expression in E. coli of CPT2 and TPS21 led to the formation of the diterpene lycosantalene, and co-expression in E. coli of CPT2, TPS21 and CYP71BN1 led to the formation of lycosantalonol, an oxidation product of lycosantalene. Here we show that maximal expression of all three genes occurs in the petiolule part of the leaf, but little expression of these genes occurs in the trichomes present on the petiolules. While lycosantalene or lycosantalonol cannot be detected in the petiolules of wild-type plants (or anywhere else in the plant), lycosantalene and lycosantalonol are detected in petiolules of transgenic tomato plants expressing CPT2 under the control of the 35S CaMV promoter. These results suggest that lycosantalene and lycosantalonol are produced in the petiolules and perhaps in other tissues of wild-type plants, but that low rate of synthesis, controlled by the rate-limiting enzyme CPT2, results in product levels that are too low for detection under our current methodology. It is also possible that these compounds are further modified in the plant. The involvement of CPT2, TPS21 and CYP71BN1 in a diterpenoid biosynthetic pathway outside the trichomes, together with the involvement of other genes in the cluster in the synthesis of monoterpenes in trichomes, indicates that this cluster is further evolving into "sub-clusters" with unique biochemical, and likely physiological, roles.

  3. Activation of thiamin diphosphate and FAD in the phosphatedependent pyruvate oxidase from Lactobacillus plantarum

    OpenAIRE

    Tittmann, Kai; Proske, Daniela; Spinka, Michael; Ghisla, Sandro; Rudolph, Rainer; Hübner, Gerhard; Kern, Gunther

    1998-01-01

    The phosphate- and oxygen-dependent pyruvate oxidase from Lactobacillus plantarum is a homotetrameric enzyme that binds 1 FAD and 1 thiamine diphosphate per subunit. A kinetic analysis of the partial reactions in the overall oxidative conversion of pyruvate to acetyl phosphate and CO2 shows an indirect activation of the thiamine diphosphate by FAD that is mediated by the protein moiety. The rate constant of the initial step, the deprotonation of C2-H of thiamine diphosphate, increases 10-fold...

  4. Molecular cloning and functional expression analysis of a new gene encoding geranylgeranyl diphosphate synthase from hazel (Corylus avellana L. Gasaway).

    Science.gov (United States)

    Wang, Yechun; Miao, Zhiqi; Tang, Kexuan

    2010-10-01

    Geranylgeranyl diphosphate synthase (GGPPS) [EC 2.5.1.29] catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP), which is a key precursor for diterpenes such as taxol. Herein, a full-length cDNA encoding GGPPS (designated as CgGGPPS) was cloned and characterized from hazel (Corylus avellana L. Gasaway), a taxol-producing angiosperms. The full-length cDNA of CgGGPPS was 1515 bp with a 1122 bp open reading frame (ORF) encoding a 373 amino acid polypeptide. The CgGGPPS genomic DNA sequence was also obtained, revealing CgGGPPS gene was not interrupted by an intron. Southern blot analysis indicated that CgGGPPS belonged to a small gene family. Tissue expression pattern analysis indicated that CgGGPPS expressed the highest in leaves. RT-PCR analysis indicated that CgGGPPS expression could be induced by exogenous methyl jasmonate acid. Furthermore, carotenoid accumulation was observed in Escherichia coli carrying pACCAR25ΔcrtE plasmid carrying CgGGPPS. The result revealed that cDNA encoded a functional GGPP synthase.

  5. Targeted cytosine deaminase-uracil phosphoribosyl transferase suicide gene therapy induces small cell lung cancer-specific cytotoxicity and tumor growth delay

    DEFF Research Database (Denmark)

    Christensen, Camilla L; Gjetting, Torben; Poulsen, Thomas Tuxen

    2010-01-01

    Small cell lung cancer (SCLC) is a highly malignant cancer for which there is no curable treatment. Novel therapies are therefore in great demand. In the present study we investigated the therapeutic effect of transcriptionally targeted suicide gene therapy for SCLC based on the yeast cytosine...... deaminase (YCD) gene alone or fused with the yeast uracil phosphoribosyl transferase (YUPRT) gene followed by administration of 5-fluorocytosine (5-FC) prodrug. Experimental design: The YCD gene or the YCD-YUPRT gene was placed under regulation of the SCLC-specific promoter insulinoma-associated 1 (INSM1...

  6. Studies of the ADP/ATP carrier of mitochondria with fluorescent ADP analogue formycin diphosphate.

    Science.gov (United States)

    Graue, C; Klingenberg, M

    1979-06-05

    The ADP/ATP carrier was studied by a fluorescent substrate, formycin diphosphate which is the only fluorescent ADP analogue to bind. Its low quantum yield, short decay time and spectral overlap with tryptophan has as yet prevented its wider use. By incorporating fluorescent acceptors of formycin diphosphate fluorescence, anthracene-maleimide and vinylanthracene, into the membrane, these difficulties were circumvented. Only bound formycin diphosphate transfers energy to the probes so that the secondary emission of these probes is a measure for membrane-bound formycin diphosphate. The fluorescent transfer is inhibited by ADP, bongkrekate and carboxyatractylate whether added before or after incubation of formycin diphosphate showing that only binding to the adenine nucleotide carrier is measured. It also shows directly that the earlier demonstrated ADP fixation by bongkrekate is indeed a displacement into the matrix. The fluorescence decay time of the bound formycin diphosphate is measured as 1.95 ns compared to 0.95 ns of the free formycin diphosphate, indicating that formycin diphosphate is bound at the carrier in a non-polar environment. The depolarization decay time was found to be larger than 15 ns, indicating that carrier-bound formycin diphosphate is immobile within this time period.

  7. PRS5, the Fifth Member of the Phosphoribosyl Pyrophosphate Synthetase Gene Family in Saccharomyces cerevisiae, Is Essential for Cell Viability in the Absence of either PRS1 or PRS3

    OpenAIRE

    Hernando, Yolanda; Parr, Adrian; Schweizer, Michael

    1998-01-01

    In Saccharomyces cerevisiae, an open reading frame, YOL061w, encodes a polypeptide with sequence similarity to the four known 5-phosphoribosyl-1(α)-pyrophosphate synthetase (PRS) genes since it contains a divalent cation binding site and a phosphoribosyl pyrophosphate binding site. We regard YOL061w as the fifth member of the PRS gene family, PRS5. Loss of Prs5p has a significant impact on PRS enzyme activity, causing it to be reduced by 84%. On the other hand, Δprs5 strains are not affected ...

  8. Nuclear magnetic resonance-based quantification of organic diphosphates.

    Science.gov (United States)

    Lenevich, Stepan; Distefano, Mark D

    2011-01-15

    Phosphorylated compounds are ubiquitous in life. Given their central role, many such substrates and analogs have been prepared for subsequent evaluation. Prior to biological experiments, it is typically necessary to determine the concentration of the target molecule in solution. Here we describe a method where concentrations of stock solutions of organic diphosphates and bisphosphonates are quantified using (31)P nuclear magnetic resonance (NMR) spectroscopy with standard instrumentation using a capillary tube with a secondary standard. The method is specific and is applicable down to a concentration of 200 μM. The capillary tube provides the reference peak for quantification and deuterated solvent for locking.

  9. Domain relationships in thiamine diphosphate-dependent enzymes.

    Science.gov (United States)

    Duggleby, Ronald G

    2006-08-01

    Three-dimensional structures have been determined for 13 different enzymes that use thiamine diphosphate (ThDP) as a cofactor. These enzymes fall into five families, where members within a family have similar structures. In different families, there are similarities between some domains that clearly point to a common ancestor for all of these enzymes. Where the enzyme structures differ, evolutionary relationships between families can be discerned. Here, I present an analysis of these families and propose an evolutionary pathway to explain the diversity of structures that are now known.

  10. Synthesis, Properties and Applications of Diazotrifluropropanoyl-Containing Photoactive Analogues of Farnesyl Diphosphate Containing Modified Linkages for Enhanced Stability

    Science.gov (United States)

    Hovlid, Marisa L.; Edelstein, Rebecca L.; Henry, Olivier; Ochocki, Joshua; DeGraw, Amanda; Lenevich, Stepan; Talbot, Trista; Young, Victor G.; Hruza, Alan W.; Lopez-Gallego, Fernando; Labello, Nicholas P.; Strickland, Corey L.; Schmidt-Dannert, Claudia; Distefano, Mark D.

    2009-01-01

    Photoactive analogues of farnesyl diphosphate (FPP) are useful probes in studies of enzymes that employ this molecule as a substrate. Here, we describe the preparation and properties of two new FPP analogues that contain diazotrifluoropropionyl photophores linked to geranyl diphosphate via amide or ester linkages. The amide-linked analogue (3) was synthesized in 32P-labeled form from geraniol in 7 steps. Experiments with Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) showed that 3 is an alternative substrate for the enzyme. Photolysis experiments with [32P]3 demonstrate that this compound labels the β-subunits of both farnesyl- and geranylgeranyltransferase (types 1 and 2). However, the amide-linked probe 3 undergoes a rearrangement to a photochemically unreactive isomeric triazolone upon long term storage making it inconvenient to use. To address this stability issue, the ester-linked analogue 4 was prepared in 6 steps from geraniol. Computational analysis and X-ray crystallographic studies suggest that 4 binds to PFTase in a similar fashion as FPP. Compound 4 is also an alternative substrate for PFTase and a 32P-labeled form selectively photocrosslinks the β-subunit of ScPFTase as well as E. coli farnesyldiphosphate synthase and a germacrene-producing sesquiterpene synthase from Nostoc sp. strain PCC7120 (a cyanobacterial source). Finally, nearly exclusive labeling of ScPFTase in crude E. coli extract was observed, suggesting that [32P]4 manifests significant selectivity and should hence be useful for identifying novel FPP utilizing enzymes in crude protein preparations. PMID:19954434

  11. Bacterial and plant HAD enzymes catalyse a missing phosphatase step in thiamin diphosphate biosynthesis.

    Science.gov (United States)

    Hasnain, Ghulam; Roje, Sanja; Sa, Na; Zallot, Rémi; Ziemak, Michael J; de Crécy-Lagard, Valérie; Gregory, Jesse F; Hanson, Andrew D

    2016-01-15

    The penultimate step of thiamin diphosphate (ThDP) synthesis in plants and many bacteria is dephosphorylation of thiamin monophosphate (ThMP). Non-specific phosphatases have been thought to mediate this step and no genes encoding specific ThMP phosphatases (ThMPases) are known. Comparative genomic analysis uncovered bacterial haloacid dehalogenase (HAD) phosphatase family genes (from subfamilies IA and IB) that cluster on the chromosome with, or are fused to, thiamin synthesis genes and are thus candidates for the missing phosphatase (ThMPase). Three typical candidates (from Anaerotruncus colihominis, Dorea longicatena and Syntrophomonas wolfei) were shown to have efficient in vivo ThMPase activity by expressing them in an Escherichia coli strain engineered to require an active ThMPase for growth. In vitro assays confirmed that these candidates all preferred ThMP to any of 45 other phosphate ester substrates tested. An Arabidopsis thaliana ThMPase homologue (At4g29530) of unknown function whose expression pattern and compartmentation fit with a role in ThDP synthesis was shown to have in vivo ThMPase activity in E. coli and to prefer ThMP to any other substrate tested. However, insertional inactivation of the At4g29530 gene did not affect growth or the levels of thiamin or its phosphates, indicating that Arabidopsis has at least one other ThMPase gene. The Zea mays orthologue of At4g29530 (GRMZM2G035134) was also shown to have ThMPase activity. These data identify HAD genes specifying the elusive ThMPase activity, indicate that ThMPases are substrate-specific rather than general phosphatases and suggest that different evolutionary lineages have recruited ThMPases independently from different branches of the HAD family.

  12. A procedure for the preparation and isolation of nucleoside-5’-diphosphates

    Directory of Open Access Journals (Sweden)

    Heidi J. Korhonen

    2015-04-01

    Full Text Available Tris[bis(triphenylphosphoranylideneammonium] pyrophosphate (PPN pyrophosphate was used in the SN2 displacements of the tosylate ion from 5’-tosylnucleosides to afford nucleoside-5’-diphosphates. Selective precipitation permitted the direct isolation of nucleoside-5’-diphosphates from crude reaction mixtures.

  13. [Interaction of pyruvate dehydrogenase complex from the heart muscle with thiamine diphosphate and its derivatives].

    Science.gov (United States)

    Strumilo, S A; Kiselevskiĭ, Iu V; Taranda, N I; Zabrodskaia, S V; Oparin, D A

    1989-01-01

    Inhibitory effects of 23 thiamin derivatives on the bovine heart pyruvate dehydrogenase complex (PDC) were studied. Oxythiamin diphosphate and tetrahydroxythiamin diphosphate exhibited the most pronounced effect on the PDC activity, affecting the complex by a competitive type of inhibition for thiamin diphosphate (TDP). The apparent affinity of TDP and the anticoenzyme derivatives for apo PDC depended on presence of phosphate and divalent metal ions. Phosphate considerably increased the Km values for TDP (up to 0.17 microM) and the Ki values for oxythiamin diphosphate (0.40 microM) as well as for tetrahydroxythiamin diphosphate (0.23 microM). In presence of Mn2+, Km value for TDP was 3.5-fold lower as compared with Mg2+ containing medium.

  14. Three-dimensional structure of transketolase, a thiamine diphosphate dependent enzyme, at 2.5 A resolution.

    OpenAIRE

    Lindqvist, Y; Schneider, G; Ermler, U.; Sundström, M

    1992-01-01

    The crystal structure of Saccharomyces cerevisiae transketolase, a thiamine diphosphate dependent enzyme, has been determined to 2.5 A resolution. The enzyme is a dimer with the active sites located at the interface between the two identical subunits. The cofactor, vitamin B1 derived thiamine diphosphate, is bound at the interface between the two subunits. The enzyme subunit is built up of three domains of the alpha/beta type. The diphosphate moiety of thiamine diphosphate is bound to the enz...

  15. A copal-8-ol diphosphate synthase from the angiosperm Cistus creticus subsp. creticus is a putative key enzyme for the formation of pharmacologically active, oxygen-containing labdane-type diterpenes.

    Science.gov (United States)

    Falara, Vasiliki; Pichersky, Eran; Kanellis, Angelos K

    2010-09-01

    The resin of Cistus creticus subsp. creticus, a plant native to Crete, is rich in labdane-type diterpenes with significant antimicrobial and cytotoxic activities. The full-length cDNA of a putative diterpene synthase was isolated from a C. creticus trichome cDNA library. The deduced amino acid sequence of this protein is highly similar (59%-70% identical) to type B diterpene synthases from other angiosperm species that catalyze a protonation-initiated cyclization. The affinity-purified recombinant Escherichia coli-expressed protein used geranylgeranyl diphosphate as substrate and catalyzed the formation of copal-8-ol diphosphate. This diterpene synthase, therefore, was named CcCLS (for C. creticus copal-8-ol diphosphate synthase). Copal-8-ol diphosphate is likely to be an intermediate in the biosynthesis of the oxygen-containing labdane-type diterpenes that are abundant in the resin of this plant. RNA gel-blot analysis revealed that CcCLS is preferentially expressed in the trichomes, with higher transcript levels found in glands on young leaves than on fully expanded leaves, while CcCLS transcript levels increased after mechanical wounding. Chemical analyses revealed that labdane-type diterpene production followed a similar pattern, with higher concentrations in trichomes of young leaves and increased accumulation upon wounding.

  16. A Copal-8-ol Diphosphate Synthase from the Angiosperm Cistus creticus subsp. creticus Is a Putative Key Enzyme for the Formation of Pharmacologically Active, Oxygen-Containing Labdane-Type Diterpenes1[OA

    Science.gov (United States)

    Falara, Vasiliki; Pichersky, Eran; Kanellis, Angelos K.

    2010-01-01

    The resin of Cistus creticus subsp. creticus, a plant native to Crete, is rich in labdane-type diterpenes with significant antimicrobial and cytotoxic activities. The full-length cDNA of a putative diterpene synthase was isolated from a C. creticus trichome cDNA library. The deduced amino acid sequence of this protein is highly similar (59%–70% identical) to type B diterpene synthases from other angiosperm species that catalyze a protonation-initiated cyclization. The affinity-purified recombinant Escherichia coli-expressed protein used geranylgeranyl diphosphate as substrate and catalyzed the formation of copal-8-ol diphosphate. This diterpene synthase, therefore, was named CcCLS (for C. creticus copal-8-ol diphosphate synthase). Copal-8-ol diphosphate is likely to be an intermediate in the biosynthesis of the oxygen-containing labdane-type diterpenes that are abundant in the resin of this plant. RNA gel-blot analysis revealed that CcCLS is preferentially expressed in the trichomes, with higher transcript levels found in glands on young leaves than on fully expanded leaves, while CcCLS transcript levels increased after mechanical wounding. Chemical analyses revealed that labdane-type diterpene production followed a similar pattern, with higher concentrations in trichomes of young leaves and increased accumulation upon wounding. PMID:20595348

  17. Towards in vivo regulon kinetics: PurR activation by 5-phosphoribosyl-a-1-pyrophosphate during purine depletion in Lactococcus lactis

    DEFF Research Database (Denmark)

    Jendresen, Christian Bille; Dimitrov, Peter; Gautier, Laurent;

    2014-01-01

    5-phosphoribosyl-a-1-pyrophosphate (PRPP) and individual mRNA levels, whereby unambiguous and homogeneous relations could be obtained for PurR regulated genes, thus linking a specific regulon to a specific metabolite. As PurR activates gene expression upon binding of PRPP, the pur mRNA curves......, for elucidating regulatory networks in vivo....

  18. Structure and Mechanism of the Farnesyl Diphosphate Synthase from Trypanosoma cruzi: Implications for Drug Design

    Energy Technology Data Exchange (ETDEWEB)

    Gabelli,S.; McLellan, J.; Montalvetti, A.; Oldfield, E.; Docampo, R.; Amzel, L.

    2006-01-01

    Typanosoma cruzi, the causative agent of Chagas disease, has recently been shown to be sensitive to the action of the bisphosphonates currently used in bone resorption therapy. These compounds target the mevalonate pathway by inhibiting farnesyl diphosphate synthase (farnesyl pyrophosphate synthase, FPPS), the enzyme that condenses the diphosphates of C{sub 5} alcohols (isopentenyl and dimethylallyl) to form C{sub 10} and C{sub 15} diphosphates (geranyl and farnesyl). The structures of the T. cruzi FPPS (TcFPPS) alone and in two complexes with substrates and inhibitors reveal that following binding of the two substrates and three Mg2+ ions, the enzyme undergoes a conformational change consisting of a hinge-like closure of the binding site. In this conformation, it would be possible for the enzyme to bind a bisphosphonate inhibitor that spans the sites usually occupied by dimethylallyl diphosphate (DMAPP) and the homoallyl moiety of isopentenyl diphosphate. This observation may lead to the design of new, more potent anti-trypanosomal bisphosphonates, because existing FPPS inhibitors occupy only the DMAPP site. In addition, the structures provide an important mechanistic insight: after its formation, geranyl diphosphate can swing without leaving the enzyme, from the product site to the substrate site to participate in the synthesis of farnesyl diphosphate.

  19. Overexpression of an isopentenyl diphosphate isomerase gene to enhance trans-polyisoprene production in Eucommia ulmoides Oliver

    Directory of Open Access Journals (Sweden)

    Chen Ren

    2012-10-01

    Full Text Available Abstract Background Natural rubber produced by plants, known as polyisoprene, is the most widely used isoprenoid polymer. Plant polyisoprenes can be classified into two types; cis-polyisoprene and trans-polyisoprene, depending on the type of polymerization of the isoprene unit. More than 2000 species of higher plants produce latex consisting of cis-polyisoprene. Hevea brasiliensis (rubber tree produces cis-polyisoprene, and is the key source of commercial rubber. In contrast, relatively few plant species produce trans-polyisoprene. Currently, trans-polyisoprene is mainly produced synthetically, and no plant species is used for its commercial production. Results To develop a plant-based system suitable for large-scale production of trans-polyisoprene, we selected a trans-polyisoprene-producing plant, Eucommia ulmoides Oliver, as the target for genetic transformation. A full-length cDNA (designated as EuIPI, Accession No. AB041629 encoding isopentenyl diphosphate isomerase (IPI was isolated from E. ulmoides. EuIPI consisted of 1028 bp with a 675-bp open reading frame encoding a protein with 224 amino acid residues. EuIPI shared high identity with other plant IPIs, and the recombinant protein expressed in Escherichia coli showed IPI enzymatic activity in vitro. EuIPI was introduced into E. ulmoides via Agrobacterium-mediated transformation. Transgenic lines of E. ulmoides overexpressing EuIPI showed increased EuIPI expression (up to 19-fold that of the wild-type and a 3- to 4-fold increase in the total content of trans-polyisoprenes, compared with the wild-type (non-transgenic root line control. Conclusions Increasing the expression level of EuIPI by overexpression increased accumulation of trans-polyisoprenes in transgenic E. ulmoides. IPI catalyzes the conversion of isopentenyl diphosphate to its highly electrophilic isomer, dimethylallyl diphosphate, which is the first step in the biosynthesis of all isoprenoids, including polyisoprene. Our

  20. Molecular cloning and characterization of three isoprenyl diphosphate synthase genes from alfalfa.

    Science.gov (United States)

    Sun, Yan; Long, Ruicai; Kang, Junmei; Zhang, Tiejun; Zhang, Ze; Zhou, He; Yang, Qingchuan

    2013-02-01

    Isoprenoid is the precursor for the biosynthesis of saponins, abscisic acid, gibberellins, chlorophylls and many other products in plants. Saponins are an important group of bioactive plant natural products. The alfalfa (Medicago sativa L.) saponins are glycosides of different triterpene aglycones and possess many biological activities. We isolated three genes (MsFPPS, MsGPPS and MsGGPPS) encoding isoprenyl diphosphate synthases (IDS) from alfalfa via a homology-based PCR approach. The enzyme activity assay of purified recombined MsFPPS and MsGGPPS expressed in Escherichia coli indicated that they all had IDS activity. Expression analysis of the three genes in different alfalfa tissues using real time PCR displayed that they were expressed in all tissues although they had a different expression patterns. MsFPPS and MsGPS displayed a significant increase in transcript level in response to methyl jasmonate, but the transcript level of MsGGPPS decreased obviously. To elucidate the functions of the three IDSs, their overexpression driven by a constitutive cauliflower mosaic virus-35S promoter in tobacco plants was applied and analyzed. The T(0) transgenic plants of MsFPPS showed high levels of squalene content when compared with control. However, no differences were detected in T(0) transgenic plants of MsGPPS and MsGGPPS. In addition, the overexpression of MsFPPS induced senescence response in transgenic plant leaves. This result may indicate that MsFPPS performs a role not only in phytosterol and triterpene biosynthesis, but also in growth regulation.

  1. Changing ribulose diphosphate carboxylase/oxygenase activity in ripening tomato fruit.

    Science.gov (United States)

    Bravdo, B A; Palgi, A; Lurie, S

    1977-08-01

    Tomato fruit (Lycopersicum esculentum Mill) from green, pink, and red stages were assayed for changes in the activity of ribulose diphosphate carboxylase and oxygenase, phosphoenolpyruvate carboxylase, changes in the levels of glycolate and respiratory gas exchange. The ribulose diphosphate carboxylase activity decreased as the fruit ripened. By comparison, the ribulose diphosphate oxygenase activity increased during the transition from the green to the pink stage, and declined afterward. The changes in the endogenous glycolate levels and the respiratory gas exchange, as observed at different stages of ripening, resembled the changes in the ribulose diphosphate oxygenase activity. The utilization of glycolate in further metabolic activity may result in the formation of peroxidases required for the onset of ripening.

  2. Acanthamoeba polyphaga mimivirus NDK: preliminary crystallographic analysis of the first viral nucleoside diphosphate kinase

    OpenAIRE

    Jeudy, Sandra; Coutard, Bruno; Lebrun, Régine; Abergel, Chantal

    2005-01-01

    A. polyphaga mimivirus, the largest known double-stranded DNA virus, is the first virus to exhibit a nucleoside diphosphate kinase gene. The expression and crystallization of the viral NDK are reported.

  3. Metabolic engineering of the Stevia rebaudiana ent-kaurene biosynthetic pathway in recombinant Escherichia coli.

    Science.gov (United States)

    Kong, Min Kyung; Kang, Hyun-Jun; Kim, Jin Ho; Oh, Soon Hwan; Lee, Pyung Cheon

    2015-11-20

    The ent-kaurene is a dedicated precursor pool and is responsible for synthesizing natural sweeteners such as steviol glycosides. In this study, to produce ent-kaurene in Escherichia coli, we modularly constructed and expressed two ent-kaurene genes encoding ent-copalyl diphosphate synthase (CPPS) and ent-kaurene synthase (KS) from Stevia rebaudiana known as a typical plant producing steviol glycoside. The CPPS and KS from S. rebaudiana were functionally expressed in a heterologous host E. coli. Furthermore, in order to enhance ent-kaurene production in E. coli, six geranylgeranyl diphosphate synthases (GGPPS) from various microorganisms and eight strains of E. coli as host were compared by measuring ent-kaurene production. The highest ent-kaurene production of approximately 41.1mg/L was demonstrated in E. coli strain MG1655 co-expressing synthetic CPPS-KS module and GGPPS from Rhodobacter sphaeroides. The ent-kaurene production was further increased up to 179.6 mg/L by overexpression of the three key enzymes for isoprenoid precursor, 1-deoxyxylulose-5-phosphate synthase (DXS), farnesyl diphosphate synthase (IspA) and isopentenyl diphosphate isomerase (IDI) from E. coli. Finally, the highest titer of ent-kaurene (578 mg/L) with a specific yield of ent-kaurene of 143.5mg/g dry cell weight was obtained by culturing E. coli strain MG1655 co-expressing the ent-kaurene module, DXS, IDI and IspA in 1L bioreactor containing 20 g/L glycerol.

  4. A standard numbering scheme for thiamine diphosphate-dependent decarboxylases

    Directory of Open Access Journals (Sweden)

    Vogel Constantin

    2012-11-01

    Full Text Available Abstract Background Standard numbering schemes for families of homologous proteins allow for the unambiguous identification of functionally and structurally relevant residues, to communicate results on mutations, and to systematically analyse sequence-function relationships in protein families. Standard numbering schemes have been successfully implemented for several protein families, including lactamases and antibodies, whereas a numbering scheme for the structural family of thiamine-diphosphate (ThDP -dependent decarboxylases, a large subfamily of the class of ThDP-dependent enzymes encompassing pyruvate-, benzoylformate-, 2-oxo acid-, indolpyruvate- and phenylpyruvate decarboxylases, benzaldehyde lyase, acetohydroxyacid synthases and 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD is still missing. Despite a high structural similarity between the members of the ThDP-dependent decarboxylases, their sequences are diverse and make a pairwise sequence comparison of protein family members difficult. Results We developed and validated a standard numbering scheme for the family of ThDP-dependent decarboxylases. A profile hidden Markov model (HMM was created using a set of representative sequences from the family of ThDP-dependent decarboxylases. The pyruvate decarboxylase from S. cerevisiae (PDB: 2VK8 was chosen as a reference because it is a well characterized enzyme. The crystal structure with the PDB identifier 2VK8 encompasses the structure of the ScPDC mutant E477Q, the cofactors ThDP and Mg2+ as well as the substrate analogue (2S-2-hydroxypropanoic acid. The absolute numbering of this reference sequence was transferred to all members of the ThDP-dependent decarboxylase protein family. Subsequently, the numbering scheme was integrated into the already established Thiamine-diphosphate dependent Enzyme Engineering Database (TEED and was used to systematically analyze functionally and structurally relevant

  5. Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W. (UIUC); (Iowa State); (Penn)

    2011-09-20

    The structure of ent-copalyl diphosphate synthase reveals three {alpha}-helical domains ({alpha}, {beta} and {gamma}), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the {beta}{gamma} domains in ent-copalyl diphosphate synthase but exclusively in the {alpha} domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.

  6. Ribulose diphosphate carboxylase of the cyanobacterium Spirulina platensis

    Energy Technology Data Exchange (ETDEWEB)

    Terekhova, I.V.; Chernyad' ev, I.I.; Doman, N.G.

    1986-11-20

    The ribulose diphosphate (RDP) carboxylase activity of the cyanobacterium Spirulina platensis is represented by two peaks when a cell homogenate is centrifuged in a sucrose density gradient. In the case of differential centrifugation (40,000 g, 1 h), the activity of the enzyme was distributed between the supernatant liquid (soluble form) and the precipitate (carboxysomal form). From the soluble fraction, in which 80-95% of the total activity of the enzyme is concentrated, electrophoretically homogeneous RDP carboxylase was isolated by precipitation with ammonium sulfate and centrifugation in a sucrose density gradient. The purified enzyme possessed greater electrophoretic mobility in comparison with the RDP carboxylase of beans Vicia faba. The molecular weight of the enzyme, determined by gel filtration, was 450,000. The enzyme consists of monotypic subunits with a molecular weight of 53,000. The small subunits were not detected in electrophoresis in polyacrylamide gel in the presence of SDS after fixation and staining of the gels by various methods.

  7. Crystal structures of phosphoketolase: thiamine diphosphate-dependent dehydration mechanism.

    Science.gov (United States)

    Suzuki, Ryuichiro; Katayama, Takane; Kim, Byung-Jun; Wakagi, Takayoshi; Shoun, Hirofumi; Ashida, Hisashi; Yamamoto, Kenji; Fushinobu, Shinya

    2010-10-29

    Thiamine diphosphate (ThDP)-dependent enzymes are ubiquitously present in all organisms and catalyze essential reactions in various metabolic pathways. ThDP-dependent phosphoketolase plays key roles in the central metabolism of heterofermentative bacteria and in the pentose catabolism of various microbes. In particular, bifidobacteria, representatives of beneficial commensal bacteria, have an effective glycolytic pathway called bifid shunt in which 2.5 mol of ATP are produced per glucose. Phosphoketolase catalyzes two steps in the bifid shunt because of its dual-substrate specificity; they are phosphorolytic cleavage of fructose 6-phosphate or xylulose 5-phosphate to produce aldose phosphate, acetyl phosphate, and H(2)O. The phosphoketolase reaction is different from other well studied ThDP-dependent enzymes because it involves a dehydration step. Although phosphoketolase was discovered more than 50 years ago, its three-dimensional structure remains unclear. In this study we report the crystal structures of xylulose 5-phosphate/fructose 6-phosphate phosphoketolase from Bifidobacterium breve. The structures of the two intermediates before and after dehydration (α,β-dihydroxyethyl ThDP and 2-acetyl-ThDP) and complex with inorganic phosphate give an insight into the mechanism of each step of the enzymatic reaction.

  8. Propioin synthesis using thiamine diphosphate-dependent enzymes.

    Science.gov (United States)

    Mikolajek, Renaud J; Spiess, Antje C; Pohl, Martina; Büchs, Jochen

    2009-01-01

    Benzaldehyde lyase (BAL, EC 4.1.2.38) from Pseudomonas fluorescens and benzoylformate decarboxylase (BFD, EC 4.1.1.7) from Pseudomonas putida are thiamine diphosphate-dependent enzymes. These enzymes share a common tetrameric structure and catalyze various C--C-bond forming and breaking reactions. Here we describe a detailed study of the asymmetric synthesis of propioin from propanal catalyzed by BAL or BFD in aqueous solution in a batch reactor. Both enzymes are deactivated in the presence of high concentration of propanal. Compared to BAL, BFD is more stable under reaction conditions as well as during storage. The kinetic studies showed a typical Michaelis-Menten kinetic for BAL with a maximal specific reaction rate of 26.2 U/mg and an unusually high K(M) of 415 mM, whereas the v/[S]-plot for BFD is almost linear in the concentration range (100-1500 mM) investigated. Both enzymes produce propioin with opposite enantiomeric excess: BAL produced the (S)-propioin (ee of 35%), whereas BFD yielded the (R)-enantiomer (ee of 67%).

  9. Thiamin diphosphate in biological chemistry: new aspects of thiamin metabolism, especially triphosphate derivatives acting other than as cofactors.

    Science.gov (United States)

    Bettendorff, Lucien; Wins, Pierre

    2009-06-01

    Prokaryotes, yeasts and plants synthesize thiamin (vitamin B1) via complex pathways. Animal cells capture the vitamin through specific high-affinity transporters essential for internal thiamin homeostasis. Inside the cells, thiamin is phosphorylated to higher phosphate derivatives. Thiamin diphosphate (ThDP) is the best-known thiamin compound because of its role as an enzymatic cofactor. However, in addition to ThDP, at least three other thiamin phosphates occur naturally in most cells: thiamin monophosphate, thiamin triphosphate (ThTP) and the recently discovered adenosine thiamin triphosphate. It has been suggested that ThTP has a specific neurophysiological role, but recent data favor a much more basic metabolic function. During amino acid starvation, Escherichia coli accumulate ThTP, possibly acting as a signal involved in the adaptation of the bacteria to changing nutritional conditions. In animal cells, ThTP can phosphorylate some proteins, but the physiological significance of this mechanism remains unknown. Adenosine thiamin triphosphate, recently discovered in E. coli, accumulates during carbon starvation and might act as an alarmone. Among the proteins involved in thiamin metabolism, thiamin transporters, thiamin pyrophosphokinase and a soluble 25-kDa thiamin triphosphatase have been characterized at the molecular level, in contrast to thiamin mono- and diphosphatases whose specificities remain to be proven. A soluble enzyme catalyzing the synthesis of adenosine thiamin triphosphate from ThDP and ADP or ATP has been partially characterized in E. coli, but the mechanism of ThTP synthesis remains elusive. The data reviewed here illustrate the complexity of thiamin biochemistry, which is not restricted to the cofactor role of ThDP.

  10. Two Eucommia farnesyl diphosphate synthases exhibit distinct enzymatic properties leading to end product preferences.

    Science.gov (United States)

    Kajiura, Hiroyuki; Suzuki, Nobuaki; Tokumoto, Yuji; Yoshizawa, Takuya; Takeno, Shinya; Fujiyama, Kazuhito; Kaneko, Yoshinobu; Matsumura, Hiroyoshi; Nakazawa, Yoshihisa

    2017-08-01

    Farnesyl diphosphate synthase (FPS) is an essential enzyme in the biosynthesis of prenyl precursors for the production of primary and secondary metabolites, including sterols, dolichols, carotenoids and ubiquinones, and for the modification of proteins. Here we identified and characterized two FPSs (EuFPS1 and EuFPS2) from the plant Eucommia ulmoides. The EuFPSs had seven highly conserved prenyltransferase-specific domains that are critical for activity. Complementation and biochemical analyses using bacterially produced recombinant EuFPS isoforms showed that the EuFPSs had FPP synthesis activities both in vivo and in vitro. In addition to the typical reaction mechanisms of FPS, EuFPSs utilized farnesyl diphosphate (FPP) as an allylic substrate and participated in further elongation of the isoprenyl chain, resulting in the synthesis of geranylgeranyl diphosphate. However, despite the high amino acid similarities between the two EuFPS isozymes, their specific activities, substrate preferences, and final reaction products were different. The use of dimethylallyl diphosphate (DMAPP) as an allylic substrate highlighted the differences between the two enzymes: depending on the pH, the metal ion cofactor, and the cofactor concentration, EuFPS2 accumulated geranyl diphosphate as an intermediate product at a constant rate, whereas EuFPS1 synthesized little geranyl diphosphate. The reaction kinetics of the EuFPSs demonstrated that isopentenyl diphosphate and DMAPP were used both as substrates and as inhibitors of EuFPS activity. Taken together, the results indicate that the biosynthesis of FPP is highly regulated by various factors indispensable for EuFPS reactions in plants. Copyright © 2017. Published by Elsevier B.V.

  11. Metal ions control product specificity of isoprenyl diphosphate synthases in the insect terpenoid pathway

    Science.gov (United States)

    Frick, Sindy; Nagel, Raimund; Schmidt, Axel; Bodemann, René R.; Rahfeld, Peter; Pauls, Gerhard; Brandt, Wolfgang; Gershenzon, Jonathan; Boland, Wilhelm; Burse, Antje

    2013-01-01

    Isoprenyl diphosphate synthases (IDSs) produce the ubiquitous branched-chain diphosphates of different lengths that are precursors of all major classes of terpenes. Typically, individual short-chain IDSs (scIDSs) make the C10, C15, and C20 isoprenyl diphosphates separately. Here, we report that the product length synthesized by a single scIDS shifts depending on the divalent metal cofactor present. This previously undescribed mechanism of carbon chain-length determination was discovered for a scIDS from juvenile horseradish leaf beetles, Phaedon cochleariae. The recombinant enzyme P. cochleariae isoprenyl diphosphate synthase 1 (PcIDS1) yields 96% C10-geranyl diphosphate (GDP) and only 4% C15-farnesyl diphosphate (FDP) in the presence of Co2+ or Mn2+ as a cofactor, whereas it yields only 18% C10 GDP but 82% C15 FDP in the presence of Mg2+. In reaction with Co2+, PcIDS1 has a Km of 11.6 μM for dimethylallyl diphosphate as a cosubstrate and 24.3 μM for GDP. However, with Mg2+, PcIDS1 has a Km of 1.18 μM for GDP, suggesting that this substrate is favored by the enzyme under such conditions. RNAi targeting PcIDS1 revealed the participation of this enzyme in the de novo synthesis of defensive monoterpenoids in the beetle larvae. As an FDP synthase, PcIDS1 could be associated with the formation of sesquiterpenes, such as juvenile hormones. Detection of Co2+, Mn2+, or Mg2+ in the beetle larvae suggests flux control into C10 vs. C15 isoprenoids could be accomplished by these ions in vivo. The dependence of product chain length of scIDSs on metal cofactor identity introduces an additional regulation for these branch point enzymes of terpene metabolism. PMID:23440195

  12. An enzyme-coupled continuous fluorescence assay for farnesyl diphosphate synthases.

    Science.gov (United States)

    Dozier, Jonathan K; Distefano, Mark D

    2012-02-01

    Farnesyl diphosphate synthase (FDPS) catalyzes the conversion of isopentenyl diphosphate and dimethylallyl diphosphate to farnesyl diphosphate, a crucial metabolic intermediate in the synthesis of cholesterol, ubiquinone, and prenylated proteins; consequently, much effort has gone into developing inhibitors that target FDPS. Currently most FDPS assays either use radiolabeled substrates and are discontinuous or monitor pyrophosphate release and not farnesyl diphosphate (FPP) creation. Here we report the development of a continuous coupled enzyme assay for FDPS activity that involves the subsequent incorporation of the FPP product of that reaction into a peptide via the action of protein farnesyltransferase (PFTase). By using a dansylated peptide whose fluorescence quantum yield increases upon farnesylation, the rate of FDPS-catalyzed FPP production can be measured. We show that this assay is more sensitive than existing coupled assays, that it can be used to conveniently monitor FDPS activity in a 96-well plate format, and that it can reproduce IC(50) values for several previously reported FDPS inhibitors. This new method offers a simple, safe, and continuous method to assay FDPS activity that should greatly facilitate the screening of inhibitors of this important target.

  13. Examination of the thiamin diphosphate binding site in yeast transketolase by site-directed mutagenesis.

    Science.gov (United States)

    Meshalkina, L; Nilsson, U; Wikner, C; Kostikowa, T; Schneider, G

    1997-03-01

    The role of two conserved amino acid residues in the thiamin diphosphate binding site of yeast transketolase has been analyzed by site-directed mutagenesis. Replacement of E162, which is part of a cluster of glutamic acid residues at the subunit interface, by alanine or glutamine results in mutant enzymes with most catalytic properties similar to wild-type enzyme. The two mutant enzymes show, however, significant increases in the K0.5 values for thiamin diphosphate in the absence of substrate and in the lag of the reaction progress curves. This suggests that the interaction of E162 with residue E418, and possibly E167, from the second subunit is important for formation and stabilization of the transketolase dimer. Replacement of the conserved residue D382, which is buried upon binding of thiamin diphosphate, by asparagine and alanine, results in mutant enzymes severely impaired in thiamin diphosphate binding and catalytic efficiency. The 25-80-fold increase in K0.5 for thiamin diphosphate suggests that D382 is involved in cofactor binding, probably by electrostatic compensation of the positive charge of the thiazolium ring and stabilization of a flexible loop at the active site. The decrease in catalytic activities in the D382 mutants indicates that this residue might also be important in subsequent steps in catalysis.

  14. The role of the C-terminal region on the oligomeric state and enzymatic activity of Trypanosoma cruzi hypoxanthine phosphoribosyl transferase.

    Science.gov (United States)

    Valsecchi, Wanda M; Cousido-Siah, Alexandra; Defelipe, Lucas A; Mitschler, André; Podjarny, Alberto; Santos, Javier; Delfino, José M

    2016-06-01

    Hypoxanthine phosphoribosyl transferase from Trypanosoma cruzi (TcHPRT) is a critical enzyme for the survival of the parasite. This work demonstrates that the full-length form in solution adopts a stable and enzymatically active tetrameric form, exhibiting large inter-subunit surfaces. Although this protein irreversibly aggregates during unfolding, oligomerization is reversible and can be modulated by low concentrations of urea. When the C-terminal region, which is predicted as a disordered stretch, is excised by proteolysis, TcHPRT adopts a dimeric state, suggesting that the C-terminal region acts as a main guide for the quaternary arrangement. These results are in agreement with X-ray crystallographic data presented in this work. On the other hand, the C-terminal region exhibits a modulatory role on the enzyme, as attested by the enhanced activity observed for the dimeric form. Bisphosphonates act as substrate-mimetics, uncovering long-range communications among the active sites. All in all, this work contributes to establish new ways applicable to the design of novel inhibitors that could eventually result in new drugs against parasitic diseases.

  15. Clinical implications of thymidylate synthetase, dihydropyrimidine dehydrogenase and orotate phosphoribosyl transferase activity levels in colorectal carcinoma following radical resection and administration of adjuvant 5-FU chemotherapy

    Directory of Open Access Journals (Sweden)

    Ishikawa Masashi

    2008-07-01

    Full Text Available Abstract Bckground A number of studies have investigated whether the activity levels of enzymes involved in 5-fluorouracil (5-FU metabolism are prognostic factors for survival in patients with colorectal carcinoma. Most reports have examined thymidylate synthetase (TS and dihydropyrimidine dehydrogenase (DPD in unresectable or metastatic cases, therefore it is unclear whether the activity of these enzymes is of prognostic value in colorectal cancer patients treated with radical resection and adjuvant chemotherapy with 5-FU. Methods This study examined fresh frozen specimens of colorectal carcinoma from 40 patients who had undergone curative operation and were orally administered adjuvant tegafur/uracil (UFT chemotherapy. TS, DPD and orotate phosphoribosyl transferase (OPRT activities were assayed in cancer tissue and adjacent normal tissue and their association with clinicopathological variables was investigated. In addition, the relationships between TS, DPD and OPRT activities and patient survival were examined to determine whether any of these enzymes could be useful prognostic factors. Results While there was no clear relationship between pathological findings and TS or DPD activity, OPRT activity was significantly lower in tumors with lymph node metastasis than in tumors lacking lymph node metastasis. Postoperative survival was significantly better in the groups with low TS activity and/or high OPRT activity. Conclusion TS and OPRT activity levels in tumor tissue may be important prognostic factors for survival in Dukes' B and C colorectal carcinoma with radical resection and adjuvant chemotherapy with UFT.

  16. Expression cloning of different bacterial phosphatase-encoding genes by histochemical screening of genomic libraries onto an indicator medium containing phenolphthalein diphosphate and methyl green.

    Science.gov (United States)

    Riccio, M L; Rossolini, G M; Lombardi, G; Chiesurin, A; Satta, G

    1997-02-01

    A system for expression cloning of bacterial phosphatase-encoding genes has been developed, and its potential has been investigated. The system is based on histochemical screening of bacterial genomic libraries, constructed in an Escherichia coli multicopy plasmid vector, for phosphatase-producing clones using an indicator medium (named TPMG) made of Tryptose-Phosphate agar supplemented with the phosphatase substrate phenolphthalein diphosphate and the stain methyl green. To test the performance of this system, three genomic libraries were constructed from bacterial strains of different species which showed different patterns of phosphatase activity, and were screened using the TPMG medium. Following a partial screening, three different phosphatase-encoding genes (respectively encoding a class A non-specific acid phosphatase, an acid-hexose phosphatase and a non-specific alkaline phosphatase) were shotgun-cloned from the above libraries, indicating that the TPMG-based expression cloning system can be useful for rapid isolation of different bacterial phosphatase-encoding genes.

  17. Structures of Bacteroides fragilis uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (BfLpxA).

    Science.gov (United States)

    Ngo, Alice; Fong, Kai T; Cox, Daniel L; Chen, Xi; Fisher, Andrew J

    2015-05-01

    Uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA) catalyzes a reversible reaction for adding an O-acyl group to the GlcNAc in UDP-GlcNAc in the first step of lipid A biosynthesis. Lipid A constitutes a major component of lipopolysaccharides, also referred to as endotoxins, which form the outer monolayer of the outer membrane of Gram-negative bacteria. Ligand-free and UDP-GlcNAc-bound crystal structures of LpxA from Bacteroides fragilis NCTC 9343, the most common pathogenic bacteria found in abdominal abscesses, have been determined and are presented here. The enzyme crystallizes in a cubic space group, with the crystallographic threefold axis generating the biological functional homotrimer and with each monomer forming a nine-rung left-handed β-helical (LβH) fold in the N-terminus followed by an α-helical motif in the C-terminus. The structure is highly similar to LpxA from other organisms. Yet, despite sharing a similar LβH structure with LpxAs from Escherichia coli and others, previously unseen calcium ions are observed on the threefold axis in B. fragilis LpxA to help stabilize the trimeric assembly.

  18. Insights into the Thiamine Diphosphate Enzyme Activation Mechanism: Computational Model for Transketolase Using a Quantum Mechanical/Molecular Mechanical Method.

    Science.gov (United States)

    Nauton, Lionel; Hélaine, Virgil; Théry, Vincent; Hecquet, Laurence

    2016-04-12

    We propose the first computational model for transketolase (TK), a thiamine diphosphate (ThDP)-dependent enzyme, using a quantum mechanical/molecular mechanical method on the basis of crystallographic TK structures from yeast and Escherichia coli, together with experimental kinetic data reported in the literature with wild-type and mutant TK. This model allowed us to define a new route for ThDP activation in the enzyme environment. We evidenced a strong interaction between ThDP and Glu418B of the TK active site, itself stabilized by Glu162A. The crucial point highlighted here is that deprotonation of ThDP C2 is not performed by ThDP N4' as reported in the literature, but by His481B, involving a HOH688A molecule bridge. Thus, ThDP N4' is converted from an amino form to an iminium form, ensuring the stabilization of the C2 carbanion or carbene. Finally, ThDP activation proceeds via an intermolecular process and not by an intramolecular one as reported in the literature. More generally, this proposed ThDP activation mechanism can be applied to some other ThDP-dependent enzymes and used to define the entire TK mechanism with donor and acceptor substrates more accurately.

  19. Metabolic engineering for improving anthranilate synthesis from glucose in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Gosset Guillermo

    2009-04-01

    Full Text Available Abstract Background Anthranilate is an aromatic amine used industrially as an intermediate for the synthesis of dyes, perfumes, pharmaceuticals and other classes of products. Chemical synthesis of anthranilate is an unsustainable process since it implies the use of nonrenewable benzene and the generation of toxic by-products. In Escherichia coli anthranilate is synthesized from chorismate by anthranilate synthase (TrpED and then converted to phosphoribosyl anthranilate by anthranilate phosphoribosyl transferase to continue the tryptophan biosynthetic pathway. With the purpose of generating a microbial strain for anthranilate production from glucose, E. coli W3110 trpD9923, a mutant in the trpD gene that displays low anthranilate producing capacity, was characterized and modified using metabolic engineering strategies. Results Sequencing of the trpED genes from E. coli W3110 trpD9923 revealed a nonsense mutation in the trpD gene, causing the loss of anthranilate phosphoribosyl transferase activity, but maintaining anthranilate synthase activity, thus causing anthranilate accumulation. The effects of expressing genes encoding a feedback inhibition resistant version of the enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (aroGfbr, transketolase (tktA, glucokinase (glk and galactose permease (galP, as well as phosphoenolpyruvate:sugar phosphotransferase system (PTS inactivation on anthranilate production capacity, were evaluated. In shake flask experiments with minimal medium, strains W3110 trpD9923 PTS- and W3110 trpD9923/pJLBaroGfbrtktA displayed the best production parameters, accumulating 0.70–0.75 g/L of anthranilate, with glucose-yields corresponding to 28–46% of the theoretical maximum. To study the effects of extending the growth phase on anthranilate production a fed-batch fermentation process was developed using complex medium, where strain W3110 trpD9923/pJLBaroGfbrtktA produced 14 g/L of anthranilate in 34 hours

  20. Fruit color mutants in tomato reveal a function of the plastidial isopentenyl diphosphate isomerase (IDI1) in carotenoid biosynthesis

    Science.gov (United States)

    Isoprenoids are a large class of compounds that are present in all living organisms. They are derived from the 5C building blocks isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In plants, IPP is synthesized in the cytoplasm from mevalonic acid via the “MVA pathway” a...

  1. Phosphorylation of nm23/nucleoside diphosphate kinase by casein kinase 2 in vitro

    DEFF Research Database (Denmark)

    Engel, M; Issinger, O G; Lascu, I;

    1994-01-01

    We have investigated phosphorylation of human nucleoside diphosphate kinase (NDPK) and of homologous NDPK from different species by human casein kinase 2 (CK-2). The human NDPK isotypes A and B were phosphorylated by CK-2 in vitro both when the purified proteins and total lysate of HL-60 leukemia...

  2. ACTIVATION OF G-PROTEINS BY RECEPTOR-STIMULATED NUCLEOSIDE DIPHOSPHATE KINASE IN DICTYOSTELIUM

    NARCIS (Netherlands)

    Bominaar, Anthony A.; Molijn, Anco C.; Pestel, Martine; Veron, Michel; Haastert, Peter J.M. van

    Recently, interest in the enzyme nucleoside diphosphate kinase (EC 2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase

  3. The effect of ticlopidine administration to humans on the binding of adenosine diphosphate to blood platelets

    NARCIS (Netherlands)

    Lips, J.P.M.; Sixma, J.J.; Schiphorst, M.E.

    1980-01-01

    Administration of Ticlopidine to human volunteers resulted in a prolonged bleeding time and decreased or absent aggregation of platelets with collagen and epinephrine. Adenosine diphosphate (ADP) induced platelet aggregation was initiated by a normal shape change, but the rate of the first wave of a

  4. Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Georgescauld, Florian; Moynie, Lucile; Habersetzer, Johann;

    2013-01-01

    Most nucleoside diphosphate kinases (NDPKs) are hexamers. The C-terminal tail interacting with the neighboring subunits is crucial for hexamer stability. In the NDPK from Mycobacterium tuberculosis (Mt) this tail is missing. The quaternary structure of Mt-NDPK is essential for full enzymatic acti...

  5. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity.

    Science.gov (United States)

    Yang, Ting; Gao, Liping; Hu, Hao; Stoopen, Geert; Wang, Caiyun; Jongsma, Maarten A

    2014-12-26

    Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1'-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 μm. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ∼100 μm, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12-0.16 μg h(-1) g(-1) fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate.

  6. Two solanesyl diphosphate synthases with different subcellular localizations and their respective physiological roles in Oryza sativa.

    Science.gov (United States)

    Ohara, Kazuaki; Sasaki, Kanako; Yazaki, Kazufumi

    2010-06-01

    Long chain prenyl diphosphates are crucial biosynthetic precursors of ubiquinone (UQ) in many organisms, ranging from bacteria to humans, as well as precursors of plastoquinone in photosynthetic organisms. The cloning and characterization of two solanesyl diphosphate synthase genes, OsSPS1 and OsSPS2, in Oryza sativa is reported here. OsSPS1 was highly expressed in root tissue whereas OsSPS2 was found to be high in both leaves and roots. Enzymatic characterization using recombinant proteins showed that both OsSPS1 and OsSPS2 could produce solanesyl diphosphates as their final product, while OsSPS1 showed stronger activity than OsSPS2. However, an important biological difference was observed between the two genes: OsSPS1 complemented the yeast coq1 disruptant, which does not form UQ, whereas OsSPS2 only very weakly complemented the growth defect of the coq1 mutant. HPLC analyses showed that both OsSPS1 and OsSPS2 yeast transformants produced UQ9 instead of UQ6, which is the native yeast UQ. According to the complementation study, the UQ9 levels in OsSPS2 transformants were much lower than that of OsSPS1. Green fluorescent protein fusion analyses showed that OsSPS1 localized to mitochondria, while OsSPS2 localized to plastids. This suggests that OsSPS1 is involved in the supply of solanesyl diphosphate for ubiquinone-9 biosynthesis in mitochondria, whereas OsSPS2 is involved in providing solanesyl diphosphate for plastoquinone-9 formation. These findings indicate that O. sativa has a different mechanism for the supply of isoprenoid precursors in UQ biosynthesis from Arabidopsis thaliana, in which SPS1 provides a prenyl moiety for UQ9 at the endoplasmic reticulum.

  7. METOVITAN PREVENTS THE ACCUMULATION OF THIAMINE DIPHOSPHATE OXIDIZED FORM IN RAT TISSUES UNDER IRRADIATION

    Directory of Open Access Journals (Sweden)

    Parkhomenko Yu. M.

    2015-08-01

    Full Text Available The aim of the research was to test the ability of the drug "Metovitan" to prevent the redox balance disturbance in the tissues and thiamine diphosphate irreversible oxidation upon exposure of ionizing radiation on the body. The rats were subjected to a single exposure of the X-ray therapeutic instrument RUM-17 to create a dose of 0.5, 1.0 and 5.0 Gray. Preparation "Metovitan" was administered at a dose of 25 mg per 1 kg body weight for 22–24 h before irradiation. Contents of thiamine diphosphate, reduced SH-groups and reactive oxygen species in blood and brain were determined using previously described methods. It has been shown that the active form of the thiamine diphosphate content in the blood was decreased depending on the doses (from 0.5 to 5.0 Gray. At the same time the content of thiamine diphosphate oxidized form was increased. Furthermore the critical changes occurred in metabolic processes redox state parameters, namely, the level of free SH-groups was reduced and the level of reactive oxygen species was increased. Similar changes were observed in the brain tissue. The Metovitan single administration to the animals the day before irradiation, at 25 mg per 1 kg of body weight dose, promotes the protection of the intracellular thiamine diphosphate and redox status in animal tissues (blood, brain tissue from the negative influence of irradiation (at 0.5; 1.0 Gray doses. These results give reason to recommend the drug for treating of the staff that is involved in the elimination of radioactive contamination. To provide the protection from the higher doses, other scheme of the treatment should be probably recommended.

  8. Characterization of an Isopentenyl Diphosphate Isomerase involved in the Juvenile Hormone pathway in Aedes aegypti

    Science.gov (United States)

    Diaz, Miguel; Mayoral, Jaime G.; Priestap, Horacio; Nouzova, Marcela; Rivera-Perez, Crisalejandra; Noriega, Fernando G.

    2012-01-01

    Isopentenyl diphosphate isomerase (IPPI) is an enzyme involved in the synthesis of juvenile hormone (JH) in the corpora allata (CA) of insects. IPPI catalyzes the conversion of isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP); afterwards IPP and DMAPP condense in a head-to-tail manner to produce geranyl diphosphate (GPP), this head-to-tail condensation can be repeated, by the further reaction of GPP with IPP, yielding the JH precursor farnesyl diphosphate. An IPPI expressed sequence tag (EST) was obtained from an Aedes aegypti corpora-allata + corpora cardiaca library. Its full-length cDNA encodes a 244-aa protein that shows a high degree of similarity with type I IPPIs from other organisms, particularly for those residues that have important roles in catalysis, metal coordination and interaction with the diphosphate moiety of the IPP. Heterologous expression produced a recombinant protein that metabolized IPP into DMAPP; treatment of DMAPP with phosphoric acid produced isoprene, a volatile compound that was measured with an assay based on a solid-phase micro extraction protocol and direct analysis by gas chromatography. A. aegypti IPPI (AaIPPI) required Mg2+ or Mn2+ but not Zn2+ for full activity and it was entirely inhibited by iodoacetamide. Real time PCR experiments showed that AaIPPI is highly expressed in the CA. Changes in AaIPPI mRNA levels in the CA in the pupal and adult female mosquito corresponded well with changes in JH synthesis (Li et al., 2003). This is the first molecular and functional characterization of an isopentenyl diphosphate isomerase involved in the production of juvenile hormone in the CA of an insect. PMID:22782071

  9. Frontalin pheromone biosynthesis in the mountain pine beetle, Dendroctonus ponderosae, and the role of isoprenyl diphosphate synthases.

    Science.gov (United States)

    Keeling, Christopher I; Chiu, Christine C; Aw, Tidiane; Li, Maria; Henderson, Hannah; Tittiger, Claus; Weng, Hong-Biao; Blomquist, Gary J; Bohlmann, Joerg

    2013-11-19

    The mountain pine beetle (Dendroctonus ponderosae Hopkins) is the most destructive pest of western North American pine forests. Adult males produce frontalin, an eight-carbon antiaggregation pheromone, via the mevalonate pathway, as part of several pheromones that initiate and modulate the mass attack of host trees. Frontalin acts as a pheromone, attractant, or kairomone in most Dendroctonus species, other insects, and even elephants. 6-Methylhept-6-en-2-one, a frontalin precursor, is hypothesized to originate from 10-carbon geranyl diphosphate (GPP), 15-carbon farnesyl diphosphate (FPP), or 20-carbon geranylgeranyl diphosphate (GGPP) via a dioxygenase- or cytochrome P450-mediated carbon-carbon bond cleavage. To investigate the role of isoprenyl diphosphate synthases in pheromone biosynthesis, we characterized a bifunctional GPP/FPP synthase and a GGPP synthase in the mountain pine beetle. The ratio of GPP to FPP produced by the GPP/FPP synthase was highly dependent on the ratio of the substrates isopentenyl diphosphate and dimethylallyl diphosphate used in the assay. Transcript levels in various tissues and life stages suggested that GGPP rather than GPP or FPP is used as a precursor to frontalin. Reduction of transcript levels by RNA interference of the isoprenyl diphosphate synthases identified GGPP synthase as having the largest effect on frontalin production, suggesting that frontalin is derived from a 20-carbon isoprenoid precursor rather than from the 10- or 15-carbon precursors.

  10. 1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IDS) is encoded by multicopy genes in gymnosperms Ginkgo biloba and Pinus taeda.

    Science.gov (United States)

    Kim, Sang-Min; Kuzuyama, Tomohisa; Kobayashi, Akio; Sando, Tomoki; Chang, Yung-Jin; Kim, Soo-Un

    2008-01-01

    Isoprenoids are synthesized through the condensation of five-carbon intermediates, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), derived from two distinct biosynthetic routes: cytosolic mevalonate (MVA) and plastidial 2-C-methyl-D: -erythritol 4-phosphate (MEP) pathways. 1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IDS; EC 1.17.1.2), which catalyzes the last step of MEP pathway, was cloned as a multicopy gene from gymnosperms Ginkgo biloba (GbIDS1, GbIDS2, and GbIDS2-1) and Pinus taeda (PtIDS1 and PtIDS2), and characterized. Phylogenetic tree constructed with other plant IDSs demonstrated gymnosperm IDSs were distinctively different from angiosperm IDSs. The gymnosperm IDS clade contained two subclades, one composed of GbIDS1 and PtIDS1, and the other composed of GbIDS2, GbIDS2-1, and PtIDS2. G. biloba IDSs, except GbIDS2-1, successfully complemented Escherichia coli DLYT1, a lytB disruptant, confirming the in vivo competency of isozymes. During the 4 weeks study period, although transcript levels of GbIDS1s were similar both in roots and leaves of cultured G. biloba embryo, the transcripts of GbIDS2 predominantly occurred in the embryo roots, where diterpene ginkgolides are biosynthesized. Levels of PtIDS2 transcripts in the diterpenoid resin-producing wood were 4-5 times higher than those in other tissues. Higher levels of GbIDS1 transcripts were induced by light, whereas those of GbIDS2 were increased by methyl jasmonate treatment. These results strongly imply GbIDS2 and PtIDS2 have high correlation with secondary metabolism. In Arabidopsis transient expression system, N-terminal 100 amino acid residues of GbIDS1 delivered fused GFP protein into chloroplast as well as cytosol and nucleus, whereas those of GbIDS2, GbIDS2-1, and two PtIDSs delivered GFP only into chloroplast.

  11. Reactions of fac-[Re(CO)3(H2O)3]+ with nucleoside diphosphates and thiamine diphosphate in aqueous solution investigated by multinuclear NMR spectroscopy.

    Science.gov (United States)

    Adams, Kristie M; Marzilli, Patricia A; Marzilli, Luigi G

    2007-10-29

    Products formed between monoester diphosphates (MDPs) and fac-[Re(CO)3(H2O)3]OTf at pH 3.6 were examined. Such adducts of the fac-[Re(CO)3]+ moiety have an uncommon combination of properties for an "inert" metal center in that sharp NMR signals can be observed, yet the products are equilibrating at rates allowing NMR EXSY cross-peaks to be observed. Thiamine diphosphate (TDP) and uridine 5'-diphosphate (5'-UDP) form 1:1 bidentate {Palpha,Pbeta} chelates, in which the MDP binds Re(I) via Palpha and Pbeta phosphate groups. Asymmetric centers are created at Re(I) (RRe/SRe) and Palpha (Delta/Lambda), leading to four diastereomers. The two mirror pairs of diastereomers (RReDelta/SReLambda) and (RReLambda/SReDelta) for TDP (no ribose) and for all four diastereomers (RReDelta, RReLambda, SReDelta, SReLambda) for 5'-UDP (asymmetric ribose) gave two and four sets of NMR signals for the bound MDP, respectively. 31Palpha-31Palpha EXSY cross-peaks indicate that the fac-[Re(CO)3(H2O)({Palpha,Pbeta}MDP)]- isomers interchange slowly on the NMR time scale, with an average k approximately equal to 0.8 s(-1) at 32 degrees C; the EXSY cross-peaks could arise from chirality changes at only Re(I) or at only Palpha. Guanosine 5'-diphosphate (5'-GDP), with a ribose moiety and a Re(I)-binding base, formed both possible diastereomers (RRe and SRe) of the fac-[Re(CO)3(H2O)({N7,Pbeta}GDP)]- macrochelate, with one slightly more abundant diastereomer suggested to be RRe by Mn2+ ion 1H NMR signal line-broadening combined with distances from molecular models. Interchange of the diastereomers requires that the coordination site of either N7 or Pbeta move to the H2O site. 31Palpha-31Palpha EXSY cross-peaks indicate a k approximately equal to 0.5 s(-1) at 32 degrees C for RRe-to-SRe interchange. The similarity of the rate constants for interchange of fac-[Re(CO)3(H2O)({Palpha,Pbeta}MDP)]- and fac-[Re(CO)3(H2O)({N7,Pbeta}GDP)]- adducts suggest strongly that interchange of Pbeta and H2O coordination

  12. A New Method of Crystallization of Octahydro Trisodium Salt of Fructose-1,6-diphosphate

    Institute of Scientific and Technical Information of China (English)

    应汉杰; 欧阳平凯

    2002-01-01

    In order to overcome the elementary heterogeneous nucleation while octahydro trisodium salt of fructose-1,6-diphosphate(FDPNaa.8 H2O) is crystallized with ethanol precipitation at low temperature, a new crystallizationmethod with alcohol precipitation combined with salt precipitation has been presented. The ethanol-sodium ac-etate system for crystallization of salt of fructose-1,6-diphosphate is based on the mechanism of crystallization ofFDPNa3.8 H2O in the ethanol-low temperature system. It is found that crystal size may be controlled by regulatingtemperature or pH value of solution in the crystallization process, and the crystal yield increases to 95% from 78%which obtained in the ethanol-low temperature system.

  13. Molecular mechanism of allosteric substrate activation in a thiamine diphosphate-dependent decarboxylase.

    Science.gov (United States)

    Versées, Wim; Spaepen, Stijn; Wood, Martin D H; Leeper, Finian J; Vanderleyden, Jos; Steyaert, Jan

    2007-11-30

    Thiamine diphosphate-dependent enzymes are involved in a wide variety of metabolic pathways. The molecular mechanism behind active site communication and substrate activation, observed in some of these enzymes, has since long been an area of debate. Here, we report the crystal structures of a phenylpyruvate decarboxylase in complex with its substrates and a covalent reaction intermediate analogue. These structures reveal the regulatory site and unveil the mechanism of allosteric substrate activation. This signal transduction relies on quaternary structure reorganizations, domain rotations, and a pathway of local conformational changes that are relayed from the regulatory site to the active site. The current findings thus uncover the molecular mechanism by which the binding of a substrate in the regulatory site is linked to the mounting of the catalytic machinery in the active site in this thiamine diphosphate-dependent enzyme.

  14. Cloning, Expression, and Purification of Nucleoside Diphosphate Kinase from Acinetobacter baumannii

    Directory of Open Access Journals (Sweden)

    Juhi Sikarwar

    2013-01-01

    Full Text Available Acinetobacter baumannii is a multidrug resistant pathogenic bacteria associated with hospital acquired infections. This bacterium possesses a variety of resistance mechanisms which makes it more difficult to control the bacterium with conventional drugs, and, so far no effective drug treatment is available against it. Nucleoside diphosphate kinase is an important enzyme, which maintains the total nucleotide triphosphate pool inside the cell by the transfer of γ-phosphate from NTPs to NDPs. The role of nucleoside diphosphate kinase (Ndk has also been observed in pathogenesis in other organisms. However, intensive studies are needed to decipher its other putative roles in Acinetobacter baumannii. In the present study, we have successfully cloned the gene encoding Ndk and achieved overexpression in bacterial host BL-21 (DE3. The overexpressed protein is further purified by nickel-nitrilotriacetic acid (Ni-NTA chromatography.

  15. Cloning, Expression, and Purification of Nucleoside Diphosphate Kinase from Acinetobacter baumannii

    Science.gov (United States)

    Sikarwar, Juhi; Kaushik, Sanket; Sinha, Mau; Kaur, Punit; Sharma, Sujata; Singh, Tej P.

    2013-01-01

    Acinetobacter baumannii is a multidrug resistant pathogenic bacteria associated with hospital acquired infections. This bacterium possesses a variety of resistance mechanisms which makes it more difficult to control the bacterium with conventional drugs, and, so far no effective drug treatment is available against it. Nucleoside diphosphate kinase is an important enzyme, which maintains the total nucleotide triphosphate pool inside the cell by the transfer of γ-phosphate from NTPs to NDPs. The role of nucleoside diphosphate kinase (Ndk) has also been observed in pathogenesis in other organisms. However, intensive studies are needed to decipher its other putative roles in Acinetobacter baumannii. In the present study, we have successfully cloned the gene encoding Ndk and achieved overexpression in bacterial host BL-21 (DE3). The overexpressed protein is further purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography. PMID:23662205

  16. Expression pattern of the coparyl diphosphate synthase gene in developing rice anthers.

    Science.gov (United States)

    Fukuda, Ari; Nemoto, Keisuke; Chono, Makiko; Yamaguchi, Shinjiro; Nakajima, Masatoshi; Yamagishi, Junko; Maekawa, Masahiko; Yamaguchi, Isomaro

    2004-08-01

    Rice anthers contain high concentrations of gibberellins A(4) and A(7). To understand their physiological roles, we examined the site of their biosynthesis by analyzing the expression pattern of a gene (OsCPS) encoding coparyl diphosphate synthase in developing rice flowers. Expression was apparent in the anthers 1-2 days before flowering, and CPS mRNA accumulated in the maturing pollen.

  17. Inclusion of thiamine diphosphate and S-adenosylmethionine at their chemically active sites.

    Science.gov (United States)

    Schrader, Thomas; Fokkens, Michael; Klärner, Frank-Gerrit; Polkowska, Jolanta; Bastkowski, Frank

    2005-12-09

    [structure: see text] Molecular clips functionalized by phosphonate or phosphate groups bind thiamine diphosphate (TPP) and S-adenosylmethionine (SAM) with high affinity in water; both sulfur-based cofactors transfer organic groups to biomolecules. For TPP, various analytical tools point toward a simultaneous insertion of both heterocyclic rings into the electron-rich clip cavity. Similarly, SAM is also embedded with its sulfonium moiety inside the receptor cavity. This paves the way for enzyme models and direct interference with enzymatic processes.

  18. A novel chloroplastic isopentenyl diphosphate isomerase gene from Jatropha curcas: Cloning, characterization and subcellular localization

    OpenAIRE

    Wei, Lei; Yin, Li; Hu,Xiaole; Xu, Ying; Chen,Fang

    2014-01-01

    Background Jatropha curcas is a rich reservoir of pharmaceutically active terpenoids. More than 25 terpenoids have been isolated from this plant, and their activities are anti-bacterial, anti-fungal, anti-cancer, insecticidal, rodenticidal, cytotoxic and molluscicidal. But not much is known about the pathway involved in the biosynthesis of terpenoids. The present investigation describes the cloning, characterization and subcellular localization of isopentenyl diphosphate isomerase (IPI) gene ...

  19. Role of Arginine-304 in the Diphosphate-Triggered Active Site Closure Mechanism of Trichodiene Synthase

    Energy Technology Data Exchange (ETDEWEB)

    Vedula,L.; Cane, D.; Christianson, D.

    2005-01-01

    The X-ray crystal structures of R304K trichodiene synthase and its complexes with inorganic pyrophosphate (PPi) and aza analogues of the bisabolyl carbocation intermediate are reported. The R304K substitution does not cause large changes in the overall structure in comparison with the wild-type enzyme. The complexes with (R)- and (S)-azabisabolenes and PPi bind three Mg2+ ions, and each undergoes a diphosphate-triggered conformational change that caps the active site cavity. This conformational change is only slightly attenuated compared to that of the wild-type enzyme complexed with Mg{sup 2+}{sub 3-}PP{sub i}, in which R304 donates hydrogen bonds to PP{sub i} and D101. In R304K trichodiene synthase, K304 does not engage in any hydrogen bond interactions in the unliganded state and it donates a hydrogen bond to only PP{sub i} in the complex with (R)-azabisabolene; K304 makes no hydrogen bond contacts in its complex with PP{sub i} and (S)-azabisabolene. Thus, although the R304-D101 hydrogen bond interaction stabilizes diphosphate-triggered active site closure, it is not required for Mg{sup 2+}{sub 3-}PP{sub i} binding. Nevertheless, since R304K trichodiene synthase generates aberrant cyclic terpenoids with a 5000-fold reduction in kcat/KM, it is clear that a properly formed R304-D101 hydrogen bond is required in the enzyme-substrate complex to stabilize the proper active site contour, which in turn facilitates cyclization of farnesyl diphosphate for the exclusive formation of trichodiene. Structural analysis of the R304K mutant and comparison with the monoterpene cyclase (+)-bornyl diphosphate synthase suggest that the significant loss in activity results from compromised activation of the PP{sub i} leaving group.

  20. Structure and Mechanism of the Diterpene Cyclase ent-Copalyl Diphosphate Synthase

    Science.gov (United States)

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W.

    2011-01-01

    The structure of ent-copalyl diphosphate synthase (CPS) reveals three α-helical domains (α, β, γ), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the βγ domains in CPS but exclusively in the α domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions. PMID:21602811

  1. Efficient diterpene production in yeast by engineering Erg20p into a geranylgeranyl diphosphate synthase.

    Science.gov (United States)

    Ignea, Codruta; Trikka, Fotini A; Nikolaidis, Alexandros K; Georgantea, Panagiota; Ioannou, Efstathia; Loupassaki, Sofia; Kefalas, Panagiotis; Kanellis, Angelos K; Roussis, Vassilios; Makris, Antonios M; Kampranis, Sotirios C

    2015-01-01

    Terpenes have numerous applications, ranging from pharmaceuticals to fragrances and biofuels. With increasing interest in producing terpenes sustainably and economically, there has been significant progress in recent years in developing methods for their production in microorganisms. In Saccharomyces cerevisiae, production of the 20-carbon diterpenes has so far proven to be significantly less efficient than production of their 15-carbon sesquiterpene counterparts. In this report, we identify the modular structure of geranylgeranyl diphosphate synthesis in yeast to be a major limitation in diterpene yields, and we engineer the yeast farnesyl diphosphate synthase Erg20p to produce geranylgeranyl diphosphate. Using a combination of protein and genetic engineering, we achieve significant improvements in the production of sclareol and several other isoprenoids, including cis-abienol, abietadiene and β-carotene. We also report the development of yeast strains carrying the engineered Erg20p, which support efficient isoprenoid production and can be used as a dedicated chassis for diterpene production or biosynthetic pathway elucidation. The design developed here can be applied to the production of any GGPP-derived isoprenoid and is compatible with other yeast terpene production platforms.

  2. Cloning and Characterization of Farnesyl Diphosphate Synthase Gene Involved in Triterpenoids Biosynthesis from Poria cocos

    Directory of Open Access Journals (Sweden)

    Jianrong Wang

    2014-12-01

    Full Text Available Poria cocos (P. cocos has long been used as traditional Chinese medicine and triterpenoids are the most important pharmacologically active constituents of this fungus. Farnesyl pyrophosphate synthase (FPS is a key enzyme of triterpenoids biosynthesis. The gene encoding FPS was cloned from P. cocos by degenerate PCR, inverse PCR and cassette PCR. The open reading frame of the gene is 1086 bp in length, corresponding to a predicted polypeptide of 361 amino acid residues with a molecular weight of 41.2 kDa. Comparison of the P. cocos FPS deduced amino acid sequence with other species showed the highest identity with Ganoderma lucidum (74%. The predicted P. cocos FPS shares at least four conserved regions involved in the enzymatic activity with the FPSs of varied species. The recombinant protein was expressed in Pichia pastoris and purified. Gas chromatography analysis showed that the recombinant FPS could catalyze the formation of farnesyl diphosphate (FPP from geranyl diphosphate (GPP and isopentenyl diphosphate (IPP. Furthermore, the expression profile of the FPS gene and content of total triterpenoids under different stages of development and methyl jasmonate treatments were determined. The results indicated that there is a positive correlation between the activity of FPS and the amount of total triterpenoids produced in P. cocos.

  3. A photoactive isoprenoid diphosphate analogue containing a stable phosphonate linkage: synthesis and biochemical studies with prenyltransferases

    Science.gov (United States)

    DeGraw, Amanda J.; Zhao, Zongbao; Strickland, Corey L.; Taban, A. Huma; Hsieh, John; Michael, Jefferies; Xie, Wenshuang; Shintani, David; McMahan, Colleen; Cornish, Katrina; Distefano, Mark D.

    2008-01-01

    A number of biochemical processes rely on isoprenoids, including the post-translational modification of signaling proteins and the biosynthesis of a wide array of compounds. Photoactivatable analogues have been developed to study isoprenoid utilizing enzymes such as the isoprenoid synthases and prenyltransferases. While these initial analogues proved to be excellent structural analogues with good cross linking capability, they lack the stability needed when the goals include isolation of cross-linked species, tryptic digestion, and subsequent peptide sequencing. Here, the synthesis of a benzophenone-based farnesyl diphosphate analogue containing a stable phosphonophosphate group is described. Inhibition kinetics, photolabeling experiments, as well as x-ray crystallographic analysis with a protein prenyltransferase are described, verifying this compound as a good isoprenoid mimetic. In addition, the utility of this new analogue was explored by using it to photoaffinity label crude protein extracts obtained from Hevea brasiliensis latex. Those experiments suggest that a small protein, Rubber Elongation Factor, interacts directly with farnesyl diphosphate during rubber biosynthesis. These results indicate that this benzophenone-based isoprenoid analogue will be useful for identifying enzymes that utilize farnesyl diphosphate as a substrate. PMID:17477573

  4. Adaptive evolution of the chrysanthemyl diphosphate synthase gene involved in irregular monoterpene metabolism

    Directory of Open Access Journals (Sweden)

    Liu Ping-Li

    2012-11-01

    Full Text Available Abstract Background Chrysanthemyl diphosphate synthase (CDS is a key enzyme in biosynthetic pathways producing pyrethrins and irregular monoterpenes. These compounds are confined to plants of the tribe Anthemideae of the Asteraceae, and play an important role in defending the plants against herbivorous insects. It has been proposed that the CDS genes arose from duplication of the farnesyl diphosphate synthase (FDS gene and have different function from FDSs. However, the duplication time toward the origin of CDS and the evolutionary force behind the functional divergence of the CDS gene are still unknown. Results Two duplication events were detected in the evolutionary history of the FDS gene family in the Asteraceae, and the second duplication led to the origin of CDS. CDS occurred after the divergence of the tribe Mutisieae from other tribes of Asteraceae but before the birth of the Anthemideae tribe. After its origin, CDS accumulated four mutations in sites homologous to the substrate-binding and catalysis sites of FDS. Of these, two sites were involved in the binding of the nucleophilic substrate isopentenyl diphosphate in FDS. Maximum likelihood analyses showed that some sites in CDS were under positive selection and were scattered throughout primary sequences, whereas in the three-dimensional structure model they clustered in the large central cavity. Conclusion Positive selection associated with gene duplication played a major role in the evolution of CDS.

  5. Cloning and characterization of farnesyl diphosphate synthase gene involved in triterpenoids biosynthesis from Poria cocos.

    Science.gov (United States)

    Wang, Jianrong; Li, Yangyuan; Liu, Danni

    2014-12-02

    Poria cocos (P. cocos) has long been used as traditional Chinese medicine and triterpenoids are the most important pharmacologically active constituents of this fungus. Farnesyl pyrophosphate synthase (FPS) is a key enzyme of triterpenoids biosynthesis. The gene encoding FPS was cloned from P. cocos by degenerate PCR, inverse PCR and cassette PCR. The open reading frame of the gene is 1086 bp in length, corresponding to a predicted polypeptide of 361 amino acid residues with a molecular weight of 41.2 kDa. Comparison of the P. cocos FPS deduced amino acid sequence with other species showed the highest identity with Ganoderma lucidum (74%). The predicted P. cocos FPS shares at least four conserved regions involved in the enzymatic activity with the FPSs of varied species. The recombinant protein was expressed in Pichia pastoris and purified. Gas chromatography analysis showed that the recombinant FPS could catalyze the formation of farnesyl diphosphate (FPP) from geranyl diphosphate (GPP) and isopentenyl diphosphate (IPP). Furthermore, the expression profile of the FPS gene and content of total triterpenoids under different stages of development and methyl jasmonate treatments were determined. The results indicated that there is a positive correlation between the activity of FPS and the amount of total triterpenoids produced in P. cocos.

  6. Escherichia Coli

    Science.gov (United States)

    Goodsell, David S.

    2009-01-01

    Diverse biological data may be used to create illustrations of molecules in their cellular context. I describe the scientific results that support a recent textbook illustration of an "Escherichia coli cell". The image magnifies a portion of the bacterium at one million times, showing the location and form of individual macromolecules. Results…

  7. A New Farnesyl Diphosphate Synthase Gene from Taxus media Rehder: Cloning, Characterization and Functional Complementation

    Institute of Scientific and Technical Information of China (English)

    Zhi-Hua Liao; Min Chen; Yi-Fu Gong; Zhu-Gang Li; Kai-Jing Zuo; Peng Wang; Feng Tan; Xiao-Fen Sun; Ke-Xuan Tang

    2006-01-01

    Farnesyl diphosphate synthase (FPS; EC 2.5.1.10) catalyzes the production of 15-carbon farnesyl diphosphate which is a branch-point intermediate for many terpenoids. This reaction is considered to be a ratelimiting step in terpenoid biosynthesis. Here we report for the first time the cloning of a new full-length cDNA encoding farnesyl diphosphate synthase from a gymnosperm plant species, Taxus media Rehder,designated as TmFPS1. The full-length cDNA of TmFPS1 (GenBank accession number: AY461811) was 1 464bp with a 1 056-bp open reading frame encoding a 351-amino acid polypeptide with a calculated molecular weight of 40.3 kDa and a theoretical pl of 5.07. Bioinformatic analysis revealed that TmFPS1 contained all five conserved domains of prenyltransferases, and showed homology to other FPSs of plant origin. Phylogenetic analysis showed that farnesyl diphosphate synthases can be divided into two groups: one of prokaryotic origin and the other of eukaryotic origin. TmFPS1 was grouped with FPSs of plant origin. Homologybased structural modeling showed that TmFPS1 had the typical spatial structure of FPS, whose most prominent structural feature is the arrangement of 13 core helices around a large central cavity in which the catalytic reaction takes place. Our bioinformatic analysis strongly suggests that TmFPS1 is a functional gene. Southern blot analysis revealed that TmFPS1 belongs to a small FPS gene family in T. media. Northern blot analysis indicated that TmFPS1 is expressed in all tested tissues, including the needles, stems and roots of T. media. Subsequently, functional complementation with TmFPS1 in a FPS-deficient mutant yeast demonstrated that TmFPS1 did encode farnesyl diphosphate synthase, which rescued the yeast mutant.This study will be helpful in future investigations aiming at understanding the detailed role of FPS in terpenoid biosynthesis flux control at the molecular genetic level.

  8. Chrysanthemyl diphosphate synthase: Isolation of the gene and characterization of the recombinant non-head-to-tail monoterpene synthase from Chrysanthemum cinerariaefolium

    OpenAIRE

    Rivera, Susan B.; Swedlund, Bradley D.; King, Gretchen J.; Bell, Russell N.; Hussey, Charles E.; Shattuck-Eidens, Donna M.; Wrobel, Wislawa M.; Peiser, Galen D.; Poulter, C. Dale

    2001-01-01

    Chrysanthemyl diphosphate synthase (CPPase) catalyzes the condensation of two molecules of dimethylallyl diphosphate to produce chrysanthemyl diphosphate (CPP), a monoterpene with a non-head-to-tail or irregular c1′-2-3 linkage between isoprenoid units. Irregular monoterpenes are common in Chrysanthemum cinerariaefolium and related members of the Asteraceae family. In C. cinerariaefolium, CPP is an intermediate in the biosynthesis of the pyrethrin ester insecticides. CPPa...

  9. Structure of trihydrated rare-earth acid diphosphates LnHP 2O 7·3H 2O ( Ln=La, Er)

    Science.gov (United States)

    Ben Moussa, S.; Ventemillas, S.; Cabeza, A.; Gutierrez-Puebla, E.; Sanz, J.

    2004-06-01

    In trihydrated lanthanum acid-diphosphates LnHP 2O 7·3H 2O, prepared from acid LnCl 3 and Na 4P 2O 7 solutions (pH=1), two crystal forms were obtained. Layered structures of two representative members of this family have been determined by single-crystal X-ray diffraction (XRD) technique. In the case of orthorhombic LaHP 2O 7·3H 2O (type I), lanthanum cations are ninefold coordinated and diphosphate groups adopt a staggered (alternated) configuration. In the case of triclinic ErHP 2O 7·3H 2O (type II), erbium cations are eightfold coordinated and diphosphate groups adopt an eclipsed configuration. In agreement with Infrared (IR) spectroscopic data, a bended configuration for diphosphate groups has been deduced. In both structures, one-dimensional chains of edge-sharing rare-earth polyhedra are linked together by diphosphate groups to form the phosphate layers. In both diphosphates, PO 4 and HPO 4 environments have been identified by 31P MAS-NMR technique. In the two compounds, OH groups of HPO 4 tetrahedra point out of diphosphate planes interacting with adjacent layers. In La-diphosphate, the interaction between HPO 4 groups and water molecules of adjacent layers is favored; however, in Er-diphosphate, the interaction between phosphate acid groups of contiguous layers is produced. Based on structural information deduced, differences detected in IR and NMR spectra of two disphosphates are discussed.

  10. Specific partial reduction of geranylgeranyl diphosphate by an enzyme from the thermoacidophilic archaeon Sulfolobus acidocaldarius yields a reactive prenyl donor, not a dead-end product.

    Science.gov (United States)

    Sato, Sho; Murakami, Motomichi; Yoshimura, Tohru; Hemmi, Hisashi

    2008-06-01

    Geranylgeranyl reductase from Sulfolobus acidocaldarius was shown to catalyze the reduction of geranylgeranyl groups in the precursors of archaeal membrane lipids, generally reducing all four double bonds. However, when geranylgeranyl diphosphate was subjected to the reductase reaction, only three of the four double bonds were reduced. Mass spectrometry and acid hydrolysis indicated that the allylic double bond was preserved in the partially reduced product derived from geranylgeranyl diphosphate. Thus, the reaction product was shown to be phytyl diphosphate, which is a substrate for archaeal prenyltransferases, unlike the completely reduced compound phytanyl diphosphate.

  11. Physiological Role of phnP-specified Phosphoribosyl Cyclic Phosphodiesterase in Catabolism of Organophosphonic Acids by the Carbon−Phosphorus Lyase Pathway

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; McSorley, Fern R.; Zechel, David L.

    2011-01-01

    In Escherichia coli , internalization and catabolism of organophosphonicacids are governed by the 14-cistron phnCDEFGHIJKLMNOP operon. The phnP gene product was previously shown to encode a phosphodiesterase with unusual specificity toward ribonucleoside 2',3'-cyclic phosphates. Furthermore, phnP...

  12. Purification, crystallization and preliminary structural analysis of nucleoside diphosphate kinase from Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Misra, Gauri [Molecular and Structural Biology Division, Central Drug Research Institute, PO Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226 001 (India); Aggarwal, Anita [Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007 (India); Mittal, Sonia [Molecular and Structural Biology Division, Central Drug Research Institute, PO Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226 001 (India); Singh, Yogendra [Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007 (India); Ramachandran, Ravishankar, E-mail: r-ravishankar@cdri.res.in [Molecular and Structural Biology Division, Central Drug Research Institute, PO Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226 001 (India)

    2007-12-01

    Nucleoside diphosphate kinase from B. anthracis has been crystallized. Preliminary crystallographic analysis shows that there is one monomer in the asymmetric unit of the crystal. Bacillus anthracis nucleoside diphosphate kinase (BaNdk) is an enzyme whose primary function is to maintain deoxynucleotide triphosphate (dNTP) pools by converting deoxynucleotide diphosphates to triphosphates using ATP as the major phosphate donor. Although the structures of Ndks from a variety of organisms have been elucidated, the enzyme from sporulating bacteria has not been structurally characterized to date. Crystals of the B. anthracis enzyme were grown using the vapour-diffusion method from a hanging drop consisting of 2 µl 10 mg ml{sup −1} protein in 50 mM Tris–HCl pH 8.0, 50 mM NaCl, 5 mM EDTA equilibrated against 500 µl reservoir solution consisting of 2.25 M ammonium formate and 0.1 M HEPES buffer pH 7.25. Diffraction data extending to 2.0 Å were collected at room temperature from a single crystal with unit-cell parameters a = b = 107.53, c = 52.3 Å. The crystals are hexagonal in shape and belong to space group P6{sub 3}22. The crystals contain a monomer in the asymmetric unit, which corresponds to a Matthews coefficient (V{sub M}) of 2.1 Å{sup 3} Da{sup −1} and a solvent content of about 36.9%.

  13. Structural Basis for Nucleotide Binding and Reaction Catalysis in Mevalonate Diphosphate Decarboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Barta, Michael L.; McWhorter, William J.; Miziorko, Henry M.; Geisbrecht, Brian V. (UMKC)

    2012-09-17

    Mevalonate diphosphate decarboxylase (MDD) catalyzes the final step of the mevalonate pathway, the Mg{sup 2+}-ATP dependent decarboxylation of mevalonate 5-diphosphate (MVAPP), producing isopentenyl diphosphate (IPP). Synthesis of IPP, an isoprenoid precursor molecule that is a critical intermediate in peptidoglycan and polyisoprenoid biosynthesis, is essential in Gram-positive bacteria (e.g., Staphylococcus, Streptococcus, and Enterococcus spp.), and thus the enzymes of the mevalonate pathway are ideal antimicrobial targets. MDD belongs to the GHMP superfamily of metabolite kinases that have been extensively studied for the past 50 years, yet the crystallization of GHMP kinase ternary complexes has proven to be difficult. To further our understanding of the catalytic mechanism of GHMP kinases with the purpose of developing broad spectrum antimicrobial agents that target the substrate and nucleotide binding sites, we report the crystal structures of wild-type and mutant (S192A and D283A) ternary complexes of Staphylococcus epidermidis MDD. Comparison of apo, MVAPP-bound, and ternary complex wild-type MDD provides structural information about the mode of substrate binding and the catalytic mechanism. Structural characterization of ternary complexes of catalytically deficient MDD S192A and D283A (k{sub cat} decreased 10{sup 3}- and 10{sup 5}-fold, respectively) provides insight into MDD function. The carboxylate side chain of invariant Asp{sup 283} functions as a catalytic base and is essential for the proper orientation of the MVAPP C3-hydroxyl group within the active site funnel. Several MDD amino acids within the conserved phosphate binding loop ('P-loop') provide key interactions, stabilizing the nucleotide triphosphoryl moiety. The crystal structures presented here provide a useful foundation for structure-based drug design.

  14. Enzymatic production of fructose 1,6-diphosphate using crude cell extract of Bacillus stearothermophilus.

    Science.gov (United States)

    Widjaja, A; Yasuda, M; Ogino, H; Nakajima, H; Ishikawa, H

    1999-01-01

    The enzymatic production of fructose 1,6-diphosphate (FDP) from glucose was performed in a batch reactor and a semibatch reactor using the crude cell extract of Bacillus stearothermophilus which contains all four enzymes required for the synthesis. The experimental results of the yield and the time courses of FDP production obtained using various enzyme concentrations were in good agreement with the theoretical predictions calculated based on the differential equations including the rate equations of the four enzymes, which were determined using the purified enzymes of B. stearothermophilus.

  15. METOVITAN PREVENTS ACCUMULATION OF THIAMIN DIPHOSPHATE OXYGENIZED FORM IN RAT TISSUES UNDER IRRADIATION

    OpenAIRE

    Yu. M. Parkhomenko; G. V. Donchenko; L. I. Chehovskaya; S. P. Stepanenko; O. A. Mejenskaya; E. N. Gorban

    2015-01-01

    The aim of the research was to test the ability of the drug “Metovitan” to prevent the redox balance disturbance in the tissues and thiamine diphosphate irreversible oxidation upon exposure of ionizing radiation on the body. The rats were undergo to a single exposure of the X-ray therapeutic instrument RUM-17 to create a dose of 0.5, 1.0 and 5.0 Gy. Preparation “Metovitan” were administered at a dose of 25 mg per 1 kg body weight for 22–24 h before irradiation. Contents of thiamin...

  16. Influence of donor substrate on kinetic parameters of thiamine diphosphate binding to transketolase.

    Science.gov (United States)

    Ospanov, R V; Kochetov, G A; Kurganov, B I

    2007-01-01

    The two-step mechanism of interaction of thiamine diphosphate (ThDP) with transketolase (TK) has been studied: TK + ThDP TK...ThDP TK*-ThDP. The scheme involves the formation of inactive intermediate complex TK...ThDP followed by its transformation into catalytically active holoenzyme, TK*-ThDP. The dissociation and kinetic constants for individual stages of this process have been determined. The values of forward and backward rate constants change in the presence of the donor substrate hydroxypyruvate. This finally leads to an increase in the overall affinity of the coenzyme to TK.

  17. Chemoenzymatic Synthesis of 4-Fluoro-N-Acetylhexosamine Uridine Diphosphate Donors: Chain Terminators in Glycosaminoglycan Synthesis.

    Science.gov (United States)

    Schultz, Victor L; Zhang, Xing; Linkens, Kathryn; Rimel, Jenna; Green, Dixy E; DeAngelis, Paul L; Linhardt, Robert J

    2017-02-17

    Unnatural uridine diphosphate (UDP)-sugar donors, UDP-4-deoxy-4-fluoro-N-acetylglucosamine (4FGlcNAc) and UDP-4-deoxy-4-fluoro-N-acetylgalactosamine (4FGalNAc), were prepared using both chemical and chemoenzymatic syntheses relying on N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU). The resulting unnatural UDP-sugar donors were then tested as substrates in glycosaminoglycan synthesis catalyzed by various synthases. UDP-4FGlcNAc was transferred onto an acceptor by Pastuerella multocida heparosan synthase 1 and subsequently served as a chain terminator.

  18. K channel activation by nucleotide diphosphates and its inhibition by glibenclamide in vascular smooth muscle cells.

    Science.gov (United States)

    Beech, D J; Zhang, H; Nakao, K; Bolton, T B

    1993-10-01

    1. Whole-cell and inside-out patch recordings were made from single smooth muscle cells that had been isolated enzymatically and mechanically from the rabbit portal vein. 2. In whole-cells the inclusion in the recording pipette solution of nucleotide diphosphates (NDPs), but not tri- or monophosphates, induced a K-current that developed gradually over 5 to 15 min. Intracellular 1 mM guanosine 5'-diphosphate (GDP) induced a slowly developing outward K-current at -37 mV that reached a maximum on average of 72 +/- 4 pA (n = 40). Half maximal effect was estimated to occur with about 0.2 mM GDP. Except for ADP, other NDPs had comparable effects. At 0.1 mM, ADP was equivalent to GDP but at higher concentration ADP was less effective. ADP induced its maximum effect at 1 mM but had almost no effect at 10 mM. 3. In 14% of inside-out patches exposed to 1 mM GDP at the intracellular surface, characteristic K channel activity was observed which showed long (> 1 s) bursts of openings separated by longer closed periods. The current-voltage relationship for the channel was linear in a 60 mM:130 mM K-gradient and the unitary conductance was 24 pS. 4. Glibenclamide applied via the extracellular solution was found to be a potent inhibitor of GDP-induced K-current (IK(GDP)) in the whole-cell. The Kd was 25 nM and the inhibition was fully reversible on wash-out. 5. IK(GDP) was not evoked if Mg ions were absent from the pipette solution. In contrast the omission of extracellular Mg ions had no effect on outward or inward IK(GDP). 6. Inclusion of 1 mM ATP in the recording pipette solution reduced IK(GDP) and also attenuated its decline during long (25 min) recordings. 7. When perforated-patch whole-cell recording was used, metabolic poisoning with cyanide and 2-deoxy-D-glucose induced a glibenclamide-sensitive K-current. This current was not observed when conventional whole-cell recording was used. Possible reasons for this difference are discussed. 8. These K channels appear similar to

  19. Coenzyme Q10 supplementation rescues renal disease in Pdss2kd/kd mice with mutations in prenyl diphosphate synthase subunit 2.

    Science.gov (United States)

    Saiki, Ryoichi; Lunceford, Adam L; Shi, Yuchen; Marbois, Beth; King, Rhonda; Pachuski, Justin; Kawamukai, Makoto; Gasser, David L; Clarke, Catherine F

    2008-11-01

    Homozygous mice carrying kd (kidney disease) mutations in the gene encoding prenyl diphosphate synthase subunit 2 (Pdss2kd/kd) develop interstitial nephritis and eventually die from end-stage renal disease. The PDSS2 polypeptide in concert with PDSS1 synthesizes the polyisoprenyl tail of coenzyme Q (Q or ubiquinone), a lipid quinone required for mitochondrial respiratory electron transport. We have shown that a deficiency in Q content is evident in Pdss2kd/kd mouse kidney lipid extracts by 40 days of age and thus precedes the onset of proteinuria and kidney disease by several weeks. The presence of the kd (V117M) mutation in PDSS2 does not prevent its association with PDSS1. However, heterologous expression of the kd mutant form of PDSS2 together with PDSS1 in Escherichia coli recapitulates the Q deficiency observed in the Pdss2kd/kd mouse. Dietary supplementation with Q10 provides a dramatic rescue of both proteinuria and interstitial nephritis in the Pdss2kd/kd mutant mice. The results presented suggest that Q may be acting as a potent lipid-soluble antioxidant, rather than by boosting kidney mitochondrial respiration. Such Q10 supplementation may have profound and beneficial effects in treatment of certain forms of focal segmental glomerulosclerosis that mirror the renal disease of the Pdss2kd/kd mouse.

  20. Amino acids allosterically regulate the thiamine diphosphate-dependent alpha-keto acid decarboxylase from Mycobacterium tuberculosis.

    Science.gov (United States)

    Werther, Tobias; Spinka, Michael; Tittmann, Kai; Schütz, Anja; Golbik, Ralph; Mrestani-Klaus, Carmen; Hübner, Gerhard; König, Stephan

    2008-02-29

    The gene rv0853c from Mycobacterium tuberculosis strain H37Rv codes for a thiamine diphosphate-dependent alpha-keto acid decarboxylase (MtKDC), an enzyme involved in the amino acid degradation via the Ehrlich pathway. Steady state kinetic experiments were performed to determine the substrate specificity of MtKDC. The mycobacterial enzyme was found to convert a broad spectrum of branched-chain and aromatic alpha-keto acids. Stopped-flow kinetics showed that MtKDC is allosterically activated by alpha-keto acids. Even more, we demonstrate that also amino acids are potent activators of this thiamine diphosphate-dependent enzyme. Thus, metabolic flow through the Ehrlich pathway can be directly regulated at the decarboxylation step. The influence of amino acids on MtKDC catalysis was investigated, and implications for other thiamine diphosphate-dependent enzymes are discussed.

  1. The subcellular localization of periwinkle farnesyl diphosphate synthase provides insight into the role of peroxisome in isoprenoid biosynthesis.

    Science.gov (United States)

    Thabet, Insaf; Guirimand, Grégory; Courdavault, Vincent; Papon, Nicolas; Godet, Stéphanie; Dutilleul, Christelle; Bouzid, Sadok; Giglioli-Guivarc'h, Nathalie; Clastre, Marc; Simkin, Andrew J

    2011-11-15

    Farnesyl diphosphate (FPP) synthase (FPS: EC.2.5.1.1, EC.2.5.1.10) catalyzes the formation of FPP from isopentenyl diphosphate and dimethylallyl diphosphate via two successive condensation reactions. A cDNA designated CrFPS, encoding a protein showing high similarities with trans-type short FPS isoforms, was isolated from the Madagascar periwinkle (Catharanthus roseus). This cDNA was shown to functionally complement the lethal FPS deletion mutant in the yeast Saccharomyces cerevisiae. At the subcellular level, while short FPS isoforms are usually described as cytosolic proteins, we showed, using transient transformations of C. roseus cells with yellow fluorescent protein-fused constructs, that CrFPS is targeted to peroxisomes. This finding is discussed in relation to the subcellular distribution of FPS isoforms in plants and animals and opens new perspectives towards the understanding of isoprenoid biosynthesis.

  2. Cloning, expression and characterization of a nucleoside diphosphate kinase (NDPK) gene from tobacco

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Nucleoside diphosphate kinase (NDPK) is a housekeeping enzyme that maintains the intracellular levels of all (d)NTPs used in biosynthesis except ATP. Here we report that a full-length cDNA encoding nucleoside diphosphate kinase (NDPK) was cloned using yeast two-hybrid approach. A tobacco NDPK gene was obtained and designated as NtNDPK1 . NtNDPK1 is 704 bp in length and encodes a putative 16.2 kD protein of 148 amino acids. Phylogenic analysis showed that NtNDPK1 is highly homologous to other plant NDPK genes and identified as type Ⅰ (NDPK1). RNA-gel blot analysis showed that there was no significant difference of NtNDPK1 expression in roots, stems, leaves and buds. And expression of NtNDPK1 was induced by ABA and PEG and repressed by NaCl, but not significantly affected by Paraquat, wounding and low temperature (4℃) treatments, indicating that NtNDPK1 may play a certain role in response to abiotic stress. In vitro phosphorylation assay demonstrated that NtNDPK1 had autophosphorylation activity.

  3. Isolation and characterization of a copalyl diphosphate synthase gene promoter from Salvia miltiorrhiza

    Directory of Open Access Journals (Sweden)

    Piotr Szymczyk

    2016-09-01

    Full Text Available The promoter, 5' UTR, and 34-nt 5' fragments of protein encoding region of the Salvia miltiorrhiza copalyl diphosphate synthase gene were cloned and characterized. No tandem repeats, miRNA binding sites, or CpNpG islands were observed in the promoter, 5' UTR, or protein encoding fragments. The entire isolated promoter and 5' UTR is 2235 bp long and contains repetitions of many cis-active elements, recognized by homologous transcription factors, found in Arabidopsis thaliana and other plant species. A pyrimidine-rich fragment with only 6 non-pyrimidine bases was localized in the 33-nt stretch from nt 2185 to 2217 in the 5' UTR. The observed cis-active sequences are potential binding sites for trans-factors that could regulate spatio-temporal CPS gene expression in response to biotic and abiotic stress conditions. Obtained results are initially verified by in silico and co-expression studies based on A. thaliana microarray data. The quantitative RT-PCR analysis confirmed that the entire 2269-bp copalyl diphosphate synthase gene fragment has the promoter activity. Quantitative RT-PCR analysis was used to study changes in CPS promoter activity occurring in response to the application of four selected biotic and abiotic regulatory factors; auxin, gibberellin, salicylic acid, and high-salt concentration.

  4. A cesium copper vanadyl-diphosphate: Synthesis, crystal structure and physical properties

    Science.gov (United States)

    Shvanskaya, Larisa; Yakubovich, Olga; Bychkov, Andrey; Shcherbakov, Vasiliy; Golovanov, Alexey; Zvereva, Elena; Volkova, Olga; Vasiliev, Alexander

    2015-02-01

    A non-centrosymmetric orthorhombic diphosphate, Cs2Cu1+x(VO)2-x(P2O7)2 (x=0.1) with a=13.7364(2) Å, b=9.2666(2) Å, c=11.5678(2) Å, Z=4, has been isolated. Its 3D framework is built from Cu atoms in square pyramidal and square planar coordination, VO5 tetragonal pyramids and P2O7 diphosphate groups, sharing vertices. Large channels are fulfilled by cesium atoms. The ESR study reveals a similarity in behaviour of two paramagnetic (Cu and V) subsystems. The temperature dependences of the ESR linewidth and static magnetic susceptibility data present evidences for a cluster type magnetic ordering in the title compound at T*=22 K. The weakness of the relevant anomalies reflects presumably obvious Cu2+ ions and (VO)2+ units disorder in the system. It is supposed that the charge and geometry of the framework are controlled by the Cu2+/(VO)2+ ratio; its variation may lead to a design of new materials.

  5. Studies on the mammary tumor-inhibiting effects of diethylstilbestrol and its mono- and diphosphate.

    Science.gov (United States)

    Schneider, M R; von Angerer, E; Prekajac, J; Brade, W P

    1986-01-01

    Diethylstilbestrol (DES), diethylstilbestrol monophosphate (DES-MP) and diethylstilbestrol diphosphate (DES-DP) were tested for their estrogen receptor affinity, estrogenic potency and mammary tumor-inhibiting activity in vitro and in vivo. DES had a much higher receptor binding affinity than its mono- or diphosphate. All three compounds inhibited the growth of the hormone-dependent MCF-7 and hormone-independent MDA-MB 231 breast cancer line only at relatively high concentrations. The estrogenic potency in the immature mouse uterine weight test decreased in the order DES greater than DES-MP much greater than DES-DP. The hormone-dependent MXT mammary tumor of the mouse was inhibited by all three compounds at a dosage of 1.0 mg/kg per week. At a dose of 0.01 mg/kg, DES, DES-MP, and DES-DP stimulated the tumor growth. Thus, for the first time, a biphasic effect on tumor growth was demonstrated in intact mature animals. As the effects of all three compounds were similar in this assay, a cleavage of the phosphate groups is likely. A decrease in estrogenic potency concomitant with a retained antitumor effect of DES-MP and DES-DP compared to DES was not demonstrable in the mature mouse using the MXT assay, only in the uterotrophic test in the immature mouse.

  6. Acanthamoeba polyphaga mimivirus NDK: preliminary crystallographic analysis of the first viral nucleoside diphosphate kinase

    Energy Technology Data Exchange (ETDEWEB)

    Jeudy, Sandra [Information Génomique et Structurale, CNRS UPR 2589, 31 Chemin Joseph Aiguier, 13402 Marseille CEDEX 20 (France); Coutard, Bruno [Architecture et Fonction des Macromolecules Biologiques, CNRS UMR 6098, 31 Chemin Joseph Aiguier, 13402 Marseille CEDEX 20 (France); Lebrun, Régine [IBSM, 31 Chemin Joseph Aiguier, 13402 Marseille CEDEX 20 (France); Abergel, Chantal, E-mail: chantal.abergel@igs.cnrs-mrs.fr [Information Génomique et Structurale, CNRS UPR 2589, 31 Chemin Joseph Aiguier, 13402 Marseille CEDEX 20 (France)

    2005-06-01

    A. polyphaga mimivirus, the largest known double-stranded DNA virus, is the first virus to exhibit a nucleoside diphosphate kinase gene. The expression and crystallization of the viral NDK are reported. The complete sequence of the largest known double-stranded DNA virus, Acanthamoeba polyphaga mimivirus, has recently been determined [Raoult et al. (2004 ▶), Science, 306, 1344–1350] and revealed numerous genes not expected to be found in a virus. A comprehensive structural and functional study of these gene products was initiated [Abergel et al. (2005 ▶), Acta Cryst. F61, 212–215] both to better understand their role in the virus physiology and to obtain some clues to the origin of DNA viruses. Here, the preliminary crystallographic analysis of the viral nucleoside diphosphate kinase protein is reported. The crystal belongs to the cubic space group P2{sub 1}3, with unit-cell parameter 99.425 Å. The self-rotation function confirms that there are two monomers per asymmetric unit related by a twofold non-crystallographic axis and that the unit cell thus contains four biological entities.

  7. The genes and enzymes involved in the biosynthesis of thiamin and thiamin diphosphate in yeasts.

    Science.gov (United States)

    Kowalska, Ewa; Kozik, Andrzej

    2008-01-01

    Thiamin (vitamin B1) is an essential molecule for all living organisms. Its major biologically active derivative is thiamin diphosphate, which serves as a cofactor for several enzymes involved in carbohydrate and amino acid metabolism. Important new functions for thiamin and its phosphate esters have recently been suggested, e.g. in gene expression regulation by influencing mRNA structure, in DNA repair after UV illumination, and in the protection of some organelles against reactive oxygen species. Unlike higher animals, which rely on nutritional thiamin intake, yeasts can synthesize thiamin de novo. The biosynthesis pathways include the separate synthesis of two precursors, 4-amino-5-hydroxymethyl-2-methylpyrimidine diphosphate and 5-(2-hydroxyethyl)-4-methylthiazole phosphate, which are then condensed into thiamin monophosphate. Additionally, yeasts evolved salvage mechanisms to utilize thiamin and its dephosphorylated late precursors, 4-amino-5-hydroxymethyl-2-methylpyrimidine and 5-(2-hydroxyethyl)-4-methylthiazole, from the environment. The current state of knowledge on the discrete steps of thiamin biosynthesis in yeasts is far from satisfactory; many intermediates are postulated only by analogy to the much better understood biosynthesis process in bacteria. On the other hand, the genetic mechanisms regulating thiamin biosynthesis in yeasts are currently under extensive exploration. Only recently, the structures of some of the yeast enzymes involved in thiamin biosynthesis, such as thiamin diphosphokinase and thiazole synthase, were determined at the atomic resolution, and mechanistic proposals for the catalysis of particular biosynthetic steps started to emerge.

  8. Reversed-phase ion-pair high-performance liquid chromatography assay of polyprenyl diphosphate oligomer homologues.

    Science.gov (United States)

    Kozlov, Vyacheslav V; Danilov, Leonid L

    2016-02-01

    A reversed-phase ion-pair high-performance liquid chromatography procedure was developed for the separation of polyprenyl diphosphate oligomer homologues obtained chemically from plant polyprenols. Tetrabutylammonium phosphate was used as the ion-pair reagent, and the dependence of the separation quality on pH of ion-pair reagent was investigated for the first time. The procedure is applicable for the control of commercial available polyprenyl monophosphates (the active components of veterinary drugs Phosprenyl and Gamapren) for the possible presence of polyprenyl diphosphate byproducts. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. [Production of coenzyme Q10 by metabolically engineered Escherichia coli].

    Science.gov (United States)

    Dai, Guanping; Miao, Liangtian; Sun, Tao; Li, Qingyan; Xiao, Dongguang; Zhang, Xueli

    2015-02-01

    Coenzyme Q10 (CoQ10) is a lipophilic antioxidant that improves human immunity, delays senility and enhances the vitality of the human body and has wide applications in pharmaceutical and cosmetic industries. Microbial fermentation is a sustainable way to produce CoQ10, and attracts increased interest. In this work, the native CoQ8 synthetic pathway of Escherichia coli was replaced by the CoQ10 synthetic pathway through integrating decaprenyl diphosphate synthase gene (dps) from Rhodobacter sphaeroides into chromosome of E. coli ATCC 8739, followed by deletion of the native octaprenyl diphosphate synthase gene (ispB). The resulting strain GD-14 produced 0.68 mg/L CoQ10 with a yield of 0.54 mg/g DCW. Modulation of dxs and idi genes of the MEP pathway and ubiCA genes in combination led to 2.46-fold increase of CoQ10 production (from 0.54 to 1.87 mg/g DCW). Recruiting glucose facilitator protein of Zymomonas mobilis to replace the native phosphoenolpyruvate: carbohydrate phosphotransferase systems (PTS) further led to a 16% increase of CoQ10 yield. Finally, fed-batch fermentation of the best strain GD-51 was performed, which produced 433 mg/L CoQ10 with a yield of 11.7 mg/g DCW. To the best of our knowledge, this was the highest CoQ10 titer and yield obtained for engineered E. coli.

  10. A functional (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase exhibits diurnal regulation of expression in Stevia rebaudiana (Bertoni).

    Science.gov (United States)

    Kumar, Hitesh; Kumar, Sanjay

    2013-09-15

    The leaves of stevia [Stevia rebaudiana (Bertoni)] are a rich source of steviol glycosides that are used as non-calorific sweetener in many countries around the world. Steviol moiety of steviol glycosides is synthesized via plastidial 2C-methyl-D-erythritol 4-phosphate pathway, where (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is the key enzyme. HDR catalyzes the simultaneous conversion of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into five carbon isoprenoid units, isopentenyl diphosphate and dimethylallyl diphosphate. Stevia HDR (SrHDR) successfully rescued HDR lethal mutant strain MG1655 araispH upon genetic complementation, suggesting SrHDR to encode a functional protein. The gene exhibited diurnal variation in expression. To identify the possible regulatory elements, upstream region of the gene was cloned and putative cis-acting elements were detected by in silico analysis. Electrophoretic mobility shift assay, using a putative light responsive element GATA showed the binding of nuclear proteins (NP) isolated from leaves during light period of the day, but not with the NP from leaves during the dark period. Data suggested the involvement of GATA box in light mediated gene regulation of SrHDR in stevia.

  11. Early Cessation of Adenosine Diphosphate Receptor Inhibitors Among Acute Myocardial Infarction Patients Treated With Percutaneous Coronary Intervention

    DEFF Research Database (Denmark)

    Fosbøl, Emil L; Ju, Christine; Anstrom, Kevin J

    2016-01-01

    BACKGROUND: Guidelines recommend the use of adenosine diphosphate receptor inhibitor (ADPri) therapy for 1 year postacute myocardial infarction; yet, early cessation of therapy occurs frequently in clinical practice. METHODS AND RESULTS: We examined 11 858 acute myocardial infarction patients tre...... adverse cardiovascular event risk. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01088503....

  12. Adenosine-diphosphate (ADP) receptor antagonists for the prevention of cardiovascular disease in type 2 diabetes mellitus (Review)

    NARCIS (Netherlands)

    Valentine, N.; Laar, F.A. van de; Driel, M.L. van

    2012-01-01

    BACKGROUND: Cardiovascular disease (CVD) is the most prevalent complication of type 2 diabetes with an estimated 65% of people with type 2 diabetes dying from a cause related to atherosclerosis. Adenosine-diphosphate (ADP) receptor antagonists like clopidogrel, ticlopidine, prasugrel and ticagrelor

  13. Structure of uridine diphosphate N-acetylglucosamine pyrophosphorylase from Entamoeba histolytica.

    Science.gov (United States)

    Edwards, Thomas E; Gardberg, Anna S; Phan, Isabelle Q H; Zhang, Yang; Staker, Bart L; Myler, Peter J; Lorimer, Donald D

    2015-05-01

    Uridine diphosphate N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the final step in the synthesis of UDP-GlcNAc, which is involved in cell-wall biogenesis in plants and fungi and in protein glycosylation. Small-molecule inhibitors have been developed against UAP from Trypanosoma brucei that target an allosteric pocket to provide selectivity over the human enzyme. A 1.8 Å resolution crystal structure was determined of UAP from Entamoeba histolytica, an anaerobic parasitic protozoan that causes amoebic dysentery. Although E. histolytica UAP exhibits the same three-domain global architecture as other UAPs, it appears to lack three α-helices at the N-terminus and contains two amino acids in the allosteric pocket that make it appear more like the enzyme from the human host than that from the other parasite T. brucei. Thus, allosteric inhibitors of T. brucei UAP are unlikely to target Entamoeba UAPs.

  14.   Adenosine-diphosphate (ADP) reduces infarct size and improves porcine heart function after myocardial infarction

    DEFF Research Database (Denmark)

    Bune, Laurids Touborg; Larsen, Jens Kjærgaard Rolighed; Thaning, Pia;

    2013-01-01

    (UTP) are both released during myocardial ischemia, influencing hemodynamics. Both mediate the release of tissue plasminogen activator (t-PA), which can reduce infarct size (IS). The objective of this study was to investigate whether exogenous ADP and UTP administration during reperfusion could reduce......Acute myocardial infarction continues to be a major cause of morbidity and mortality. Timely reperfusion can substantially improve outcomes and the administration of cardioprotective substances during reperfusion is therefore highly attractive. Adenosine diphosphate (ADP) and uridine-5-triphoshate...... myocardial IS and whether this correlated to t-PA release or improvements in hemodynamic responses. Hemodynamic variables and t-PA were measured in 22 pigs before, during, and after 45 min of left anterior coronary artery occlusion. During reperfusion, the pigs were randomized to 240 min of intracoronary...

  15. Preliminary crystallographic data for the thiamin diphosphate-dependent enzyme pyruvate decarboxylase from brewers' yeast.

    Science.gov (United States)

    Dyda, F; Furey, W; Swaminathan, S; Sax, M; Farrenkopf, B; Jordan, F

    1990-10-15

    Single crystals of the thiamin diphosphate (the vitamin B1 coenzyme)-dependent enzyme pyruvate decarboxylase (EC 4.1.1.1) from brewers' yeast have been grown using polyethylene glycol as a precipitating agent. Crystals of the homotetrameric version alpha 4 of the holoenzyme are triclinic, space group P1, with cell constants a = 81.0, b = 82.4, c = 116.6 A, alpha = 69.5 beta = 72.6, gamma = 62.4 degrees. The crystals are reasonably stable in a rotating anode x-ray beam and diffract to at least 2.5 A resolution. The Vm value of 2.55 A/dalton is consistent with a unit cell containing four subunits with mass of approximately 60 kDa each. Rotation function results with native data indicate strong non-crystallographic 222 symmetry relating the four identical subunits, thus density averaging methods are likely to play a role in the structure determination.

  16. Interaction of thiamin diphosphate with phosphorylated and dephosphorylated mammalian pyruvate dehydrogenase complex.

    Science.gov (United States)

    Liu, Xiaoqing; Bisswanger, Hans

    2005-01-01

    Kinetic and binding studies were carried out on substrate and cofactor interaction with the pyruvate dehydrogenase complex from bovine heart. Fluoropyruvate and pyruvamide, previously described as irreversible and allosteric inhibitors, respectively, are strong competitive inhibitors with respect to pyruvate. Binding of thiamin diphosphate was used to study differences between the active dephosphorylated and inactive phosphorylated enzyme states by spectroscopic methods. The change in both the intrinsic tryptophan fluorescence and the fluorescence of the 6-bromoacetyl-2-dimethylaminonaphthalene-labelled enzyme complex produced on addition of the cofactor showed similar binding behaviour for both enzyme forms, with slightly higher affinity for the phosphorylated form. Changes in the CD spectrum, especially the negative Cotton effect at 330 nm as a function of cofactor concentration, both in the absence and presence of pyruvate, also revealed no drastic differences between the two enzyme forms. Thus, inactivation of the enzyme activity of the pyruvate dehydrogenase complex is not caused by impeding the binding of substrate or cofactor.

  17. Acanthamoeba polyphaga mimivirus NDK: preliminary crystallographic analysis of the first viral nucleoside diphosphate kinase.

    Science.gov (United States)

    Jeudy, Sandra; Coutard, Bruno; Lebrun, Régine; Abergel, Chantal

    2005-06-01

    The complete sequence of the largest known double-stranded DNA virus, Acanthamoeba polyphaga mimivirus, has recently been determined [Raoult et al. (2004), Science, 306, 1344-1350] and revealed numerous genes not expected to be found in a virus. A comprehensive structural and functional study of these gene products was initiated [Abergel et al. (2005), Acta Cryst. F61, 212-215] both to better understand their role in the virus physiology and to obtain some clues to the origin of DNA viruses. Here, the preliminary crystallographic analysis of the viral nucleoside diphosphate kinase protein is reported. The crystal belongs to the cubic space group P2(1)3, with unit-cell parameter 99.425 A. The self-rotation function confirms that there are two monomers per asymmetric unit related by a twofold non-crystallographic axis and that the unit cell thus contains four biological entities.

  18. Structure activity relationship of uridine 5′-diphosphate analogues at the human P2Y6 receptor

    Science.gov (United States)

    Besada, Pedro; Shin, Dae Hong; Costanzi, Stefano; Ko, Hyojin; Mathé, Christophe; Gagneron, Julien; Gosselin, Gilles; Maddileti, Savitri; Harden, T. Kendall; Jacobsona, Kenneth A.

    2012-01-01

    The structure activity relationships and molecular modeling of the uracil nucleotide-activated P2Y6 receptor have been studied. A series of UDP analogues bearing substitutions of the ribose moiety, the uracil ring, and the diphosphate group was synthesized and assayed for activity at the human P2Y6 receptor. The uracil ring was modified at the 4-position, with the synthesis of 4-substituted-thiouridine-5′-diphosphate analogues, as well as at positions 3 and 5. The effect of modifications at the level of the phosphate chain was studied by preparing a cyclic 3′,5′-diphosphate analogue, a 3′-diphosphate analogue and several dinucleotide diphosphates. 5-Iodo-UDP 32 (EC50 0.15 μM) was equipotent to UDP, while substitutions of the 2′-hydroxyl (amino, azido) greatly reduce potency. 2- and 4-Thio analogues, 20 and 21, respectively, were also relatively potent in comparison to UDP. However, most other modifications greatly reduced potency. Molecular modeling indicates that the β-phosphate of 5′-UDP and analogs is essential for the establishment of electrostatic interactions with two of the three conserved cationic residues of the receptor. Among 4-thioether derivatives, a 4-ethylthio analogue 23 displayed an EC50 of 0.28 μM, indicative of favorable interactions predicted for a small 4-alkylthio moiety with the aromatic ring of Y33 in TM1. The activity of analogue 19 in which the ribose was substituted with a 2-oxabicyclohexane ring in a rigid (S) conformation (P= 126°, 1′-exo) was consistent with molecular modeling. These results provide a better understanding of molecular recognition at the P2Y6 receptor and will be helpful in designing selective and potent P2Y6 receptor ligands PMID:16942026

  19. Crystal Structures of Staphylococcus epidermidis Mevalonate Diphosphate Decarboxylase Bound to Inhibitory Analogs Reveal New Insight into Substrate Binding and Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Barta, Michael L.; Skaff, D. Andrew; McWhorter, William J.; Herdendorf, Timothy J.; Miziorko, Henry M.; Geisbrecht, Brian V. (UMKC)

    2011-10-28

    The polyisoprenoid compound undecaprenyl phosphate is required for biosynthesis of cell wall peptidoglycans in Gram-positive bacteria, including pathogenic Enterococcus, Streptococcus, and Staphylococcus spp. In these organisms, the mevalonate pathway is used to produce the precursor isoprenoid, isopentenyl 5-diphosphate. Mevalonate diphosphate decarboxylase (MDD) catalyzes formation of isopentenyl 5-diphosphate in an ATP-dependent irreversible reaction and is therefore an attractive target for inhibitor development that could lead to new antimicrobial agents. To facilitate exploration of this possibility, we report the crystal structure of Staphylococcus epidermidis MDD (1.85 {angstrom} resolution) and, to the best of our knowledge, the first structures of liganded MDD. These structures include MDD bound to the mevalonate 5-diphosphate analogs diphosphoglycolyl proline (2.05 {angstrom} resolution) and 6-fluoromevalonate diphosphate (FMVAPP; 2.2 {angstrom} resolution). Comparison of these structures provides a physical basis for the significant differences in K{sub i} values observed for these inhibitors. Inspection of enzyme/inhibitor structures identified the side chain of invariant Ser{sup 192} as making potential contributions to catalysis. Significantly, Ser {yields} Ala substitution of this side chain decreases k{sub cat} by {approx}10{sup 3}-fold, even though binding interactions between FMVAPP and this mutant are similar to those observed with wild type MDD, as judged by the 2.1 {angstrom} cocrystal structure of S192A with FMVAPP. Comparison of microbial MDD structures with those of mammalian counterparts reveals potential targets at the active site periphery that may be exploited to selectively target the microbial enzymes. These studies provide a structural basis for previous observations regarding the MDD mechanism and inform future work toward rational inhibitor design.

  20. Stathmin slows down guanosine diphosphate dissociation from tubulin in a phosphorylation-controlled fashion.

    Science.gov (United States)

    Amayed, P; Carlier, M F; Pantaloni, D

    2000-10-10

    Stathmin is an important protein that interacts with tubulin and regulates microtubule dynamics in a phosphorylation-controlled fashion. Here we show that the dissociation of guanosine 5'-diphosphate (GDP) from beta-tubulin is slowed 20-fold in the (tubulin)(2)-stathmin ternary complex (T(2)S). The kinetics of GDP or guanosine 5'-triphosphate (GTP) dissociation from tubulin have been monitored by the change in tryptophan fluorescence of tubulin upon exchanging 2-amino-6-mercapto-9-beta-ribofuranosylpurine 5'-diphosphate (S6-GDP) for tubulin-bound guanine nucleotide. At molar ratios of stathmin to tubulin lower than 0.5, biphasic kinetics were observed, indicating that the dynamics of the complex is extremely slow, consistent with its high stability. The method was used to characterize the effects of phosphorylation of stathmin on its interaction with tubulin. The serine-to-glutamate substitution of all four phosphorylatable serines of stathmin (4E-stathmin) weakens the stability of the T(2)S complex by about 2 orders of magnitude. The phosphorylation of serines 16 and 63 in stathmin has a more severe effect and weakens the stability of T(2)S 10(4)-fold. The rate of GDP dissociation is lowered only 7-fold and 4-fold in the complexes of tubulin with 4E-stathmin and diphosphostathmin, respectively. Sedimentation velocity studies support the conclusions of nucleotide exchange data and show that the T(2)S complexes formed between tubulin and 4E-stathmin or diphosphostathmin are less compact than the highly stable T(2)S complex. The correlation between the effect of phosphorylation of stathmin on the stability of T(2)S complex measured in vitro and on the function of stathmin in vivo is discussed.

  1. A STUDY OF THE NUCLEOSIDE TRI- AND DIPHOSPHATE ACTIVITIES OF RAT LIVER MICROSOMES

    Science.gov (United States)

    Ernster, Lars; Jones, Lois C.

    1962-01-01

    Rat liver microsomes catalyze the hydrolysis of the triphosphates of adenosine, guanosine, uridine, cytidine, and inosine into the corresponding diphosphates and inorganic orthophosphate. The activities are stimulated by Na2S2O4, and inhibited by atebrin, chlorpromazine, sodium azide, and deaminothyroxine. Sodium deoxycholate inhibits the ATPase activity in a progressive manner; the release of orthophosphate from GTP and UTP is stimulated by low, and inhibited by high, concentrations of deoxycholate, and that from CTP and ITP is unaffected by low, and inhibited by high, concentrations of deoxycholate. Subfractionation of microsomes with deoxycholate into ribosomal, membrane, and soluble fractions reveals a concentration of the triphosphatase activity in the membrane fraction. Rat liver microsomes also catalyze the hydrolysis of the diphosphates of the above nucleosides into the corresponding monophosphates and inorganic orthophosphate. Deoxycholate strongly enhances the GDPase, UDPase, and IDPase activities while causing no activation or even inhibition of the ADPase and CDPase activities. The diphosphatase is unaffected by Na2S2O4 and is inhibited by azide and deaminothyroxine but not by atebrin or chlorpromazine. Upon fractionation of the microsomes with deoxycholate, a large part of the GDPase, UDPase, and IDPase activities is recovered in the soluble fraction. Mechanical disruption of the microsomes with an Ultra Turrax Blender both activates and releases the GDPase, UDPase, and IDPase activities, and the former effect occurs more readily than the latter. The GDPase, UDPase, and IDPase activities of the rat liver cell reside almost exclusively in the microsomal fraction, as revealed by comparative assays of the mitochondrial, microsomal, and final supernatant fractions of the homogenate. The microsomes exhibit relatively low nucleoside monophosphatase and inorganic pyrophosphatase activities, and these are unaffected by deoxycholate or mechanical treatment

  2. Improving monoterpene geraniol production through geranyl diphosphate synthesis regulation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Zhao, Jianzhi; Bao, Xiaoming; Li, Chen; Shen, Yu; Hou, Jin

    2016-05-01

    Monoterpenes have wide applications in the food, cosmetics, and medicine industries and have recently received increased attention as advanced biofuels. However, compared with sesquiterpenes, monoterpene production is still lagging in Saccharomyces cerevisiae. In this study, geraniol, a valuable acyclic monoterpene alcohol, was synthesized in S. cerevisiae. We evaluated three geraniol synthases in S. cerevisiae, and the geraniol synthase Valeriana officinalis (tVoGES), which lacked a plastid-targeting peptide, yielded the highest geraniol production. To improve geraniol production, synthesis of the precursor geranyl diphosphate (GPP) was regulated by comparing three specific GPP synthase genes derived from different plants and the endogenous farnesyl diphosphate synthase gene variants ERG20 (G) (ERG20 (K197G) ) and ERG20 (WW) (ERG20 (F96W-N127W) ), and controlling endogenous ERG20 expression, coupled with increasing the expression of the mevalonate pathway by co-overexpressing IDI1, tHMG1, and UPC2-1. The results showed that overexpressing ERG20 (WW) and strengthening the mevalonate pathway significantly improved geraniol production, while expressing heterologous GPP synthase genes or down-regulating endogenous ERG20 expression did not show positive effect. In addition, we constructed an Erg20p(F96W-N127W)-tVoGES fusion protein, and geraniol production reached 66.2 mg/L after optimizing the amino acid linker and the order of the proteins. The best strain yielded 293 mg/L geraniol in a fed-batch cultivation, a sevenfold improvement over the highest titer previously reported in an engineered S. cerevisiae strain. Finally, we showed that the toxicity of geraniol limited its production. The platform developed here can be readily used to synthesize other monoterpenes.

  3. 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.

  4. Repeated febrile convulsions impair hippocampal neurons and cause synaptic damage in immature rats:neuroprotective effect of fructose-1,6-diphosphate

    Institute of Scientific and Technical Information of China (English)

    Jianping Zhou; Fan Wang; Jun Zhang; Hui Gao; Yufeng Yang; Rongguo Fu

    2014-01-01

    Fructose-1,6-diphosphate is a metabolic intermediate that promotes cell metabolism. We hy-pothesize that fructose-1,6-diphosphate can protect against neuronal damage induced by febrile convulsions. Hot-water bathing was used to establish a repetitive febrile convulsion model in rats aged 21 days, equivalent to 3-5 years in humans. Ninety minutes before each seizure induc-tion, rats received an intraperitoneal injection of low- or high-dose fructose-1,6-diphosphate (500 or 1,000 mg/kg, respectively). Low- and high-dose fructose-1,6-diphosphate prolonged the latency and shortened the duration of seizures. Furthermore, high-dose fructose-1,6-di-phosphate effectively reduced seizure severity. Transmission electron microscopy revealed that 24 hours after the last seizure, high-dose fructose-1,6-diphosphate reduced mitochondrial swelling, rough endoplasmic reticulum degranulation, Golgi dilation and synaptic cleft size, and increased synaptic active zone length, postsynaptic density thickness, and synaptic interface cur-vature in the hippocampal CA1 area. The present findings suggest that fructose-1,6-diphosphate is a neuroprotectant against hippocampal neuron and synapse damage induced by repeated fe-brile convulsion in immature rats.

  5. Evaluation of the sorption of Eu(III) in titanium diphosphate; Evaluacion de la sorcion de Eu(III) en difosfato de titanio

    Energy Technology Data Exchange (ETDEWEB)

    Ortiz O, H.B.; Ordonez R, E.; Fernandez V, S.M. [ININ, Carretera Mexico-Toluca Km 36.5, Salazar, Estado de Mexico (Mexico)]. e-mail: hortiz@nuclear.inin.mx

    2007-07-01

    In this work its are presented: the synthesis, physicochemical characterization and the surface parameters estimation that can be related with the retention properties of the titanium diphosphate for the actinides of valence III (Pu, Am, Cm among others), using the Eu{sup 3+} like a chemical analog. The surface area, hydration time, zero charge point, density of active sites and the surface species distribution in the titanium diphosphate are reported. This information was used to explain the retention of the Eu(lll) in the surface of the titanium diphosphate. (Author)

  6. E. Coli and Pregnancy

    Science.gov (United States)

    ... best live chat Live Help Fact Sheets Share E. coli and Pregnancy Thursday, 20 November 2014 In every ... risk. This sheet talks about whether exposure to E. coli may increase the risk for birth defects over ...

  7. E. coli enteritis

    Science.gov (United States)

    ... medlineplus.gov/ency/article/000296.htm E. coli enteritis To use the sharing features on this page, please enable JavaScript. E. coli enteritis is swelling (inflammation) of the small intestine from ...

  8. Organization of monoterpene biosynthesis in Mentha. Immunocytochemical localizations of geranyl diphosphate synthase, limonene-6-hydroxylase, isopiperitenol dehydrogenase, and pulegone reductase.

    Science.gov (United States)

    Turner, Glenn W; Croteau, Rodney

    2004-12-01

    We present immunocytochemical localizations of four enzymes involved in p-menthane monoterpene biosynthesis in mint: the large and small subunits of peppermint (Mentha x piperita) geranyl diphosphate synthase, spearmint (Mentha spicata) (-)-(4S)-limonene-6-hydroxylase, peppermint (-)-trans-isopiperitenol dehydrogenase, and peppermint (+)-pulegone reductase. All were localized to the secretory cells of peltate glandular trichomes with abundant labeling corresponding to the secretory phase of gland development. Immunogold labeling of geranyl diphosphate synthase occurred within secretory cell leucoplasts, (-)-4S-limonene-6-hydroxylase labeling was associated with gland cell endoplasmic reticulum, (-)-trans-isopiperitenol dehydrogenase labeling was restricted to secretory cell mitochondria, while (+)-pulegone reductase labeling occurred only in secretory cell cytoplasm. We discuss this pathway compartmentalization in relation to possible mechanisms for the intracellular movement of monoterpene metabolites, and for monoterpene secretion into the extracellular essential oil storage cavity.

  9. Discrimination between acid and alkali-labile phosphorylated residues on Immobilon: phosphorylation studies of nucleoside diphosphate kinase

    DEFF Research Database (Denmark)

    Biondi, R M; Walz, K; Issinger, O G

    1996-01-01

    to deplete phosphate from membranes incubated successively under acid and basic conditions. The technique was applied to the study of nucleoside diphosphate kinase (NDP kinase) phosphorylation. In this enzyme, autophosphorylation of active site histidine is an accepted intermediate step in the catalytic...... phosphate transfer activity of nucleoside diphosphate kinase (NDP kinase). Nonetheless, a significant degree of autophosphorylation on other residues has been reported by several laboratories, and the hypothesis has been advanced that this nonhistidine phosphorylation may play an important role in NDP...... of phosphoserine after strong acid hydrolysis of the histidine autophosphorylated enzyme is in fact a nonenzymatic transphosphorylation from phosphohistidine due to the harsh acid treatment. This methodology was also applied to in vivo phosphorylation studies of C. albicans NDP kinase. We believe...

  10. Peculiarities of the inhibition of the pyruvate dehydrogenase complex by thiamine thiazolone diphosphate in vitro and in intact mitochondria

    Energy Technology Data Exchange (ETDEWEB)

    Yakovleva, G.M.; Strumilo, S.A.; Gorenshtein, B.I.; Ostrovskii, Yu.M.

    1986-07-10

    Thiamine thiazolone diphosphate (TTPP) possesses the ability to penetrate through the mitochondrial membrane and inhibit the pyruvate dehydrogenase complex in intact mitochondria, TTPP inhibits the activity of the complex of animal origin according to a mixed type (K/sub i/ 5 x 10/sup -8/ M) and yeast pyruvate decarboxylase according to a competitive type (K/sub i/ 5 x 10/sup -6/ M) with respect to thiamine diphosphate (TPP). Decarboxylation of pyruvate in intact and lysed rat liver and brain mitochondria is inhibited in the presence of TTPP significantly more weakly than the total activity of the pyruvate dehydrogenase complex, determined according to the formation of acetyl-CoA. It is suggested that TTPP, as an analog of the transition state, acts only in dehydrogenase reactions but not at the stage of simple decarboxylation of pyruvate.

  11. Activation and modulation of cardiac poly-adenosine diphosphate ribose polymerase activity in a rat model of brain death.

    Science.gov (United States)

    Brain, John G; Rostron, Anthony J; Dark, John H; Kirby, John A

    2008-05-15

    DNA damage during transplantation can activate poly-adenosine diphosphate ribose polymerase (PARP) resulting in the generation of polymers of adenosine diphosphate-ribose (PAR). Excessive linkage of PAR to nuclear proteins can induce cell death, thereby limiting the function of transplanted organs. This study uses a rat model of brain death to determine the profile of PARP activation and whether mechanisms that lead to cell death can be ameliorated by appropriate donor resuscitation. The expression of PAR-linked nuclear proteins within cardiac myocytes was greatly increased after the induction of donor brain death. Importantly, infusion of noradrenaline or vasopressin to normalize the chronic hypotension produced by brain death reduced the expression of PAR to a level below baseline. These data suggest that chronic hypotension after donor brain death has the potential to limit cardiac function through the activation of PARP; however, this early cause of graft damage can be mitigated by appropriate donor resuscitation.

  12. E. Coli Infections

    Science.gov (United States)

    E. coli is the name of a type of bacteria that lives in your intestines. Most types of E. coli are harmless. However, some types can make you ... type causes travelers' diarrhea. The worst type of E. coli causes bloody diarrhea, and can sometimes cause kidney ...

  13. Enhancement of cytidine production by coexpression of gnd, zwf, and prs genes in recombinant Escherichia coli CYT15.

    Science.gov (United States)

    Fang, Haitian; Xie, Xixian; Xu, Qingyang; Zhang, Chenglin; Chen, Ning

    2013-02-01

    Cytidine is a precursor of several antiviral drugs. The pentose phosphate pathway (PPP) is primarily responsible for NADPH and 5-phospho-α-D-ribose 1-diphosphate as an important precursor of cytidine biosynthesis in Escherichia coli. To enhance cytidine production, we obtained the recombinant E. coli CYT15-gnd-prs-zwf that co-expressed the prs, zwf, and gnd genes encoding phosphoribosylpyrophosphate synthetase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (three key enzymes in PPP) respectively. In fermentation experiments, strain CYT15-gnd-prs-zwf produced 735 mg cytidine/l using glucose as substrate, which was approx. 128 % higher than the cytidine production by the parental strain (CYT15). Co-expression of zwf, gnd, and prs decreased growth (3.2 %) slightly and increased glucose uptake (72 %). This is the first study to report increased cytidine production by increasing metabolic flux through the PPP in E. coli.

  14. Saccharomyces pastorianus as cell factory to improve production of fructose 1,6-diphosphate using novel fermentation strategies

    Directory of Open Access Journals (Sweden)

    Chiara Schiraldi

    2015-08-01

    Full Text Available Enzymatic phosphorylation of glucose with inorganic phosphate, mediated by permeabilized yeast cells, is one of the methods commonly used to manufacture fructose 1,6-diphosphate, a compound of pharmaceutical interest. This process requires high concentrations of yeast active biomass, that is the catalyst of bioconversion of glucose and inorganic phosphate into fructose 1,6-diphosphate. In this study we firstly describe the high cell density production of a brewer's Saccharomyces strain (Saccharomyces pastorianus DSM 6581, focusing on the optimization of medium composition and exploiting fed-batch strategies and novel technologies based on membrane bioreactors. In fed-batch fermentation an appropriate exponential feed profile was set up to maintain the glucose concentration in the bioreactor below 0.9 g·L-1, thus yielding reproducibly 58 g dry weight biomass per liter in 80 h fermentation, improving eight-fold batch processes output. In addition a higher final biomass density was reached when implementing a microfiltration strategy (70 g dry weight biomass, that led to a productivity of 2.1 gcdw·L-1·h-1, 2.4-fold the fed-batch one. Successively, this biomass was opportunely permeabilized and proved capable of catalyzing the bioconversion of glucose into fructose 1,6-diphosphate. Acting on critical parameters of the bioconversion (substrates molar ratio, catalyst concentration and permeabilization agent, fructose 1,6-diphosphate was produced, after 3 h of process, at 56.3 ± 1 g·L-1 with a yield of 80% of the theoretical value.

  15. Feasibility of gas/solid carboligation: conversion of benzaldehyde to benzoin using thiamine diphosphate-dependent enzymes.

    Science.gov (United States)

    Mikolajek, R; Spiess, A C; Büchs, J

    2007-05-10

    A carboligation was investigated for the first time as an enzymatic gas phase reaction, where benzaldehyde was converted to benzoin using thiamine diphosphate (ThDP)-dependent enzymes, namely benzaldehyde lyase (BAL) and benzoylformate decarboxylase (BFD). The biocatalyst was immobilized per deposition on non-porous support. Some limitations of the gas/solid biocatalysis are discussed based on this carboligation and it is also demonstrated that the solid/gas system is an interesting tool for more volatile products.

  16. A tailor-made chimeric thiamine diphosphate dependent enzyme for the direct asymmetric synthesis of (S)-benzoins.

    Science.gov (United States)

    Westphal, Robert; Vogel, Constantin; Schmitz, Carlo; Pleiss, Jürgen; Müller, Michael; Pohl, Martina; Rother, Dörte

    2014-08-25

    Thiamine diphosphate dependent enzymes are well known for catalyzing the asymmetric synthesis of chiral α-hydroxy ketones from simple prochiral substrates. The steric and chemical properties of the enzyme active site define the product spectrum. Enzymes catalyzing the carboligation of aromatic aldehydes to (S)-benzoins have not so far been identified. We were able to close this gap by constructing a chimeric enzyme, which catalyzes the synthesis of various (S)-benzoins with excellent enantiomeric excess (>99%) and very good conversion.

  17. Mitochondria from cultured cells derived from normal and thiamine-responsive megaloblastic anemia individuals efficiently import thiamine diphosphate

    OpenAIRE

    Singleton Charles K; Song Qilin

    2002-01-01

    Abstract Background Thiamine diphosphate (ThDP) is the active form of thiamine, and it serves as a cofactor for several enzymes, both cytosolic and mitochondrial. Isolated mitochondria have been shown to take up thiamine yet thiamine diphosphokinase is cytosolic and not present in mitochondria. Previous reports indicate that ThDP can also be taken up by rat mitochondria, but the kinetic constants associated with such uptake seemed not to be physiologically relevant. Results Here we examine Th...

  18. Biosynthesis of anthecotuloide, an irregular sesquiterpene lactone from Anthemis cotula L. (Asteraceae) via a non-farnesyl diphosphate route.

    Science.gov (United States)

    van Klink, John; Becker, Hans; Andersson, Susannah; Boland, Wilhelm

    2003-05-07

    Retrobiosynthetic analysis of the allergenic sesquiterpene lactone, anthecotuloide, suggested that this natural product could be formed either by head to head condensation of geranyl diphosphate with dimethylallyl diphosphate, or from farnesyl diphosphate (FPP), the accepted regular sesquiterpene precursor via the rearrangement of a germacranolide precursor. Isotopic labelling of anthecotuloide has now been achieved by feeding [1-(13)C]-glucose, [U-13C6]-glucose and [6,6-(2)H2]-glucose to aseptically grown plantlets of Anthemis cotula(family Asteraceae). Analysis of labelling patterns and absolute 13C abundances using quantitative 13C NMR spectroscopy showed that the isoprene building blocks of this sesquiterpene are formed exclusively via the MEP terpene biosynthetic pathway. This was supported by results from an experiment using [U-13C6]-glucose. A deuterium labelling experiment using [6,6-(2)H2]-glucose supported the original proposal and showed that anthecotuloide is formed from a non FPP precursor. Isotope ratio mass spectrometry suggested that there were two pathways for sesquiterpene biosynthesis in A. cotula.

  19. Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

    Science.gov (United States)

    Bominaar, A A; Molijn, A C; Pestel, M; Veron, M; Van Haastert, P J

    1993-01-01

    Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins. Images PMID:8389692

  20. Evaluation of serum nucleoside diphosphate kinase A for the detection of colorectal cancer

    Science.gov (United States)

    Otero-Estévez, Olalla; De Chiara, Loretta; Barcia-Castro, Leticia; Páez de la Cadena, María; Rodríguez-Berrocal, Francisco Javier; Cubiella, Joaquín; Hernández, Vicent; Martínez-Zorzano, Vicenta Soledad

    2016-01-01

    We previously described the over-expression of nucleoside diphosphate kinase A (NDKA) in tumours and serum from colorectal cancer (CRC) patients, suggesting its use as biomarker. In this study we evaluated the diagnostic accuracy of serum NDKA to detect advanced neoplasia (CRC or advanced adenomas). Furthermore, the performance of NDKA was compared with the faecal immunochemical test (FIT). The study population included a case-control cohort and a screening cohort (511 asymptomatic first-degree relatives of CRC patients that underwent a colonoscopy and a FIT). Serum NDKA was elevated in CRC patients in the case-control cohort (p = 0.002). In the screening cohort, NDKA levels were higher for advanced adenomas (p = 0.010) and advanced neoplasia (p = 0.006) compared to no neoplasia. Moreover, elevated NDKA was associated with severe characteristics of adenomas (≥3 lesions, size ≥ 1 cm or villous component). Setting specificity to 85%, NDKA showed a sensitivity of 30.19% and 29.82% for advanced adenomas and advanced neoplasia, respectively. NDKA combined with FIT (100 ng/mL cut-off) detected advanced adenomas and advanced neoplasia with 45.28% and 49.12% sensitivity, with specificity close to 90%. The combination of serum NDKA and FIT can improve the detection of advanced neoplasia, mainly for lesions located on the proximal colon, in asymptomatic individuals with CRC family-risk. PMID:27222072

  1. New function of the amino group of thiamine diphosphate in thiamine catalysis.

    Science.gov (United States)

    Meshalkina, L E; Kochetov, G A; Hübner, G; Tittmann, K; Golbik, R

    2009-03-01

    In this work, we investigated the rate of formation of the central intermediate of the transketolase reaction with thiamine diphosphate (ThDP) or 4'-methylamino-ThDP as cofactors and its stability using stopped-flow spectroscopy and circular dichroism (CD) spectroscopy. The intermediates of the transketolase reaction were analyzed by NMR spectroscopy. The kinetic stability of the intermediate was shown to be dependent on the state of the amino group of the coenzyme. The rates of the intermediate formation were the same in the case of the native and methylated ThDP, but the rates of the protonation or oxidation of the complex in the ferricyanide reaction were significantly higher in the complex with methylated ThDP. A new negative band was detected in the CD spectrum of the complex transketolase--4'-methylamino-ThDP corresponding to the protonated dihydroxyethyl-4'-methylamino-ThDP released from the active sites of the enzyme. These data suggest that transketolase in the complex with the NH2-methylated ThDP exhibits dihydroxyethyl-4'-methylamino-ThDP-synthase activity. Thus, the 4'-amino group of the coenzyme provides kinetic stability of the central intermediate of the transketolase reaction, dihydroxyethyl-ThDP.

  2. Isolation and functional analysis of two Cistus creticus cDNAs encoding geranylgeranyl diphosphate synthase.

    Science.gov (United States)

    Pateraki, Irene; Kanellis, Angelos K

    2008-05-01

    Cistus creticus ssp. creticus is an indigenous shrub of the Mediterranean area. The glandular trichomes covering its leaf surfaces secrete a resin called "ladanum", which among others contains a number of specific labdane-type diterpenes that exhibit antibacterial and antifungal action as well as in vitro and in vivo cytotoxic and cytostatic activity against human cancer cell lines. In view of the properties and possible future exploitation of these metabolites, it was deemed necessary to study the geranylgeranyl diphosphate synthase enzyme (GGDPS, EC 2.5.1.30), a short chain prenyltransferase responsible for the synthesis of the precursor molecule of all diterpenes. In this work, we present the cloning, functional characterisation and expression profile at the gene and protein levels of two differentially expressed C. creticus full-length cDNAs, CcGGDPS1 and CcGGDPS2. Heterologous yeast cell expression system showed that these cDNAs exhibited GGDPS enzyme activity. Gene and protein expression analyses suggest that this enzyme is developmentally and tissue-regulated showing maximum expression in trichomes and smallest leaves (0.5-1.0cm). This work is the first attempt to study the terpenoid biosynthesis at the molecular level in C. creticus ssp. creticus.

  3. Roles and mechanisms of the CD38/cyclic adenosine diphosphate ribose/Ca2+ signaling pathway

    Institute of Scientific and Technical Information of China (English)

    Wenjie; Wei; Richard; Graeff; Jianbo; Yue

    2014-01-01

    Mobilization of intracellular Ca2+ stores is involved inmany diverse cell functions, including: cell proliferation;differentiation; fertilization; muscle contraction; secre-tion of neurotransmitters, hormones and enzymes;and lymphocyte activation and proliferation. Cyclic ad-enosine diphosphate ribose(cADPR) is an endogenousCa2+ mobilizing nucleotide present in many cell typesand species, from plants to animals. cADPR is formedby ADP-ribosyl cyclases from nicotinamide adenine di-nucleotide. The main ADP-ribosyl cyclase in mammalsis CD38, a multi-functional enzyme and a type Ⅱ mem-brane protein. It has been shown that many extracel-lular stimuli can induce cADPR production that leadsto calcium release or influx, establishing cADPR as asecond messenger. cADPR has been linked to a widevariety of cellular processes, but the molecular mecha-nisms regarding cADPR signaling remain elusive. Theaim of this review is to summarize the CD38/cADPR/Ca2+ signaling pathway, focusing on the recent advanc-es involving the mechanism and physiological functionsof cADPR-mediated Ca2+ mobilization.

  4. Geosmin biosynthesis. Streptomyces coelicolor germacradienol/germacrene D synthase converts farnesyl diphosphate to geosmin.

    Science.gov (United States)

    Jiang, Jiaoyang; He, Xiaofei; Cane, David E

    2006-06-28

    Geosmin is responsible for the characteristic odor of moist soil. Incubation of recombinant germacradienol synthase, encoded by the SCO6073 (SC9B1.20) gene of the Gram-positive soil bacterium Streptomyces coelicolor, with farnesyl diphosphate (2, FPP) in the presence of Mg2+ gave a mixture of (4S,7R)-germacra-1(10)E,5E-diene-11-ol (3) (74%), (-)-(7S)-germacrene D (4) (10%), geosmin (1) (13%), and a hydrocarbon, tentatively assigned the structure of octalin 5 (3%). Individual incubations of recombinant germacradienol synthase with [1,1-2H2]FPP (2a), (1R)-[1-2H]-FPP (2b), and (1S)-[1-2H]-FPP (2c), as well as with FPP (2) in D2O, and GC-MS analysis of the resulting deuterated products supported a mechanism of geosmin formation involving proton-initiated cyclization and retro-Prins fragmentation of the initially formed germacradienol to give intermediate 5, followed by protonation of 5, 1,2-hydride shift, and capture of water.

  5. Solubility of triuranyl diphosphate tetrahydrate (TDT) and Na autunite at 23 and 50 degrees C

    Energy Technology Data Exchange (ETDEWEB)

    Armstrong, Christopher R.; Felmy, Andrew R.; Clark, Sue B.

    2010-11-01

    In this report we present experimental solubility data for well-characterized triuranyl diphosphate tetrahydrate (TDT: (UO2)(3)(PO4)(2)center dot 4H(2)O) and Na autunite (Na[UO2PO4]center dot xH(2)O) at 23 and 50 degrees C in NaClO4-HClO4 solutions at pC(H+) = 2. Duplicate samples of TDT in 0.1, 0.5, 1.0, 2.0 and 5.0 in solutions were equilibrated at 23 and 50 degrees C. TDT solid was synthesized and characterized with ICP-OES, ATR-IR and powder XRD before and after solubility experiments. The pH of the suspensions were monitored throughout the experiments. Equilibrium was achieved from undersaturation with respect to TDT and oversaturation for Na autunite. Steady-state conditions were achieved in all cases within 82 d. TDT was unstable at ionic strengths above 0.1 m, where its complete conversion to Na autunite was observed. The ion-interaction model was used to interpret the experimental solubility data. The solubility product, log K-sp, for TDT was determined to be -49.7 and -51.3 at 23 and 50 degrees C respectively. log K for Na autunite was determined to be -24.4 (23 degrees C) and -24.1 +/- 0.2 (50 degrees C).

  6. Solubility of triuranyl diphosphate tetrahydrate (TDT) and Na autunite at 23 and 50 C

    Energy Technology Data Exchange (ETDEWEB)

    Armstrong, C.R.; Clark, S.B. [Washington State Univ., Pullman, WA (United States); Felmy, A.R. [Pacific Northwest National Lab., Richland, WA (United States)

    2010-07-01

    In this report we present experimental solubility data for well-characterized triuranyl diphosphate tetrahydrate (TDT: (UO{sub 2}){sub 3}(PO{sub 4}){sub 2} . 4H{sub 2}O) and Na autunite (Na[UO{sub 2}PO{sub 4}] . xH{sub 2}O) at 23 and 50 C in NaClO{sub 4}-HClO{sub 4} solutions at pC{sub H1} = 2. Duplicate samples of TDT in 0.1, 0.5, 1.0, 2.0 and 5.0 m solutions were equilibrated at 23 and 50 C. TDT solid was synthesized and characterized with ICP-OES, ATR-IR and powder XRD before and after solubility experiments. The pH of the suspensions were monitored throughout the experiments. Equilibrium was achieved from undersaturation with respect to TDT and oversaturation for Na autunite. Steady-state conditions were achieved in all cases within 82 d. TDT was unstable at ionic strengths above 0.1 m, where its complete conversion to Na autunite was observed. The ion-interaction model was used to interpret the experimental solubility data. The solubility product, log K{sub sp}, for TDT was determined to be -49.7 and -51.3 at 23 and 50 C respectively. log K{sub sp} for Na autunite was determined to be -24.4 (23 C) and -24.1 {+-} 0.2 (50 C). (orig.)

  7. The uridine diphosphate glucuronosyltransferases: quantitative structure-activity relationships for hydroxyl polychlorinated biphenyl substrates

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Degao [Dalian University of Technology, Department of Environmental Science and Technology, Dalian (China)

    2005-10-01

    Quantitative structure-activity relationships (QSARs), which relate the glucuronidation of hydroxyl polychlorinated biphenyls (OH-PCBs) - catalyzed by the uridine diphosphate glucuronosyltransferases (UGTs) - to their physicochemical properties and molecular structural parameters, can be used to predict the rate constants and interpret the mechanism of glucuronidation. In this study, QSARs have been developed that use 23 semi-empirical calculated quantum chemical descriptors to predict the logarithms of the constants 1/K{sub m} and V{sub max}, related to enzyme kinetics. A partial least squares regression method was used to select the optimal set of descriptors to minimize the multicollinearity between the descriptors, as well as to maximize the cross-validated coefficient (Q{sup 2} {sub cum}) values. The key descriptors affecting log(1/K{sub m}) were E{sub lumo}- E{sub homo} (the energy gap between the lowest unoccupied molecular orbital and the highest occupied molecular orbital) and q{sub C}{sup -} (the largest negative net atomic charge on a carbon atom), while the key descriptors affecting log V{sub max} were the polarizability {alpha}, the Connolly solvent-excluded volume (CSEV), and logP (the logarithm of the partition coefficient for octanol/water). From the results obtained it can be concluded that hydrophobic and electronic aspects of OH-PCBs are important in the glucuronidation of OH-PCBs. (orig.)

  8. Metabolism of farnesyl diphosphate in tobacco BY-2 cells treated with squalestatin.

    Science.gov (United States)

    Hartmann, M A; Wentzinger, L; Hemmerlin, A; Bach, T J

    2000-12-01

    Plant isoprenoids represent a large group of compounds with a wide range of physiological functions. In the cytosol, isoprenoids are synthesized via the classical acetate/mevalonate pathway. In this pathway, farnesyl diphosphate (FPP) occupies a central position, from which isoprene units are dispatched to the different classes of isoprenoids, with sterols as the major end products. The present work deals with effects of squalestatin (SQ) on the metabolism of FPP in proliferating and synchronized cultured tobacco cv. Bright Yellow-2 cells. SQ is a potent inhibitor of squalene synthase (SQS), the first committed enzyme in the sterol pathway. At nanomolar concentrations, SQ severely impaired cell growth and sterol biosynthesis, as attested by the rapid decrease in SQS activity. At the same time, it triggered a several-fold increase in both the enzymic activity and mRNA levels of 3-hydroxy-3-methylglutaryl CoA reductase. When SQ was added to cells synchronized by aphidicolin treatment, it was found to block the cell cycle at the end of G(1) phase, but no cell death was induced. Tobacco cells were also fed exogenous tritiated trans-trans farnesol, the allylic alcohol derived from FPP, in the presence and absence of SQ. Evidence is presented that this compound was incorporated into sterols and ubiquinone Q(10). In the presence of SQ, the sterol pathway was inhibited, but no increase in the radioactivity of ubiquinone was observed, suggesting that this metabolic channel was already saturated under normal conditions.

  9. In vitro synthesis and characterization of guanosine 3',5'-bis(diphosphate).

    Science.gov (United States)

    Hardiman, Timo; Windeisen, Volker; Ewald, Jennifer C; Zibek, Susanne; Schlack, Petra; Rebell, Jochen; Reuss, Matthias; Siemann-Herzberg, Martin

    2008-12-15

    The intracellular alarmone guanosine 3',5'-bis(diphosphate) (ppGpp) has been thoroughly investigated over the past 40 years and has become one of the best-known effectors in bacterial physiology. ppGpp is also of great importance for biotechnological applications. Systems biology research, involving experimental and mathematical approaches, has contributed a great deal to uncovering the alarmone's complex regulatory effects. HPLC analysis and UV detection are used to quantify intracellular ppGpp. The samples analyzed also contain other phosphorylated guanine nucleotides and, therefore, are spiked with a standard ppGpp solution. A rapidly growing number of laboratories are turning to synthesizing the nucleotide in vitro involving time-consuming protocols and yielding only low amounts of ppGpp. The current article provides a protocol for the preparation of large quantities of a ribosome extract that contains high ppGpp synthesis activity. The demonstrated upscaling from shaking flask to bioreactor cultivation involves the continuous and refrigerated harvest of the biomass. (13)C NMR analysis enabled the structural characterization of the synthesis product and was complemented by mass spectrometry and methods that are commonly used to identify ppGpp.

  10. 烟酰胺磷酸核糖转移酶在大鼠涎腺的表达及定位%Expression and localization of nicotinamide phosphoribosyl transferase in salivary glands of rat

    Institute of Scientific and Technical Information of China (English)

    赵西博; 张歆; 李秀秀; 高媛; 向彬; 张福胤

    2011-01-01

    [Objective] To investigate the expression and distribution of nicotinamide phosphoribosyl transferase (Nampt)in rat salivary glands. [ Methods ] The expressions of Nampt mRNA and protein were investigated by reverse transcription polymerase chain reaction (RT- PCR) and Western Blot in the parotid,submandibular and major sublingual glands of rat.Immunohistochemical assay was applied to detect the distribution of Nampt in these glands too. [ Results] The results of RT- PCR showed that the optical density of the expression of Nampt mRNA was 82.04% ,50.89% and 30.53% respectively in the parotid, submandibular and major sublingual glands of rat ( P < 0.05, compared with actin). The results of Western blot showed that the optical density of the expression of Nampt protein was 77.34% ,47.61% and 31.78% ,respectively in these glands (P < 0.05, compared with actin). Nampt protein was widely distributed in them, mainly localized in the cytoplasm and nucleus of both serous acinar cells and ductal cells. [ Conclusions] Nampt mRNA and protein are expressed in the parotid,submandibular and major sublingual glands of rat,and may be involved in underlying mechanisms of physiology and patho- physiology in the salivary glands.%[目的]研究烟酰胺磷酸核糖转移酶(nicotinamide phosphoribosyl transferase,Nampt)在大鼠涎腺的表达和分布.[方法]应用半定量逆转录聚合酶链法(RT-PCR)和免疫印迹法检测大鼠正常腮腺、颌下腺和大舌下腺组织Nampt mRNA及蛋白质的表达.采用免疫组织化学法检测大鼠腮腺、颌下腺和大舌下腺组织Nampt的分布.[结果]大鼠腮腺、颌下腺和大舌下腺组织均有Nampt的mRNA及蛋白质表达,RT-PCR结果显示其表达相对光密度值分别为:腮腺82.04%,颌下腺50.89%,大舌下腺30.53%(与aetin比较,P<0.05);Western Blot结果显示其表达相对光密度值分别为:腮腺77.34%,颌下腺47.61%,大舌下腺31.78%(与actin比较,P<0.05).Nampt均主要分布于

  11. Morin, a dietary bioflavonol suppresses monosodium urate crystal-induced inflammation in an animal model of acute gouty arthritis with reference to NLRP3 inflammasome, hypo-xanthine phospho-ribosyl transferase, and inflammatory mediators.

    Science.gov (United States)

    Dhanasekar, Chitra; Rasool, Mahaboobkhan

    2016-09-05

    The anti-inflammatory effect of morin, a dietary bioflavanol was explored on monosodium urate (MSU) crystal-induced inflammation in rats, an experimental model for acute gouty arthritis. Morin treatment (30mg/kg b.wt) significantly attenuated the ankle swelling and the levels of lipid peroxidation, nitric oxide, serum pro-inflammatory cytokines (tumor necrosis factor (TNF) -α, interleukin (IL)-1β, and IL-6), monocyte chemoattractant protein (MCP)-1, vascular endothelial growth factor (VEGF), prostaglandin E2 (PGE2), and articular elastase along with an increased anti-oxidant status (catalase (CAT) and superoxide dismutase (SOD)) in the joint homogenate of MSU crystal-induced rats. Histological assessment revealed that morin limited the diffusion of joint space, synovial hyperplasia, and inflammatory cell infiltrations. The mRNA expression of NLRP3 (nucleotide oligomerization domain (NOD)-like receptor family, pyrin domain containing 3) inflammasome, caspase-1, pro-inflammatory cytokines, MCP-1, inflammatory enzymes (inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2)), and nuclear factor-kappa B (NF-κB) p65 was found downregulated and HPRT (hypo-xanthine phospho-ribosyl transferase) mRNA expression was upregulated in morin treated MSU crystal-induced rats. In addition, morin treatment reduced the protein expression of NF-κB p65, p-NF-κB p65, iNOS, COX-2, and TNF-α. The results clearly demonstrated that morin exert a potent anti-inflammatory effect on MSU crystal-induced inflammation in rats.

  12. Mycobacterium tuberculosis nucleoside diphosphate kinase inactivates small GTPases leading to evasion of innate immunity.

    Directory of Open Access Journals (Sweden)

    Jim Sun

    Full Text Available Defining the mechanisms of Mycobacterium tuberculosis (Mtb persistence in the host macrophage and identifying mycobacterial factors responsible for it are keys to better understand tuberculosis pathogenesis. The emerging picture from ongoing studies of macrophage deactivation by Mtb suggests that ingested bacilli secrete various virulence determinants that alter phagosome biogenesis, leading to arrest of Mtb vacuole interaction with late endosomes and lysosomes. While most studies focused on Mtb interference with various regulators of the endosomal compartment, little attention was paid to mechanisms by which Mtb neutralizes early macrophage responses such as the NADPH oxidase (NOX2 dependent oxidative burst. Here we applied an antisense strategy to knock down Mtb nucleoside diphosphate kinase (Ndk and obtained a stable mutant (Mtb Ndk-AS that displayed attenuated intracellular survival along with reduced persistence in the lungs of infected mice. At the molecular level, pull-down experiments showed that Ndk binds to and inactivates the small GTPase Rac1 in the macrophage. This resulted in the exclusion of the Rac1 binding partner p67(phox from phagosomes containing Mtb or Ndk-coated latex beads. Exclusion of p67(phox was associated with a defect of both NOX2 assembly and production of reactive oxygen species (ROS in response to wild type Mtb. In contrast, Mtb Ndk-AS, which lost the capacity to disrupt Rac1-p67(phox interaction, induced a strong ROS production. Given the established link between NOX2 activation and apoptosis, the proportion of Annexin V positive cells and levels of intracellular active caspase 3 were significantly higher in cells infected with Mtb Ndk-AS compared to wild type Mtb. Thus, knock down of Ndk converted Mtb into a pro-apoptotic mutant strain that has a phenotype of increased susceptibility to intracellular killing and reduced virulence in vivo. Taken together, our in vitro and in vivo data revealed that Ndk

  13. Thiamin diphosphate-dependent enzymes: from enzymology to metabolic regulation, drug design and disease models.

    Science.gov (United States)

    Bunik, Victoria I; Tylicki, Adam; Lukashev, Nikolay V

    2013-12-01

    Bringing a knowledge of enzymology into research in vivo and in situ is of great importance in understanding systems biology and metabolic regulation. The central metabolic significance of thiamin (vitamin B1 ) and its diphosphorylated derivative (thiamin diphosphate; ThDP), and the fundamental differences in the ThDP-dependent enzymes of metabolic networks in mammals versus plants, fungi and bacteria, or in health versus disease, suggest that these enzymes are promising targets for biotechnological and medical applications. Here, the in vivo action of known regulators of ThDP-dependent enzymes, such as synthetic structural analogs of the enzyme substrates and thiamin, is analyzed in light of the enzymological data accumulated during half a century of research. Mimicking the enzyme-specific catalytic intermediates, the phosphonate analogs of 2-oxo acids selectively inhibit particular ThDP-dependent enzymes. Because of their selectivity, use of these compounds in cellular and animal models of ThDP-dependent enzyme malfunctions improves the validity of the model and its predictive power when compared with the nonselective and enzymatically less characterized oxythiamin and pyrithiamin. In vitro studies of the interaction of thiamin analogs and their biological derivatives with potential in vivo targets are necessary to identify and attenuate the analog selectivity. For both the substrate and thiamin synthetic analogs, in vitro reactivities with potential targets are highly relevant in vivo. However, effective concentrations in vivo are often higher than in vitro studies would suggest. The significance of specific inihibition of the ThDP-dependent enzymes for the development of herbicides, antibiotics, anticancer and neuroprotective strategies is discussed.

  14. Flagellar Radial Spokes Contain a Ca2+-stimulated Nucleoside Diphosphate Kinase

    Science.gov (United States)

    Patel-King, Ramila S.; Gorbatyuk, Oksana; Takebe, Sachiko; King, Stephen M.

    2004-01-01

    The radial spokes are required for Ca2+-initiated intraflagellar signaling, resulting in modulation of inner and outer arm dynein activity. However, the mechanochemical properties of this signaling pathway remain unknown. Here, we describe a novel nucleoside diphosphate kinase (NDK) from the Chlamydomonas flagellum. This protein (termed p61 or RSP23) consists of an N-terminal catalytic NDK domain followed by a repetitive region that includes three IQ motifs and a highly acidic C-terminal segment. We find that p61 is missing in axonemes derived from the mutants pf14 (lacks radial spokes) and pf24 (lacks the spoke head and several stalk components) but not in those from pf17 (lacking only the spoke head). The p61 protein can be extracted from oda1 (lacks outer dynein arms) and pf17 axonemes with 0.5 M KI, and copurifies with radial spokes in sucrose density gradients. Furthermore, p61 contains two classes of calmodulin binding site: IQ1 interacts with calmodulin-Sepharose beads in a Ca2+-independent manner, whereas IQ2 and IQ3 show Ca2+-sensitive associations. Wild-type axonemes exhibit two distinct NDKase activities, at least one of which is stimulated by Ca2+. This Ca2+-responsive enzyme, which accounts for ∼45% of total axonemal NDKase, is missing from pf14 axonemes. We found that purified radial spokes also exhibit NDKase activity. Thus, we conclude that p61 is an integral component of the radial spoke stalk that binds calmodulin and exhibits Ca2+-controlled NDKase activity. These observations suggest that nucleotides other than ATP may play an important role in the signal transduction pathway that underlies the regulatory mechanism defined by the radial spokes. PMID:15194815

  15. Mechanism of reconstitution of brewers' yeast pyruvate decarboxylase with thiamin diphosphate and magnesium.

    Science.gov (United States)

    Vaccaro, J A; Crane, E J; Harris, T K; Washabaugh, M W

    1995-10-01

    Reconstitution of apo-pyruvate decarboxylase isozymes (PDC, EC 4.1.1.1) from Saccharomyces carlsbergensis was investigated by determination of the steady-state kinetics of the reaction with thiamin diphosphate (TDP) and Mg2+ in the presence and absence of substrate (pyruvate) or allosteric effector (pyruvamide). Reconstitution of the PDC isozyme mixture and alpha 4 isozyme (alpha 4-PDC) exhibits biphasic kinetics with 52 +/- 11% of the PDC reacting with k1 = (1.0 +/- 0.3) x 10(-2) s-1 and 48 +/- 12% of the PDC reacting with k2 = (1.1 +/- 0.6) x 10(-1) s-1 when TDP (KTDP = 0.5 +/- 0.2 mM) is added to apo-PDC equilibrated with saturating Mg2+. PDC reconstitution exhibits first-order kinetics with k1 = (1.6 +/- 0.5) x 10(-2) s-1 upon addition of Mg2+ (KMg2+ = 0.2 +/- 0.1 mM) to apo-PDC equilibrated with saturating TDP. Biphasic kinetics for the PDC isozymes provides evidence that apo-PDC reconstitution with TDP and Mg2+ involves two pathways, TDP binding followed by Mg2+ (k1) or Mg2+ binding followed by TDP (k2). This is supported by a change in reconstitution pathway with the order of cofactor addition and is inconsistent with a single pathway involving ordered binding of the metal ion followed by TDP. The presence of pyruvamide has no significant effect on the rate constants for apo-PDC reconstitution and favors the k2 pathway; pyruvate decreases the value of k2 < or = 3-fold and has no effect on the value of k1.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Heterologous expression of active human uridine diphosphate glucuronosyltransferase 1A3 in Chinese hamster lung cells

    Institute of Scientific and Technical Information of China (English)

    Ya-Kun Chen; Xin Li; Shu-Qing Chen; Su Zeng

    2005-01-01

    AIM: To obtain the active human recombinant uridine diphosphate glucuronosyltransferase 1A3 (UGT1A3) enzyme from Chinese hamster lung (CHL) cells.METHODS: The full-length UGT1A3 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR)using total RNA from human liver as template. The correct fragment confirmed by sequencing was subcloned into the mammalian expression vector pcDNA3.1 (+), and the recombinant vector was transfected into CHL cells using a calcium phosphate method. Expressed UGT1A3 protein was prepared from CHL cells resistant to neomycin (G418). Then the protein was added into a reaction mixture for glucuronidation of quercetin. The glucuronidation activity of UGT1A3 was determined by reverse phase-high performance liquid chromatography (RP-HPLC) coupled with a diode array detector (DAD). The quercetin glucuronide was confirmed by hydrolysis with β-glucuronidase. Control experiments were performed in parallel. The transcriptions of recombinants were also determined by RT-PCR.RESULTS: The gene was confirmed to be an allele (UGT1A3-3) of UGT1A3 by DNA sequencing. The fragment was introduced into pcDNA3.1 (+) successfully. Several colonies were obtained under the selection pressure of G418.The result of RT-PCR showed transcription of recombinants in mRNA level. Glucuronidation assay and HPLC analysis indicated UGT1A3 expressed heterologously in CHL cells was in an active form, and one of the gulcuronides corresponding to quercetin was also detected.CONCLUSION: Correct sequence of UGT1A3 gene can be obtained, and active UGT1A3 enzyme is expressed heterologously in CHL cells.

  17. Mycobacterium tuberculosis nucleoside diphosphate kinase inactivates small GTPases leading to evasion of innate immunity.

    Directory of Open Access Journals (Sweden)

    Jim Sun

    Full Text Available Defining the mechanisms of Mycobacterium tuberculosis (Mtb persistence in the host macrophage and identifying mycobacterial factors responsible for it are keys to better understand tuberculosis pathogenesis. The emerging picture from ongoing studies of macrophage deactivation by Mtb suggests that ingested bacilli secrete various virulence determinants that alter phagosome biogenesis, leading to arrest of Mtb vacuole interaction with late endosomes and lysosomes. While most studies focused on Mtb interference with various regulators of the endosomal compartment, little attention was paid to mechanisms by which Mtb neutralizes early macrophage responses such as the NADPH oxidase (NOX2 dependent oxidative burst. Here we applied an antisense strategy to knock down Mtb nucleoside diphosphate kinase (Ndk and obtained a stable mutant (Mtb Ndk-AS that displayed attenuated intracellular survival along with reduced persistence in the lungs of infected mice. At the molecular level, pull-down experiments showed that Ndk binds to and inactivates the small GTPase Rac1 in the macrophage. This resulted in the exclusion of the Rac1 binding partner p67(phox from phagosomes containing Mtb or Ndk-coated latex beads. Exclusion of p67(phox was associated with a defect of both NOX2 assembly and production of reactive oxygen species (ROS in response to wild type Mtb. In contrast, Mtb Ndk-AS, which lost the capacity to disrupt Rac1-p67(phox interaction, induced a strong ROS production. Given the established link between NOX2 activation and apoptosis, the proportion of Annexin V positive cells and levels of intracellular active caspase 3 were significantly higher in cells infected with Mtb Ndk-AS compared to wild type Mtb. Thus, knock down of Ndk converted Mtb into a pro-apoptotic mutant strain that has a phenotype of increased susceptibility to intracellular killing and reduced virulence in vivo. Taken together, our in vitro and in vivo data revealed that Ndk

  18. Observation of an Acryloyl–Thiamin Diphosphate Adduct in the First Step of Clavulanic Acid Biosynthesis

    Science.gov (United States)

    Merski, Matthew

    2011-01-01

    The first committed biosynthetic step toward clavulanic acid, the clinically-important β-lactamase inhibitor, is catalyzed by the thiamin diphosphate (ThDP)-dependent enzyme N2-(2-carboxyethyl)arginine synthase (CEAS). This protein carries out a unique reaction among ThDP-dependent processes in which a C–N bond is formed, and an electrophilic acryloyl–thiazolium intermediate of ThDP is proposed to be involved, unlike the nucleophilic enamine species typically generated by this class of enzymes. Here we present evidence for the existence of the putative acryloyl adduct, and report the unexpected observation of a long-wavelength chromophore (λ = 433 nm), which we attribute to this enzyme bound species. Chemical models were synthesized that both confirm its expected absorption (λ = 310–320 nm), and exclude self-condensation and intramolecular imine formation with the cofactor as its cause. Circular dichroism experiments and others discount charge transfer as a likely explanation for the ~120 nm red shift of the chromophore (~25 kcal). Examples are well-known of charged molecules that exhibit significantly red-shifted UV-visible spectra compared to their neutral forms as, for example, polyene cations and dyes such as indigo and the cyanines. Rhodopsin is the classic biochemical example where the protein (opsin)-bound protonated Schiff base of retinal displays a remarkable range of red-shifted absorptions modulated by the protein environment. Similar tuning of the chromophoric behavior of the enzyme-bound CEAS acryloyl•ThDP species may be occurring. PMID:18052280

  19. P2X receptors regulate adenosine diphosphate release from hepatic cells.

    Science.gov (United States)

    Chatterjee, Cynthia; Sparks, Daniel L

    2014-12-01

    Extracellular nucleotides act as paracrine regulators of cellular signaling and metabolic pathways. Adenosine polyphosphate (adenosine triphosphate (ATP) and adenosine diphosphate (ADP)) release and metabolism by human hepatic carcinoma cells was therefore evaluated. Hepatic cells maintain static nanomolar concentrations of extracellular ATP and ADP levels until stress or nutrient deprivation stimulates a rapid burst of nucleotide release. Reducing the levels of media serum or glucose has no effect on ATP levels, but stimulates ADP release by up to 10-fold. Extracellular ADP is then metabolized or degraded and media ADP levels fall to basal levels within 2-4 h. Nucleotide release from hepatic cells is stimulated by the Ca(2+) ionophore, ionomycin, and by the P2 receptor agonist, 2'3'-O-(4-benzoyl-benzoyl)-adenosine 5'-triphosphate (BzATP). Ionomycin (10 μM) has a minimal effect on ATP release, but doubles media ADP levels at 5 min. In contrast, BzATP (10-100 μM) increases both ATP and ADP levels by over 100-fold at 5 min. Ion channel purinergic receptor P2X7 and P2X4 gene silencing with small interference RNA (siRNA) and treatment with the P2X inhibitor, A438079 (100 μM), decrease ADP release from hepatic cells, but have no effect on ATP. P2X inhibitors and siRNA have no effect on BzATP-stimulated nucleotide release. ADP release from human hepatic carcinoma cells is therefore regulated by P2X receptors and intracellular Ca(2+) levels. Extracellular ADP levels increase as a consequence of a cellular stress response resulting from serum or glucose deprivation.

  20. The antihyperalgesic effect of cytidine-5'-diphosphate-choline in neuropathic and inflammatory pain models.

    Science.gov (United States)

    Bagdas, Deniz; Sonat, Fusun Ak; Hamurtekin, Emre; Sonal, Songul; Gurun, Mine Sibel

    2011-09-01

    This study was designed to test the effects of intracerebroventricularly (i.c.v.) administered CDP-choline (cytidine-5'-diphosphate-choline; citicoline) and its metabolites in rat models of inflammatory and neuropathic pain. The i.c.v. administration of CDP-choline (0.5, 1.0 and 2.0 µmol) produced a dose and time-dependent reversal of mechanical hyperalgesia in both carrageenan-induced inflammatory and chronic constriction injury-induced neuropathic pain models in rats. The antihyperalgesic effect of CDP-choline was similar to that observed with an equimolar dose of choline (1 µmol). The CDP-choline-induced antihyperalgesic effect was prevented by central administration of the neuronal high-affinity choline uptake inhibitor hemicholinium-3 (1 µg), the nonselective nicotinic receptor antagonist mecamylamine (50 µg), the α7-selective nicotinic ACh receptor antagonist, α-bungarotoxin (2 µg) and the γ-aminobutyric acid B receptor antagonist CGP-35348 (20 µg). In contrast, i.c.v. pretreatment with the nonselective opioid receptor antagonist naloxone (10 µg) only prevented the CDP-choline-induced antihyperalgesic effect in the neuropathic pain model while the nonselective muscarinic receptor antagonist atropine (10 µg) did not alter the antihyperalgesic effect in the two models. These results indicate that CDP-choline-elicited antihyperalgesic effect in different models of pain occurs through mechanisms that seem to involve an interaction with supraspinal α7-selective nicotinic ACh receptors, and γ-aminobutyric acid B receptors, whereas central opioid receptors have a role only in the neuropathic pain model.

  1. Elementary steps in the reaction of the pyruvate dehydrogenase complex from pig heart. Kinetics of thiamine diphosphate binding to the complex.

    Science.gov (United States)

    Sümegi, B; Alkonyi, I

    1983-11-02

    In the progress curve of the reaction of the pyruvate dehydrogenase complex, a lag phase was observed when the concentration of thiamin diphosphate was lower than usual (about 0.2-1 mM) in the enzyme assay. The length of the lag phase was dependent on thiamin diphosphate concentration, ranging from 0.2 min to 2 min as the thiamin diphosphate concentration varied from 800 nM to 22 nM. The lag phase was also observed in the elementary steps catalyzed by the pyruvate dehydrogenase component. A Km value of 107 nM was found for thiamin diphosphate with respect to the steady-state reaction rate following the lag phase. The pre-steady-state kinetic data indicate that the resulting lag phase was the consequence of a slow holoenzyme formation from apoenzyme and thiamin diphosphate. The thiamin diphosphate can bind to the pyruvate dehydrogenase complex in the absence of pyruvate, but the presence of 2 mM pyruvate increases the rate constant of binding from 1.4 X 10(4) M-1 S-1 to 1.3 X 10(5) M-1 S-1 and decreases the rate constant of dissociation from 2.3 X 10(-2) S-1 to 4.1 X 10(-3) S-1. On the other hand, the effect of pyruvate on the thiamin diphosphate binding revealed the existence of a thiamin-diphosphate-independent pyruvate-binding site in the pyruvate dehydrogenase complex. Direct evidence was also obtained with fluorescence techniques for the existence of this binding site and the dissociation constant of pyruvate was found to be 0.38 mM. On the basis of these data we have proposed a random mechanism for the binding of pyruvate and thiamin diphosphate to the complex. Binding of substrates to the enzyme complex caused an increase in the fluorescence of the dansylaziridine-labelled pyruvate dehydrogenase complex, showing that binding of substrates to the complex is accompanied by structural changes.

  2. ANALISIS CEMARAN BAKTERI Escherichia coli ANALISIS CEMARAN BAKTERI Escherichia coli ANALISIS CEMARAN BAKTERI Escherichia coli

    OpenAIRE

    ANGGREINI, RAHAYU

    2015-01-01

    2015 RAHAYU ANGGREINI coli Penelitian ini bertujuan untuk melakukan identifikasi cemaran bakteri E. coli O157:H7 pada daging sapi di kota Makassar. Sampel pada penelitian ini sebanyak 72 sampel Kata Kunci : Daging sapi, pasar tradisional, E. coli, E. coli O157:H7, kontaminasi bakteri, identifikasi E. coli O157:H7.

  3. ANALISIS CEMARAN BAKTERI Escherichia coli ANALISIS CEMARAN BAKTERI Escherichia coli ANALISIS CEMARAN BAKTERI Escherichia coli

    OpenAIRE

    ANGGREINI, RAHAYU

    2015-01-01

    2015 RAHAYU ANGGREINI coli Penelitian ini bertujuan untuk melakukan identifikasi cemaran bakteri E. coli O157:H7 pada daging sapi di kota Makassar. Sampel pada penelitian ini sebanyak 72 sampel Kata Kunci : Daging sapi, pasar tradisional, E. coli, E. coli O157:H7, kontaminasi bakteri, identifikasi E. coli O157:H7.

  4. Structural and thermodynamic basis of the inhibition of Leishmania major farnesyl diphosphate synthase by nitrogen-containing bisphosphonates

    Energy Technology Data Exchange (ETDEWEB)

    Aripirala, Srinivas [Johns Hopkins University, 725 North Wolfe Street WBSB 605, Baltimore, MD 21210 (United States); Gonzalez-Pacanowska, Dolores [López-Neyra Institute of Parasitology and Biomedicine, 18001 Granada (Spain); Oldfield, Eric [University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States); Kaiser, Marcel [University of Basel, Petersplatz 1, CH-4003 Basel (Switzerland); Amzel, L. Mario, E-mail: mamzel@jhmi.edu [Johns Hopkins University School of Medicine, 725 N. Wolfe Street WBSB 604, Baltimore, MD 21205 (United States); Gabelli, Sandra B., E-mail: mamzel@jhmi.edu [Johns Hopkins University School of Medicine, 725 N. Wolfe Street WBSB 604, Baltimore, MD 21205 (United States); Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Johns Hopkins University, 725 North Wolfe Street WBSB 605, Baltimore, MD 21210 (United States)

    2014-03-01

    Structural insights into L. major farnesyl diphosphate synthase, a key enzyme in the mevalonate pathway, are described. Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray crystallographic structures of complexes of FPPS from Leishmania major (the causative agent of cutaneous leishmaniasis) with three bisphosphonates determined at resolutions of 1.8, 1.9 and 2.3 Å are reported. Two of the inhibitors, 1-(2-hydroxy-2,2-diphosphonoethyl)-3-phenylpyridinium (300B) and 3-butyl-1-(2,2-diphosphonoethyl)pyridinium (476A), co-crystallize with the homoallylic substrate isopentenyl diphosphate (IPP) and three Ca{sup 2+} ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg{sup 2+} ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of L. major FPPS (LmFPPS) and human FPPS provides new information for the design of bisphosphonates that will be more specific for inhibition of LmFPPS. The asymmetric structure of the LmFPPS–46I homodimer indicates that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. It is proposed that IPP first binds to the open site, which then closes, opening the site on the other monomer, which closes after binding the second IPP, leading to the symmetric fully occupied FPPS dimer observed in other structures.

  5. Involvement of an ent-copalyl diphosphate synthase in tissue-specific accumulation of specialized diterpenes in Andrographis paniculata.

    Science.gov (United States)

    Misra, Rajesh Chandra; Garg, Anchal; Roy, Sudeep; Chanotiya, Chandan Singh; Vasudev, Prema G; Ghosh, Sumit

    2015-11-01

    Ent-labdane-related diterpene (ent-LRD) specialized (i.e. secondary) metabolites of the medicinal plant kalmegh (Andrographis paniculata) have long been known for several pharmacological activities. However, our understanding of the ent-LRD biosynthetic pathway has remained largely incomplete. Since ent-LRDs accumulate in leaves, we carried out a comparative transcriptional analysis using leaf and root tissues, and identified 389 differentially expressed transcripts, including 223 transcripts that were preferentially expressed in leaf tissue. Analysis of the transcripts revealed various specialized metabolic pathways, including transcripts of the ent-LRD biosynthetic pathway. Two class II diterpene synthases (ApCPS1 and ApCPS2) along with one (ApCPS1') and two (ApCPS2' and ApCPS2″) transcriptional variants that were the outcomes of alternative splicing of the precursor mRNA and alternative transcriptional termination, respectively, were identified. ApCPS1 and ApCPS2 encode for 832- and 817-amino acids proteins, respectively, and are phylogenetically related to the dicotyledons ent-copalyl diphosphate synthases (ent-CPSs). The spatio-temporal patterns of ent-LRD metabolites accumulation and gene expression suggested a likely role for ApCPS1 in general (i.e. primary) metabolism, perhaps by providing precursor for the biosynthesis of phytohormone gibberellin (GA). However, ApCPS2 is potentially involved in tissue-specific accumulation of ent-LRD specialized metabolites. Bacterially expressed recombinant ApCPS2 catalyzed the conversion of (E,E,E)-geranylgeranyl diphosphate (GGPP), the general precursor of diterpenes to ent-copalyl diphosphate (ent-CPP), the precursor of ent-LRDs. Taken together, these results advance our understanding of the tissue-specific accumulation of specialized ent-LRDs of medicinal importance.

  6. Regeneration of hemopoietic and lymphoid tissues following total-body irradiation and therapeutic administration of thiamin diphosphate. [Mice, gamma radiation

    Energy Technology Data Exchange (ETDEWEB)

    Vavrova, J.; Nouza, K.; Petjrek, P.

    1977-01-01

    Analysis was made of the mechanism of therapeutic application of thiamine diphosphate (TDP) in radiation sickness in mice. This agent increased the number of endogenous colonies in the spleen and incorporation of /sup 59/Fe in spleen and bone marrow with sublethal doses of radiation (500 and 600 R) and has no effect with a lethal dose (750 R). After administration of TDP to mice exposed to 500 R radiation, there is faster DNA synthesis in the spleen, thymus and bone marrow, as well as reliable increase in number of nuclear cells in femoral marrow.

  7. Zoonotic Escherichia coli

    Directory of Open Access Journals (Sweden)

    Wasteson Yngvild

    2002-03-01

    Full Text Available Escherichia coli is a normal inhabitant of the gastrointestinal tract of all warm-blooded animals, but variants of this species is also among the important etiological agents of enteritis and several extraintestinal diseases. The E. coli strains that cause diarrhoeal illness are categorised into pathogenicity groups based on virulence properties, mechanisms of pathogenicity, clinical symptoms and serology. The five main categories include enterotoxinogenic E. coli (ETEC, enteropathogenic E. coli (EPEC, enteroaggregative E. coli (EAggEC, enteroinvasive E. coli (EIEC and Shiga (Vero toxin-producing E. coli (STEC/VTEC. From a zoonotic point of view, STEC is the only E. coli pathogenicity group of major interest, as the shiga toxin-producing strains are able to cause severe disease in humans when being transmitted through the food chain from their animal reservoirs. The focus of this manuscript is therefore on STEC; pathogenicity factors, disease, the reservoirs and on-farm ecology, transmission into the food chain, growth and survival in food and in the environment, and the shiga toxin-encoding bacteriophages.

  8. PATHOGENIC ESCHERICHIA COLI

    Science.gov (United States)

    Escherichia coli is a bacterial species which inhabits the gastrointestinal tract of man and warm-blooded animals. Because of the ubiquity of this bacterium in the intestinal flora, it serves as an important indicator organism of fecal contamination. E. coli, aside from serving a...

  9. Diarrheagenic Escherichia coli.

    Science.gov (United States)

    Gomes, Tânia A T; Elias, Waldir P; Scaletsky, Isabel C A; Guth, Beatriz E C; Rodrigues, Juliana F; Piazza, Roxane M F; Ferreira, Luís C S; Martinez, Marina B

    2016-12-01

    Most Escherichia coli strains live harmlessly in the intestines and rarely cause disease in healthy individuals. Nonetheless, a number of pathogenic strains can cause diarrhea or extraintestinal diseases both in healthy and immunocompromised individuals. Diarrheal illnesses are a severe public health problem and a major cause of morbidity and mortality in infants and young children, especially in developing countries. E. coli strains that cause diarrhea have evolved by acquiring, through horizontal gene transfer, a particular set of characteristics that have successfully persisted in the host. According to the group of virulence determinants acquired, specific combinations were formed determining the currently known E. coli pathotypes, which are collectively known as diarrheagenic E. coli. In this review, we have gathered information on current definitions, serotypes, lineages, virulence mechanisms, epidemiology, and diagnosis of the major diarrheagenic E. coli pathotypes.

  10. Engineering E. coli-E. coli cocultures for production of muconic acid from glycerol

    National Research Council Canada - National Science Library

    Zhang, Haoran; Li, Zhengjun; Pereira, Brian; Stephanopoulos, Gregory

    2015-01-01

    .... In this study, we aim to explore the potential of the E. coli-E. coli coculture system to use a single renewable carbon source, glycerol, for the production of value-added product cis, cis-muconic acid...

  11. Farnesol production in Escherichia coli through the construction of a farnesol biosynthesis pathway - application of PgpB and YbjG phosphatases.

    Science.gov (United States)

    Wang, Chonglong; Park, Ju-Eon; Choi, Eui-Sung; Kim, Seon-Won

    2016-10-01

    Farnesol is a sesquiterpenoid alcohol that has important industrial and medical potential. It is usually synthesized from farnesyl diphosphate (FPP) by farnesol synthase in plants. FPP accumulation can cause up-regulation of phosphatases capable of FPP hydrolysis, resulting in farnesol production in Escherichia coli. We found that PgpB and YbjG, two integral membrane phosphatases, can hydrolyze FPP into farnesol. Overexpression of FPP synthase (IspA) and PgpB, along with a heterologous mevalonate pathway, enabled recombinant E. coli to produce 526.1 mg/L of farnesol. This result indicates that the phosphatases PgpB and YbjG can be used to construct a novel farnesol synthesis pathway for mass production in E. coli.

  12. Binding of nitrogen-containing bisphosphonates (N-BPs) to the Trypanosoma cruzi farnesyl diphosphate synthase homodimer

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Chuan-Hsiang; Gabelli, Sandra B.; Oldfield, Eric; Amzel, L. Mario (UIUC); (JHU-MED)

    2010-11-15

    Bisphosphonates (BPs) are a class of compounds that have been used extensively in the treatment of osteoporosis and malignancy-related hypercalcemia. Some of these compounds act through inhibition of farnesyl diphosphate synthase (FPPS), a key enzyme in the synthesis of isoprenoids. Recently, nitrogen-containing bisphosphonates (N-BPs) used in bone resorption therapy have been shown to be active against Trypanosoma cruzi, the parasite that causes American trypanosomiasis (Chagas disease), suggesting that they may be used as anti-trypanosomal agents. The crystal structures of TcFPPS in complex with substrate (isopentenyl diphosphate, IPP) and five N-BP inhibitors show that the C-1 hydroxyl and the nitrogen-containing groups of the inhibitors alter the binding of IPP and the conformation of two TcFPPS residues, Tyr94 and Gln167. Isothermal titration calorimetry experiments suggest that binding of the first N-BPs to the homodimeric TcFPPS changes the binding properties of the second site. This mechanism of binding of N-BPs to TcFPPS is different to that reported for the binding of the same compounds to human FPPS.

  13. Active site binding modes of inhibitors of Staphylococcus aureus mevalonate diphosphate decarboxylase from docking and molecular dynamics simulations.

    Science.gov (United States)

    Addo, James K; Skaff, D Andrew; Miziorko, Henry M

    2016-01-01

    Bacterial mevalonate diphosphate decarboxylase (MDD) is an attractive therapeutic target for antibacterial drug development. In this work, we discuss a combined docking and molecular dynamics strategy toward inhibitor binding to bacterial MDD. The docking parameters utilized in this study were first validated with observations for the inhibitors 6-fluoromevalonate diphosphate (FMVAPP) and diphosphoglycolylproline (DPGP) using existing structures for the Staphylococcus epidermidis enzyme. The validated docking protocol was then used to predict structures of the inhibitors bound to Staphylococcus aureus MDD using the unliganded crystal structure of Staphylococcus aureus MDD. We also investigated a possible interactions improvement by combining this docking method with molecular dynamics simulations. Thus, the predicted docking structures were analyzed in a molecular dynamics trajectory to generate dynamic models and reinforce the predicted binding modes. FMVAPP is predicted to make more extensive contacts with S. aureus MDD, forming stable hydrogen bonds with Arg144, Arg193, Lys21, Ser107, and Tyr18, as well as making stable hydrophobic interactions with Tyr18, Trp19, and Met196. The differences in predicted binding are supported by experimentally determined Ki values of 0.23 ± 0.02 and 34 ± 8 μM, for FMVAPP and DPGP, respectively. The structural information coupled with the kinetic characterization obtained from this study should be useful in defining the requirements for inhibition as well as in guiding the selection of active compounds for inhibitor optimization.

  14. The Thiamine diphosphate dependent Enzyme Engineering Database: A tool for the systematic analysis of sequence and structure relations

    Directory of Open Access Journals (Sweden)

    Radloff Robert

    2010-02-01

    Full Text Available Abstract Background Thiamine diphosphate (ThDP-dependent enzymes form a vast and diverse class of proteins, catalyzing a wide variety of enzymatic reactions including the formation or cleavage of carbon-sulfur, carbon-oxygen, carbon-nitrogen, and especially carbon-carbon bonds. Although very diverse in sequence and domain organisation, they share two common protein domains, the pyrophosphate (PP and the pyrimidine (PYR domain. For the comprehensive and systematic comparison of protein sequences and structures the Thiamine diphosphate (ThDP-dependent Enzyme Engineering Database (TEED was established. Description The TEED http://www.teed.uni-stuttgart.de contains 12048 sequence entries which were assigned to 9443 different proteins and 379 structure entries. Proteins were assigned to 8 different superfamilies and 63 homologous protein families. For each family, the TEED offers multisequence alignments, phylogenetic trees, and family-specific HMM profiles. The conserved pyrophosphate (PP and pyrimidine (PYR domains have been annotated, which allows the analysis of sequence similarities for a broad variety of proteins. Human ThDP-dependent enzymes are known to be involved in many diseases. 20 different proteins and over 40 single nucleotide polymorphisms (SNPs of human ThDP-dependent enzymes were identified in the TEED. Conclusions The online accessible version of the TEED has been designed to serve as a navigation and analysis tool for the large and diverse family of ThDP-dependent enzymes.

  15. Molecular cloning and catalytic activity of a membrane-bound prenyl diphosphate phosphatase from Croton stellatopilosus Ohba.

    Science.gov (United States)

    Nualkaew, Natsajee; Guennewich, Nils; Springob, Karin; Klamrak, Anuwatchakit; De-Eknamkul, Wanchai; Kutchan, Toni M

    2013-07-01

    Geranylgeraniol (GGOH), a bioactive acyclic diterpene with apoptotic induction activity, is the immediate precursor of the commercial anti-peptic, plaunotol (18-hydroxy geranylgeraniol), which is found in Croton stellatopilosus (Ohba). From this plant, a cDNA encoding a prenyl diphosphate phosphatase (CsPDP), which catalyses the dephosphorylation of geranylgeranyl diphosphate (GGPP) to GGOH, was isolated using a PCR approach. The full-length cDNA contained 888bp and encoded a 33.6 kDa protein (295 amino acids) that was phylogenetically grouped into the phosphatidic acid phosphatase (PAP) enzyme family. The deduced amino acid sequence showed 6 hydrophobic transmembrane regions with 57-85% homology to the sequences of other plant PAPs. The recombinant CsPDP and its 4 truncated constructs exhibited decreasing dephosphorylation activities relative to the lengths of the N-terminal deletions. While the full-length CsPDP successfully performed the two sequential monodephosphorylation steps on GGPP to form GGOH, the larger N-terminal deletion in the truncated enzymes appeared to specifically decrease the catalytic efficiency of the second monodephosphorylation step. The information presented here on the CsPDP cDNA and factors affecting the dephosphorylation activity of its recombinant protein may eventually lead to the discovery of the specific GGPP phosphatase gene and enzyme that are involved in the formation of GGOH in the biosynthetic pathway of plaunotol in C. stellatopilosus.

  16. Engineering of a plasmid-free Escherichia coli strain for improved in vivo biosynthesis of astaxanthin

    Directory of Open Access Journals (Sweden)

    Steuer Kristin

    2011-04-01

    Full Text Available Abstract Background The xanthophyll astaxanthin is a high-value compound with applications in the nutraceutical, cosmetic, food, and animal feed industries. Besides chemical synthesis and extraction from naturally producing organisms like Haematococcus pluvialis, heterologous biosynthesis in non-carotenogenic microorganisms like Escherichia coli, is a promising alternative for sustainable production of natural astaxanthin. Recent achievements in the metabolic engineering of E. coli strains have led to a significant increase in the productivity of carotenoids like lycopene or β-carotene by increasing the metabolic flux towards the isoprenoid precursors. For the heterologous biosynthesis of astaxanthin in E. coli, however, the conversion of β-carotene to astaxanthin is obviously the most critical step towards an efficient biosynthesis of astaxanthin. Results Here we report the construction of the first plasmid-free E. coli strain that produces astaxanthin as the sole carotenoid compound with a yield of 1.4 mg/g cdw (E. coli BW-ASTA. This engineered E. coli strain harbors xanthophyll biosynthetic genes from Pantoea ananatis and Nostoc punctiforme as individual expression cassettes on the chromosome and is based on a β-carotene-producing strain (E. coli BW-CARO recently developed in our lab. E. coli BW-CARO has an enhanced biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP and produces β-carotene in a concentration of 6.2 mg/g cdw. The expression of crtEBIY along with the β-carotene-ketolase gene crtW148 (NpF4798 and the β-carotene-hydroxylase gene (crtZ under controlled expression conditions in E. coli BW-ASTA directed the pathway exclusively towards the desired product astaxanthin (1.4 mg/g cdw. Conclusions By using the λ-Red recombineering technique, genes encoding for the astaxanthin biosynthesis pathway were stably integrated into the chromosome of E. coli. The expression levels of chromosomal integrated recombinant

  17. Study of the thorium phosphate-diphosphate (TPD) dissolution: kinetic aspect - thermodynamic aspect: analysis of the neo-formed phases; Etude de la dissolution du phosphate diphosphate de thorium: - aspect cinetique - aspect thermodynamique: analyse des phases neoformees

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, A.Ch

    2000-10-06

    The aim of this work is to study the aqueous corrosion of the thorium phosphate-diphosphate (TPD), of the formula Th{sub 4}(PO{sub 4}){sub 4}P{sub 2}O{sub 7}, in the framework of the actinides immobilization. In order to complete the anterior studies concerning solid solutions where thorium is substituted by a tetravalent ion (uranium (IV) or plutonium (IV)) in the TPD structure, compounds of thorium and neptunium phosphate-diphosphate, of formula Th{sub 4-x}Np{sub x}(PO{sub 4}){sub 4}P{sub 2}O{sub 7}, have been prepared. Furthermore, a new chemical way of synthesis has been investigated in order to sinter solids solution of thorium and uranium phosphate-diphosphate (TUPD) in good conditions. The TPD dissolution study showed two principals steps. The first one corresponds to the control of element concentration by the material dissolution whereas the second corresponds to the formation of secondary precipitates for which thermodynamic equilibrium controls the concentration of the species in solution. Leaching tests have been performed varying several independent parameters in order to determine the TPD dissolution rate. The partial orders related to the protons or to the hydroxide ions have been found between 0.35 and 0.45 whereas the apparent dissolution rate constants are in the range 1.10{sup -5} for 9.10{sup -5} g.m{sup -2}.j{sup -1} for acidic and basic media. The neo-formed phases have been characterized after the dissolution of TPD and TUPD. We found that the TPD leaching in acidic medium leads to the formation of the crystallized thorium phosphate-hydrogen-phosphate (TPHP), of formula Th{sub 2}(PO{sub 4}){sub 2}(HPO{sub 4}), x H{sub 2}O, whereas the TUPD dissolution leads to the TPHP and an other compound, of formula (UO{sub 2}){sub 3}(PO{sub 4}){sub 2}, 5 H{sub 2}O. We calculated its solubility product which is in good agreement with those found in the literature. The phases formed during the leaching of solids containing plutonium; americium or curium (Th

  18. E. Coli and Pregnancy

    Science.gov (United States)

    ... best live chat Live Help Fact Sheets Share E. coli and Pregnancy Thursday, 20 November 2014 In ... pregnancy and while breastfeeding. Donate Sign Up For E-Newsletter Full Name * Email Address * Enter The Code: ...

  19. Chronic alcoholism in rats induces a compensatory response, preserving brain thiamine diphosphate, but the brain 2-oxo acid dehydrogenases are inactivated despite unchanged coenzyme levels.

    Science.gov (United States)

    Parkhomenko, Yulia M; Kudryavtsev, Pavel A; Pylypchuk, Svetlana Yu; Chekhivska, Lilia I; Stepanenko, Svetlana P; Sergiichuk, Andrej A; Bunik, Victoria I

    2011-06-01

    Thiamine-dependent changes in alcoholic brain were studied using a rat model. Brain thiamine and its mono- and diphosphates were not reduced after 20 weeks of alcohol exposure. However, alcoholism increased both synaptosomal thiamine uptake and thiamine diphosphate synthesis in brain, pointing to mechanisms preserving thiamine diphosphate in the alcoholic brain. In spite of the unchanged level of the coenzyme thiamine diphosphate, activities of the mitochondrial 2-oxoglutarate and pyruvate dehydrogenase complexes decreased in alcoholic brain. The inactivation of pyruvate dehydrogenase complex was caused by its increased phosphorylation. The inactivation of 2-oxoglutarate dehydrogenase complex (OGDHC) correlated with a decrease in free thiols resulting from an elevation of reactive oxygen species. Abstinence from alcohol following exposure to alcohol reactivated OGDHC along with restoration of the free thiol content. However, restoration of enzyme activity occurred before normalization of reactive oxygen species levels. Hence, the redox status of cellular thiols mediates the action of oxidative stress on OGDHC in alcoholic brain. As a result, upon chronic alcohol consumption, physiological mechanisms to counteract the thiamine deficiency and silence pyruvate dehydrogenase are activated in rat brain, whereas OGDHC is inactivated due to impaired antioxidant ability.

  20. New role of flavin as a general acid-base catalyst with no redox function in type 2 isopentenyl-diphosphate isomerase.

    Science.gov (United States)

    Unno, Hideaki; Yamashita, Satoshi; Ikeda, Yosuke; Sekiguchi, Shin-Ya; Yoshida, Norie; Yoshimura, Tohru; Kusunoki, Masami; Nakayama, Toru; Nishino, Tokuzo; Hemmi, Hisashi

    2009-04-03

    Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes.

  1. Synthesis and antitumor activities of nicotiamide phosphoribosyl transferase inhibitors%烟酰胺磷酸核糖转移酶抑制剂的设计合成及抗肿瘤细胞增殖活性

    Institute of Scientific and Technical Information of China (English)

    罗飚; 洪新福; 姚郑林; 占文虎; 赵砚瑾; 李庶心

    2012-01-01

    Nowadays.nicotinamide phosphoribosyl transferase(Nampt) has studied as an important target in the area of tumor inhibition. As a result,it is necessary to find some better Nampt inhibitors. A Nampt inhibitor APO866(also known as FK866) which entered clinical trials was chosen as a lead compound,eight Nampt inhibitors derivatives were designed and synthesized. Using piperazine as raw materials,by seven steps including substitution reaction,SN2 reaction,Gabriel reaction,synthesis of guanidine,cyclization,deprotection and acyla-don,the target compounds were synthesized. Moreover,the structures of compounds were verified by MS and 1H-NMR,and preliminary in vitro antitumor assessment showed that three compounds (H1, H5.H7) had a certain antitumor activity (the IC50, value of HI on MCF-7, HL-60 and A549 were 20.25 20. 64 μmol·L-1 and 13.45 μmol·L-1 ;the IC50, value of H5 on MCF-7,HL-60 and A549 were 23.88 25.23 μmol·L-1 and 18.52 μmol·L-1; the IC50 value of H7 on MCF-7, HL-60 and A549 were 22.99 μmol·L-1,28.72 μmol·L-1 and 10. 57 uμmol·L-1) ,in which the biological activity of them nearly approaches the APO866 (IC50, value of APO866 on MCF-7, HL-60 and A549 were 20.18 μmol·L-1, 12. 92 μmol·L-1and 7. 15 μmol·L-1). These data will provide certain reference for APO866 further structure transformation.%目的 寻找新的烟酰胺磷酸核糖转移酶( Nampt)抑制剂,为进一步药物设计提供依据.方法 以进入临床研究的APO866为先导化合物,设计合成一系列Nampt抑制剂.以哌嗪为原料,经取代、SN2反应、Gabriel反应、成胍、环合等多步反应得到关键中间体(7a,7b),该中间体再与酰氯衍生物反应,最终合成目标化合物.结果与结论 合成了8个全新的Nampt抑制剂,化合物的结构经质谱和核磁共振氢谱确证;初步体外抗肿瘤实验结果表明,其中3个化合物(H1、H5、H7)具有一定的抗肿瘤活性,其活性与阳性对照APO866相当.

  2. Taxonomy Icon Data: Escherichia coli [Taxonomy Icon

    Lifescience Database Archive (English)

    Full Text Available Escherichia coli Escherichia coli Escherichia_coli_L.png Escherichia_coli_NL.png Escherich...ia_coli_S.png Escherichia_coli_NS.png http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Escherichia+co...li&t=L http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Escherichia+coli&t=NL http://biosciencedbc.jp/taxono...my_icon/icon.cgi?i=Escherichia+coli&t=S http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Escherichia+coli&t=NS ...

  3. Interaction of hexa-His tag with acidic amino acids results in facilitated refolding of halophilic nucleoside diphosphate kinase.

    Science.gov (United States)

    Ishibashi, Matsujiro; Ida, Keiko; Tatsuda, Shuhei; Arakawa, Tsutomu; Tokunaga, Masao

    2011-11-01

    We have previously reported that amino-terminal extension sequence containing hexa-His facilitated refolding and assembly of hexameric nucleoside diphosphate kinase from extremely halophilic archaeon Halobacterium salinarum (NDK). In this study, we made various mutations in both the tag sequence and within NDK molecule. SerNDK, in which hexa-His was replaced with hexa-Ser, showed no facilitated folding. In addition, HisD58GD63G, in which both Asp58 and Asp63 in NDK were replaced with Gly, also showed no refolding enhancement. These results suggest that hexa-His in His-tag interact cooperatively with either Asp58 or Asp63 or both. Furthermore, G114D mutant, which formed a dimer in low salt solution, was strongly stabilized by His-tag to form a stable hexamer.

  4. THE REGULATORY EFFECT OF NUCLEOSIDE DIPHOSPHATE KINASE ON G-PROTEIN AND G-PROTEIN MEDIATED PHOSPHOLIPASE C

    Institute of Scientific and Technical Information of China (English)

    张德昌; 张宽仁

    1995-01-01

    The effect of nueleoside diphosphate kinase (NDPK) on the activity of guanine nueleotide regulatory protein (G-protein) mediated phospholipase C (PLC) and on the [35S ] GTPTτS binding of G-protein was investigated in this work in order to demonstrate the mechanism behind the regulation of G-protein and its effector PLC by NDPK. The stimulation of PLC in turkey erythrocyte membrane by both GTP and GTPτS indicated that the PLC stimulation was msdiated by G-protein, NDPK alone stimulated PLC activity, as well as the stimulation in the presence of GTP and GDP, in a dose-dependent manner. However, NDPK inhibited GTPτS-stimulated PLC, Furthermore, NDPK inhibited [35S] GTPτS binding of purified Gi-protein in a non-competitive manner. A hypothesis implying an important role of direct interaction of G-protein and NDPK in the regulation of their functions is suggested and discussed.

  5. Diethylstilbestrol-diphosphate induces chromosomal aberrations but not sister chromatid exchanges in murine bone marrow cells in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Ivett, J.L. (North Carolina State Univ., Raleigh); Tice, R.R.

    1981-01-01

    Diethylstilbestrol diphosphate (DES-dp) clastogenesis was examined in the bone marrow of C57B1/6 male and female mice. Significant and sex-related dose effects were observed for the induction of chromatid-type chromosomal aberrations and for the inhibition of cellular proliferation. Females were more sensitive to the effects of DES-dp than males when assessed for either induced chromosomal aberrations or proliferative inhibition. Contrary to other published results, we did not observe either an increase in sister chromatid exchanges or an increased incidence of aneuploidy. Ovariectomy reduced the ability of DES-dp to inhibit cellular proliferation and decreased the high degree of variability between animals at high doses of DES-dp. The results of our studies show that DES is a clastogenic agent in vivo which may relate to its carcinogenicity.

  6. α-Hydroxy-β-keto acid rearrangement-decarboxylation: impact on thiamine diphosphate-dependent enzymatic transformations.

    Science.gov (United States)

    Beigi, Maryam; Loschonsky, Sabrina; Lehwald, Patrizia; Brecht, Volker; Andrade, Susana L A; Leeper, Finian J; Hummel, Werner; Müller, Michael

    2013-01-14

    The thiamine diphosphate (ThDP) dependent MenD catalyzes the reaction of α-ketoglutarate with pyruvate to selectively form 4-hydroxy-5-oxohexanoic acid 2, which seems to be inconsistent with the assumed acyl donor role of the physiological substrate α-KG. In contrast the reaction of α-ketoglutarate with acetaldehyde gives exclusively the expected 5-hydroxy-4-oxo regioisomer 1. These reactions were studied by NMR and CD spectroscopy, which revealed that with pyruvate the observed regioselectivity is due to the rearrangement-decarboxylation of the initially formed α-hydroxy-β-keto acid rather than a donor-acceptor substrate role variation. Further experiments with other ThDP-dependent enzymes, YerE, SucA, and CDH, verified that this degenerate decarboxylation can be linked to the reduced enantioselectivity of acyloins often observed in ThDP-dependent enzymatic transformations.

  7. Theoretical pKa prediction of the α-phosphate moiety of uridine 5‧-diphosphate-GlcNAc

    Science.gov (United States)

    Vipperla, Bhavaniprasad; Griffiths, Thomas M.; Wang, Xingyong; Yu, Haibo

    2017-01-01

    The pKa value of the α-phosphate moiety of uridine 5‧-diphosphate-GlcNAc (UDP-GlcNAc) has been successfully calculated using density functional theory methods in conjunction with the Polarizable Continuum Models. Theoretical methods were benchmarked over a dataset comprising of alkyl phosphates. B3LYP/6-31+G(d,p) calculations using SMD solvation model provide excellent agreement with the experimental data. The predicted pKa for UDP-GlcNAc is consistent with most recent NMR studies but much higher than what it has long been thought to be. The importance of this study is evident that the predicted pKa for UDP-GlcNAc supports its potential role as a catalytic base in the substrate-assisted biocatalysis.

  8. Mutational analysis of the nucleotide binding site of Escherichia coli dCTP deaminase

    DEFF Research Database (Denmark)

    Thymark, Majbritt; Johansson, Eva; Larsen, Sine;

    2007-01-01

    In Escherichia coli and Salmonella typhimurium about 80% of the dUMP used for dTMP synthesis is derived from deamination of dCTP. The dCTP deaminase produces dUTP that subsequently is hydrolyzed by dUTPase to dUMP and diphosphate. The dCTP deaminase is regulated by dTTP that inhibits the enzyme...... of E138D in complex with dUTP showed a hydrogen bonding network in the active site similar to wild-type enzyme. However, changes in the hydrogen bond lengths between the carboxylate and a catalytic water molecule as well as a slightly different orientation of the pyrimidine ring of the bound nucleotide...

  9. Distribution of the thiamin diphosphate C(2)-proton during catalysis of acetaldehyde formation by brewers' yeast pyruvate decarboxylase.

    Science.gov (United States)

    Harris, T K; Washabaugh, M W

    1995-10-31

    The distribution of tritium derived from enzyme-bound [thiazole-2-T]thiamin diphosphate (TDP) during the reaction of pyruvate to form acetaldehyde catalyzed by pyruvate decarboxylase isozymes (PDC; EC 4.1.1.1) from Saccharomyces carlsbergensis was determined under single-turnover conditions ([E] > [S]) in the presence of the nonsubstrate allosteric effector pyruvamide. The specific radioactivity of the [1-L]acetaldehyde product and solvent ([L]H2O) was 43 +/- 4% and 54 +/- 2%, respectively, of the initial specific radioactivity of PDC-bound [thiazole-2-T]TDP and was independent of the extent of the single-turnover reaction. There is little (< or = 3%) or no return of the abstracted C(2)-hydron to the C(2) position of PDC-bound TDP. This provides evidence that the abstracted C(2)-hydron is involved in the specific protonation of the C(alpha) position of the PDC-bound intermediate 2-(1-hydroxyethyl)thiamin diphosphate (HETDP), which is cleaved to form [1-L]acetaldehyde and PDC-bound [thiazole-2-H]TDP. The partial exchange of C(2)-derived tritium into solvent requires that (1) hydron transfer from C(2) occurs to a catalytic-base in which the conjugate catalytic acid is partially shielded from hydron exchange with the solvent, (2) the conjugate catalytic acid transfers the C(2)-derived hydron to the C(alpha) position of HETDP, and (3) hydron transfer to C(2) to regenerate the coenzyme occurs either from solvent directly or from a second catalytic acid of the enzyme that undergoes rapid hydron exchange with the solvent.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Characterization of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS) gene from Ginkgo biloba.

    Science.gov (United States)

    Kim, Sang-Min; Kim, Soo-Un

    2010-02-01

    Diterpene trilactone ginkgolides, one of the major constituents of Ginkgo biloba extract, have shown interesting bioactivities including platelet-activating factor antagonistic activity. 1-Hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS), converting 2-C-methyl-d-erythritol-2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate, is the penultimate enzyme of the seven-step 2-C-methyl-d-erythritol 4-phosphate pathway that supplies building blocks for plant isoprenoids of plastid origin such as ginkgolides and carotenoids. Here, we report on the isolation and characterization of the full-length cDNA encoding HDS (GbHDS, GenBank accession number: DQ251630) from G. biloba. Full-length cDNA of GbHDS, 2,763 bp long, contained an ORF of 2,226 bp encoding a protein composed of 741 amino acids. The theoretical molecular weight and pI of the deduced mature GbHDS of 679 amino acid residues are 75.6 kDa and 5.5, respectively. From 2 weeks after initiation of the culture onward, transcription level of this gene in the ginkgo embryo roots increased to about two times higher than that in the leaves. GbHDS was predicted to possess chloroplast transit peptide of 62 amino acid residues, suggesting its putative localization in the plastids. The transient gene expression in Arabidopsis protoplasts confirmed that the transit peptide was capable of delivering the GbHDS protein from the cytosol into the chloroplasts. The isolation and characterization of GbHDS gene enabled us to further understand the role of GbHDS in the terpenoid biosynthesis in G. biloba.

  11. Nature of the main contaminant in the drug primaquine diphosphate: SFC and SFC-MS methods of analysis.

    Science.gov (United States)

    Brondz, Ilia; Ekeberg, Dag; Bell, David S; Annino, Amy R; Hustad, Jan Arild; Svendsen, Robert; Vlachos, Vaso; Oakley, Paul; Langley, G John; Mohini, Thite; Amaury, Cazenave-Gassiot; Mikhalitsyn, Felix

    2007-02-19

    The drug primaquine diphosphate is used for causative treatment of malaria. Using HPLC-MS and GC-MS, this research group was previously able to show that the main contaminant of primaquine is the positional isomer quinocide [I. Brondz, D. Mantzilas, U. Klein, D. Ekeberg, E. Hvattum, M.N. Lebedeva, F.S. Mikhailitsyn, G.D. Soulimanov, J. Roe, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 800 (2004) 211-223; I. Brondz, U. Klein, D. Ekeberg, D. Mantzilas, E. Hvattum, H. Schultz, F. S. Mikhailitsyn, Asian J. Chem. 17 (2005) 1678-1688]. Primaquine and quinocide are highly toxic substances which can have a number of side effects upon use in medical treatment. A standard for quinocide is not typically commercially available. In the present work, supercritical fluid chromatography-mass spectrometry (SFC-MS) with two different columns was used to achieve a shorter analysis time for the separation between the positional isomers quinocide and primaquine in primaquine diphosphate and to elucidate additional information about differences in their MS fragmentation. Unlike using HPLC-MS, it was possible to achieve the differential fragmentation of positional isomers at branching points using the SFC-MS technique. The desired short analysis time was achieved using SFC equipped with a Discovery HS F5 column and the differential fragmentation of positional isomers during SFC-MS provides information on the differences in the structure of these substances. Using a Chiralpak AD-H chiral column, it was possible to resolve the enantiomers in primaquine and separate quinocide from those enantiomers.

  12. Biosynthesis of the psychotropic plant diterpene salvinorin A: Discovery and characterization of the Salvia divinorum clerodienyl diphosphate synthase.

    Science.gov (United States)

    Pelot, Kyle A; Mitchell, Rod; Kwon, Moonhyuk; Hagelthorn, David M; Wardman, Jacob F; Chiang, Angela; Bohlmann, Jörg; Ro, Dae-Kyun; Zerbe, Philipp

    2017-03-01

    Salvia divinorum commonly known as diviner's sage, is an ethnomedicinal plant of the mint family (Lamiaceae). Salvia divinorum is rich in clerodane-type diterpenoids, which accumulate predominantly in leaf glandular trichomes. The main bioactive metabolite, salvinorin A, is the first non-nitrogenous natural compound known to function as an opioid-receptor agonist, and is undergoing clinical trials for potential use in treating neuropsychiatric diseases and drug addictions. We report here the discovery and functional characterization of two S. divinorum diterpene synthases (diTPSs), the ent-copalyl diphosphate (ent-CPP) synthase SdCPS1, and the clerodienyl diphosphate (CLPP) synthase SdCPS2. Mining of leaf- and trichome-specific transcriptomes revealed five diTPSs, two of which are class II diTPSs (SdCPS1-2) and three are class I enzymes (SdKSL1-3). Of the class II diTPSs, transient expression in Nicotiana benthamiana identified SdCPS1 as an ent-CPP synthase, which is prevalent in roots and, together with SdKSL1, exhibits a possible dual role in general and specialized metabolism. In vivo co-expression and in vitro assays combined with nuclear magnetic resonance (NMR) analysis identified SdCPS2 as a CLPP synthase. A role of SdCPS2 in catalyzing the committed step in salvinorin A biosynthesis is supported by its biochemical function, trichome-specific expression and absence of additional class II diTPSs in S. divinorum. Structure-guided mutagenesis revealed four catalytic residues that enabled the re-programming of SdCPS2 activity to afford four distinct products, thus advancing our understanding of how neo-functionalization events have shaped the array of different class II diTPS functions in plants, and may promote synthetic biology platforms for a broader spectrum of diterpenoid bioproducts.

  13. Zoledronate derivatives as potential inhibitors of uridine diphosphate-galactose ceramide galactosyltransferase 8: A combined molecular docking and dynamic study.

    Science.gov (United States)

    Pannuzzo, Giovanna; Graziano, Adriana Carol Eleonora; Pannuzzo, Martina; Masman, Marcelo Fabricio; Avola, Rosanna; Cardile, Venera

    2016-11-01

    Krabbe's disease is a neurodegenerative disorder caused by deficiency of galactocerebrosidase activity that affects the myelin sheath of the nervous system, involving dysfunctional metabolism of sphingolipids. It has no cure. Because substrate inhibition therapy has been shown to be effective in some human lysosomal storage diseases, we hypothesize that a substrate inhibition therapeutic approach might be appropriate to allow correction of the imbalance between formation and breakdown of glycosphingolipids and to prevent pathological storage of psychosine. The enzyme responsible for the biosynthesis of galactosylceramide and psychosine is uridine diphosphate-galactose ceramide galactosyltransferase (2-hydroxyacylsphingosine 1-β-galactosyltransferase; UGT8; EC 2.4.1.45), which catalyzes the transferring of galactose from uridine diphosphate-galactose to ceramide or sphingosine, an important step of the biosynthesis of galactosphingolipids. Because some bisphosphonates have been identified as selective galactosyltransferase inhibitors, we verify the binding affinity to a generated model of the enzyme UGT8 and investigate the molecular mechanisms of UGT8-ligand interactions of the bisphosphonate zoledronate by a multistep framework combining homology modeling, molecular docking, and molecular dynamics simulations. From structural information on UGTs' active site stereochemistry, charge density, and access through the hydrophobic environment, the molecular docking procedure allowed us to identify zoledronate as a potential inhibitor of human ceramide galactosyltransferase. More importantly, zoledronate derivates were designed through computational modeling as putative new inhibitors. Experiments in vivo and in vitro have been planned to verify the possibility of using zoledronate and/or the newly identified inhibitors of UGT8 for a substrate inhibition therapy useful for treatment of Krabbe's disease and/or other lysosomal disorders. © 2016 Wiley Periodicals, Inc.

  14. A moonlighting enzyme links Escherichia coli cell size with central metabolism.

    Directory of Open Access Journals (Sweden)

    Norbert S Hill

    Full Text Available Growth rate and nutrient availability are the primary determinants of size in single-celled organisms: rapidly growing Escherichia coli cells are more than twice as large as their slow growing counterparts. Here we report the identification of the glucosyltransferase OpgH as a nutrient-dependent regulator of E. coli cell size. During growth under nutrient-rich conditions, OpgH localizes to the nascent septal site, where it antagonizes assembly of the tubulin-like cell division protein FtsZ, delaying division and increasing cell size. Biochemical analysis is consistent with OpgH sequestering FtsZ from growing polymers. OpgH is functionally analogous to UgtP, a Bacillus subtilis glucosyltransferase that inhibits cell division in a growth rate-dependent fashion. In a striking example of convergent evolution, OpgH and UgtP share no homology, have distinct enzymatic activities, and appear to inhibit FtsZ assembly through different mechanisms. Comparative analysis of E. coli and B. subtilis reveals conserved aspects of growth rate regulation and cell size control that are likely to be broadly applicable. These include the conservation of uridine diphosphate glucose as a proxy for nutrient status and the use of moonlighting enzymes to couple growth rate-dependent phenomena to central metabolism.

  15. Adenylate kinase-independent thiamine triphosphate accumulation under severe energy stress in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Wins Pierre

    2008-01-01

    Full Text Available Abstract Background Thiamine triphosphate (ThTP exists in most organisms and might play a role in cellular stress responses. In E. coli, ThTP is accumulated in response to amino acid starvation but the mechanism of its synthesis is still a matter of controversy. It has been suggested that ThTP is synthesized by an ATP-dependent specific thiamine diphosphate kinase. However, it is also known that vertebrate adenylate kinase 1 catalyzes ThTP synthesis at a very low rate and it has been postulated that this enzyme is responsible for ThTP synthesis in vivo. Results Here we show that bacterial, as vertebrate adenylate kinases are able to catalyze ThTP synthesis, but at a rate more than 106-fold lower than ATP synthesis. This activity is too low to explain the high rate of ThTP accumulation observed in E. coli during amino acid starvation. Moreover, bacteria from the heat-sensitive CV2 strain accumulate high amounts of ThTP (>50% of total thiamine at 37°C despite complete inactivation of adenylate kinase and a subsequent drop in cellular ATP. Conclusion These results clearly demonstrate that adenylate kinase is not responsible for ThTP synthesis in vivo. Furthermore, they show that E. coli accumulate large amounts of ThTP under severe energy stress when ATP levels are very low, an observation not in favor of an ATP-dependent mechanisms for ThTP synthesis.

  16. taenia coli de cobaye

    African Journals Online (AJOL)

    AKA BOKO

    of the rhythmical contraction of guinea pig Taenia coli smooth muscle. ... déterminer le mécanisme d'action de ces composés. 2. Matériel et ..... [34, 35]. Il rappelle les effets de l'acétylcholine sur les muscles lisses viscéraux [36- 38]. Pour cela.

  17. Characterization of a cytosolic nucleoside diphosphate kinase associated with cell division and growth in potato.

    Science.gov (United States)

    Dorion, Sonia; Matton, Daniel P; Rivoal, Jean

    2006-06-01

    A cDNA encoding Solanum chacoense cytosolic NDPK (NDPK1, EC 2.7.4.6) was isolated. The open reading frame encoded a 148 amino acid protein that shares homology with other cytosolic NDPKs including a conserved N-terminal domain. S. chacoense NDPK1 was expressed in Escherichia coli as a 6xHis-tagged protein and purified by affinity chromatography. The recombinant protein exhibited a pattern of abortive complex formation suggesting that the enzyme is strongly regulated by the NTP/NDP ratio. A polyclonal antibody generated against recombinant NDPK1 was specific for the cytosolic isoform in Solanum tuberosum as shown from immunoprecipitation experiments and immunoblot analysis of chloroplasts and mitochondria preparations. NDPK activity and NDPK1 protein were found at different levels in various vegetative and reproductive tissues. DEAE fractogel analyses of NDPK activity in root tips, leaves, tubers and cell cultures suggest that NDPK1 constitutes the bulk of extractable NDPK activity in all these organs. NDPK activity and NDPK1 protein levels raised during the exponential growth phase of potato cell cultures whereas no rise in activity or NDPK1 protein was observed when sucrose concentration in the culture was manipulated to limit growth. Activity measurements, immunoblot analysis as well as immunolocalization experiments performed on potato root tips and shoot apical buds demonstrated that NDPK1 was predominantly localized in the meristematic zones and provascular tissues of the apical regions. These data suggest that NDPK1 plays a specific role in the supply of UTP during early growth of plant meristematic and provascular tissues.

  18. Blocking Cyclic Adenosine Diphosphate Ribose-mediated Calcium Overload Attenuates Sepsis-induced Acute Lung Injury in Rats

    Institute of Scientific and Technical Information of China (English)

    Qian-yi Peng; Yu Zou; Li-Na Zhang; Mei-Lin Ai; Wei Liu; Yu-Hang Ai

    2016-01-01

    Background:Acute lung injury (ALI) is a common complication of sepsis that is associated with high mortality.Intracellular Ca2+ overload plays an important role in the pathophysiology of sepsis-induced ALI,and cyclic adenosine diphosphate ribose (cADPR) is an important regulator of intracellular Ca2+ mobilization.The cluster of differentiation 38 (CD38)/cADPR pathway has been found to play roles in multiple inflammatory processes but its role in sepsis-induced ALI is still unknown.This study aimed to investigate whether the CD38/cADPR signaling pathway is activated in sepsis-induced ALI and whether blocking cADPR-mediated calcium overload attenuates ALI.Methods:Septic rat models were established by cecal ligation and puncture (CLP).Rats were divided into the sham group,the CLP group,and the CLP+ 8-bromo-cyclic adenosine diphosphate ribose (8-Br-cADPR) group.Nicotinamide adenine dinucleotide (NAD+),cADPR,CD38,and intracellular Ca2+ levels in the lung tissues were measured at 6,12,24,and 48 h after CLP surgery.Lung histologic injury,tumor necrosis factor (TNF)-α,malondialdehyde (MDA) levels,and superoxide dismutase (SOD) activities were measured.Results:NAD+,cADPR,CD38,and intracellular Ca2+ levels in the lungs of septic rats increased significantly at 24 h after CLP surgery.Treatment with 8-Br-cADPR,a specific inhibitor of cADPR,significantly reduced intracellular Ca2+ levels (P =0.007),attenuated lung histological injury (P =0.023),reduced TNF-α and MDA levels (P < 0.001 and P =0.002,respectively) and recovered SOD activity (P =0.031) in the lungs of septic rats.Conclusions:The CD38/cADPR pathway is activated in the lungs of septic rats,and blocking cADPR-mediated calcium overload with 8-Br-cADPR protects against sepsis-induced ALI.

  19. Organization of Monoterpene Biosynthesis in Mentha. Immunocytochemical Localizations of Geranyl Diphosphate Synthase, Limonene-6-Hydroxylase, Isopiperitenol Dehydrogenase, and Pulegone Reductase1

    Science.gov (United States)

    Turner, Glenn W.; Croteau, Rodney

    2004-01-01

    We present immunocytochemical localizations of four enzymes involved in p-menthane monoterpene biosynthesis in mint: the large and small subunits of peppermint (Mentha x piperita) geranyl diphosphate synthase, spearmint (Mentha spicata) (−)-(4S)-limonene-6-hydroxylase, peppermint (−)-trans-isopiperitenol dehydrogenase, and peppermint (+)-pulegone reductase. All were localized to the secretory cells of peltate glandular trichomes with abundant labeling corresponding to the secretory phase of gland development. Immunogold labeling of geranyl diphosphate synthase occurred within secretory cell leucoplasts, (−)-4S-limonene-6-hydroxylase labeling was associated with gland cell endoplasmic reticulum, (−)-trans-isopiperitenol dehydrogenase labeling was restricted to secretory cell mitochondria, while (+)-pulegone reductase labeling occurred only in secretory cell cytoplasm. We discuss this pathway compartmentalization in relation to possible mechanisms for the intracellular movement of monoterpene metabolites, and for monoterpene secretion into the extracellular essential oil storage cavity. PMID:15542490

  20. Temperature effects on the interaction mechanisms between the europium (III) and uranyl ions and zirconium diphosphate; Effets de la temperature sur les mecanismes d'interaction entre les ions europium (3) et uranyle et le diphosphate de zirconium

    Energy Technology Data Exchange (ETDEWEB)

    Finck, N

    2006-10-15

    Temperature should remain higher than 25 C in the near field environment of a nuclear waste repository for thousands years. In this context, the aim of this work is to study the temperature influence on the interaction mechanisms between europium (III) and uranyl ions and zirconium diphosphate, as well as the influence of a complexing medium (nitrate) on the sorption of the lanthanide. The experimental definition of the equilibria was achieved by combining a structural investigation with the macroscopic sorption data. Surface complexes were characterized at all temperatures (25 C to 90 C) by TRLFS experiments carried out on dry and in situ samples using an oven. This characterization was completed by XPS experiments carried out at 25 C on samples prepared at 25 C and 90 C. The reaction constants (surface hydration and cations sorption) were obtained by simulating the experimental data with the constant capacitance surface complexation model. The reaction constants temperature dependency allowed one to characterize thermodynamically the different reactions by application of the van't Hoff relation. The validity of this law was tested by performing microcalorimetric measurements of the sorption heat for both cations. (author)

  1. Suppressing Farnesyl Diphosphate Synthase Alters Chloroplast Development and Triggers Sterol-Dependent Induction of Jasmonate- and Fe-Related Responses1[OPEN

    Science.gov (United States)

    Andrade, Paola; Caudepón, Daniel; Arró, Montserrat

    2016-01-01

    Farnesyl diphosphate synthase (FPS) catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. Arabidopsis (Arabidopsis thaliana) contains two genes (FPS1 and FPS2) encoding FPS. Single fps1 and fps2 knockout mutants are phenotypically indistinguishable from wild-type plants, while fps1/fps2 double mutants are embryo lethal. To assess the effect of FPS down-regulation at postembryonic developmental stages, we generated Arabidopsis conditional knockdown mutants expressing artificial microRNAs devised to simultaneously silence both FPS genes. Induction of silencing from germination rapidly caused chlorosis and a strong developmental phenotype that led to seedling lethality. However, silencing of FPS after seed germination resulted in a slight developmental delay only, although leaves and cotyledons continued to show chlorosis and altered chloroplasts. Metabolomic analyses also revealed drastic changes in the profile of sterols, ubiquinones, and plastidial isoprenoids. RNA sequencing and reverse transcription-quantitative polymerase chain reaction transcriptomic analysis showed that a reduction in FPS activity levels triggers the misregulation of genes involved in biotic and abiotic stress responses, the most prominent one being the rapid induction of a set of genes related to the jasmonic acid pathway. Down-regulation of FPS also triggered an iron-deficiency transcriptional response that is consistent with the iron-deficient phenotype observed in FPS-silenced plants. The specific inhibition of the sterol biosynthesis pathway by chemical and genetic blockage mimicked these transcriptional responses, indicating that sterol depletion is the primary cause of the observed alterations. Our results highlight the importance of sterol homeostasis for normal chloroplast development and function and reveal important clues about how isoprenoid and sterol metabolism is integrated within plant physiology and development. PMID

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

    Science.gov (United States)

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

    2015-11-15

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

  3. Exploring diterpene metabolism in non-model species: transcriptome-enabled discovery and functional characterization of labda-7,13E-dienyl diphosphate synthase from Grindelia robusta.

    Science.gov (United States)

    Zerbe, Philipp; Rodriguez, Selina M; Mafu, Sibongile; Chiang, Angela; Sandhu, Harpreet K; O'Neil-Johnson, Mark; Starks, Courtney M; Bohlmann, Jörg

    2015-09-01

    Grindelia robusta or gumweed, is a medicinal herb of the sunflower family that forms a diverse suite of diterpenoid natural products. Its major constituents, grindelic acid and related grindelane diterpenoids accumulate in a resinous exudate covering the plants' surfaces, most prominently the unopened composite flower. Recent studies demonstrated potential pharmaceutical applications for grindelic acid and its synthetic derivatives. Mining of the previously published transcriptome of G. robusta flower tissue identified two additional diterpene synthases (diTPSs). We report the in vitro and in vivo functional characterization of an ent-kaurene synthase of general metabolism (GrTPS4) and a class II diTPS (GrTPS2) of specialized metabolism that converts geranylgeranyl diphosphate (GGPP) into labda-7,13E-dienyl diphosphate as verified by nuclear magnetic resonance (NMR) analysis. Tissue-specific transcript abundance of GrTPS2 in leaves and flowers accompanied by the presence of an endocyclic 7,13 double bond in labda-7,13E-dienyl diphosphate suggest that GrTPS2 catalyzes the first committed reaction in the biosynthesis of grindelic acid and related grindelane metabolites. With the formation of labda-7,13E-dienyl diphosphate, GrTPS2 adds an additional function to the portfolio of monofunctional class II diTPSs, which catalytically most closely resembles the bifunctional labda-7,13E-dien-15-ol synthase of the lycopod Selaginella moellendorffii. Together with a recently identified functional diTPS pair of G. robusta producing manoyl oxide, GrTPS2 lays the biosynthetic foundation of the diverse array of labdane-related diterpenoids in the genus Grindelia. Knowledge of these natural diterpenoid metabolic pathways paves the way for developing biotechnology approaches toward producing grindelic acid and related bioproducts.

  4. 7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity.

    Science.gov (United States)

    Wypijewska, Anna; Bojarska, Elzbieta; Lukaszewicz, Maciej; Stepinski, Janusz; Jemielity, Jacek; Davis, Richard E; Darzynkiewicz, Edward

    2012-10-09

    Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' → 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' → 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

  5. Structure of the ent-Copalyl Diphosphate Synthase PtmT2 from Streptomyces platensis CB00739, a Bacterial Type II Diterpene Synthase.

    Science.gov (United States)

    Rudolf, Jeffrey D; Dong, Liao-Bin; Cao, Hongnan; Hatzos-Skintges, Catherine; Osipiuk, Jerzy; Endres, Michael; Chang, Chin-Yuan; Ma, Ming; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N; Shen, Ben

    2016-08-31

    Terpenoids are the largest and most structurally diverse family of natural products found in nature, yet their presence in bacteria is underappreciated. The carbon skeletons of terpenoids are generated through carbocation-dependent cyclization cascades catalyzed by terpene synthases (TSs). Type I and type II TSs initiate cyclization via diphosphate ionization and protonation, respectively, and protein structures of both types are known. Most plant diterpene synthases (DTSs) possess three α-helical domains (αβγ), which are thought to have arisen from the fusion of discrete, ancestral bacterial type I TSs (α) and type II TSs (βγ). Type II DTSs of bacterial origin, of which there are no structurally characterized members, are a missing piece in the structural evolution of TSs. Here, we report the first crystal structure of a type II DTS from bacteria. PtmT2 from Streptomyces platensis CB00739 was verified as an ent-copalyl diphosphate synthase involved in the biosynthesis of platensimycin and platencin. The crystal structure of PtmT2 was solved at a resolution of 1.80 Å, and docking studies suggest the catalytically active conformation of geranylgeranyl diphosphate (GGPP). Site-directed mutagenesis confirmed residues involved in binding the diphosphate moiety of GGPP and identified DxxxxE as a potential Mg(2+)-binding motif for type II DTSs of bacterial origin. Finally, both the shape and physicochemical properties of the active sites are responsible for determining specific catalytic outcomes of TSs. The structure of PtmT2 fundamentally advances the knowledge of bacterial TSs, their mechanisms, and their role in the evolution of TSs.

  6. tRNA thiolated pyrimidines as targets for near-ultraviolet-induced synthesis of guanosine tetraphosphate in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, G.; Thiam, K.; Favre, A.

    1981-10-01

    Illumination with near-ultraviolet light triggers synthesis of ppGpp (guanosine 3'-diphosphate 5'-diphosphate) not only in growing Escherichia coli cells containing the putative chromophore 4-thiouridine in their tRNAs but also in nuv/sup -/ cells which lack 4-thiouridine. The burst of ppGpp in nuv/sup -/ cells is, however, induced exclusively by light of wavelenghts shorter than 350 nm. Its maximum level is half that obtained in the parental strain. This ppGpp synthesis is also under the control of the relA gene, indicating that it is due to the accumulation of uncharged tRNAs. A candidate likely to trigger this effect is a 5-methylaminomethyl-2-thiouracil residues present in the first position of the anticodon loop of tRNAsup(Glu), tRNAsup(Lys) and one tRNAsup(Glu) isoacceptor. In conditions in vitro, this base is highly photoreactive at wavelenghts shorter than 350 nm. Furthermore, near-ultraviolet-photomodified tRNAsup(Glu) and tRNAsup(Lys) become poor substrates of their acylation enzyme.

  7. Cloning and functional analysis of the second geranylgeranyl diphosphate synthase gene influencing helvolic acid biosynthesis in Metarhizium anisopliae.

    Science.gov (United States)

    Singkaravanit, Suthitar; Kinoshita, Hiroshi; Ihara, Fumio; Nihira, Takuya

    2010-07-01

    A gene (ggs2) having high similarity to the geranylgeranyl diphosphate synthase (GGPP synthase) gene was cloned from Metarhizium anisopliae NAFF635007. The ggs2 gene (1,239-bp open reading frame with no intron) encoded a protein of 412 amino acids, and the transcription occurred only after late log-phase during the growth. Gene disruption of ggs2, performed to clarify the function in M. anisopliae, resulted in decreased GGPP synthase activity together with a slight delay of sporulation. An high performance liquid chromatography (HPLC) comparison of compound profiles between the wild-type strain and the disruptant revealed that a compound was abolished by the ggs2 disruption. Purification and structural elucidation by 1H-NMR and mass spectrometry analyses revealed that the lost compound is helvolic acid. Furthermore, the pathogenicity assay against two species of insect larvae revealed that the ggs2-disruptant possessed much weaker toxicity than the wild-type strain. Based on these results, it was concluded that ggs2 encodes the GGPP synthase influencing the biosynthesis of secondary metabolites in various species, including helvolic acid in M. anisopliae. To the best of our knowledge, this is the first report to identify a GGPP synthase gene related to secondary metabolism in entomopathogenic fungi.

  8. Synthesis and Crystal Structure of a New Potassium Yttrium Diphosphate Dihydrate KYP2O7·2H2O

    Institute of Scientific and Technical Information of China (English)

    Férid M. Horchani-Naifer K

    2008-01-01

    The dihydrated potassium yttrium diphosphate, KYP2O7·2H2O, has been synthe-sized for the first time. Single-crystal XRD study has allowed the determination of its structure and correct formula. It crystallizes in the monoclinic system, space group P21/c, with the cell parameters a = 7.7069(3), b = 10.5801(4), c = 10.0204(5) ?, β = 93.24(3)°, V = 815.76(6) ?3, Z = 4, Mr = 337.98, F(000) = 656, Dc = 2.752 g/cm3 and μ = 8.073 mm-1. The single-crystal structure was solved from 1856 unique reflections with the final R = 0.0365 and wR = 0.0772, refined with 119 parameters. The atomic arrangement of KYP2O7·2H2O is built by the P2O7 groups, the YO7 and the KO10 polyhedrons which are connected by corner and edge-sharing to form a three-dimensional framework. Water molecules are directly bonded to the Y and K atoms, and they are located in channels running along the a direction.

  9. Vanadium-Binding Ability of Nucleoside Diphosphate Kinase from the Vanadium-Rich Fan Worm, Pseudopotamilla occelata.

    Science.gov (United States)

    Yamaguchi, Nobuo; Yoshinaga, Masafumi; Kamino, Kei; Ueki, Tatsuya

    2016-06-01

    Polychaete fan worms and ascidians accumulate high levels of vanadium ions. Several vanadiumbinding proteins, known as vanabins, have been found in ascidians. However, no vanadium-binding factors have been isolated from the fan worm. In the present study, we sought to identify vanadiumbinding proteins in the branchial crown of the fan worm using immobilized metal ion affinity chromatography. A nucleoside diphosphate kinase (NDK) homolog was isolated and determined to be a vanadium-binding protein. Kinase activity of the NDK homologue, PoNDK, was suppressed by the addition of V(IV), but was unaffected by V(V). The effect of V(IV) on PoNDK precedes its activation by Mg(II). This is the first report to describe the relationship between NDK and V(IV). PoNDK is located in the epidermis of the branchial crown, and its distribution is very similar to that of vanadium. These results suggest that PoNDK is associated with vanadium accumulation and metabolism in P. occelata.

  10. Molecular cloning and functional characterization of Catharanthus roseus hydroxymethylbutenyl 4-diphosphate synthase gene promoter from the methyl erythritol phosphate pathway.

    Science.gov (United States)

    Ginis, Olivia; Courdavault, Vincent; Melin, Céline; Lanoue, Arnaud; Giglioli-Guivarc'h, Nathalie; St-Pierre, Benoit; Courtois, Martine; Oudin, Audrey

    2012-05-01

    The Madagascar periwinkle produces monoterpenoid indole alkaloids (MIA) of high interest due to their therapeutical values. The terpenoid moiety of MIA is derived from the methyl erythritol phosphate (MEP) and seco-iridoid pathways. These pathways are regarded as the limiting branch for MIA biosynthesis in C. roseus cell and tissue cultures. In previous studies, we demonstrated a coordinated regulation at the transcriptional and spatial levels of genes from both pathways. We report here on the isolation of the 5'-flanking region (1,049 bp) of the hydroxymethylbutenyl 4-diphosphate synthase (HDS) gene from the MEP pathway. To investigate promoter transcriptional activities, the HDS promoter was fused to GUS reporter gene. Agrobacterium-mediated transformation of young tobacco leaves revealed that the cloned HDS promoter displays a tissue-specific GUS staining restricted to the vascular region of the leaves and limited to a part of the vein that encompasses the phloem in agreement with the previous localization of HDS transcripts in C. roseus aerial organs. Further functional characterizations in stably or transiently transformed C. roseus cells allowed us to identify the region that can be consider as the minimal promoter and to demonstrate the induction of HDS promoter by several hormonal signals (auxin, cytokinin, methyljasmonate and ethylene) leading to MIA production. These results, and the bioinformatic analysis of the HDS 5'-region, suggest that the HDS promoter harbours a number of cis-elements binding specific transcription factors that would regulate the flux of terpenoid precursors involved in MIA biosynthesis.

  11. Consensus and future directions on the definition of high on-treatment platelet reactivity to adenosine diphosphate.

    Science.gov (United States)

    Bonello, Laurent; Tantry, Udaya S; Marcucci, Rossella; Blindt, Ruediger; Angiolillo, Dominick J; Becker, Richard; Bhatt, Deepak L; Cattaneo, Marco; Collet, Jean Philippe; Cuisset, Thomas; Gachet, Christian; Montalescot, Gilles; Jennings, Lisa K; Kereiakes, Dean; Sibbing, Dirk; Trenk, Dietmar; Van Werkum, Jochem W; Paganelli, Franck; Price, Matthew J; Waksman, Ron; Gurbel, Paul A

    2010-09-14

    The addition of clopidogrel to aspirin treatment reduces ischemic events in a wide range of patients with cardiovascular disease. However, recurrent ischemic event occurrence during dual antiplatelet therapy, including stent thrombosis, remains a major concern. Platelet function measurements during clopidogrel treatment demonstrated a variable and overall modest level of P2Y(12) inhibition. High on-treatment platelet reactivity to adenosine diphosphate (ADP) was observed in selected patients. Multiple studies have now demonstrated a clear association between high on-treatment platelet reactivity to ADP measured by multiple methods and adverse clinical event occurrence. However, the routine measurement of platelet reactivity has not been widely implemented and recommended in the guidelines. Reasons for the latter include: 1) a lack of consensus on the optimal method to quantify high on-treatment platelet reactivity and the cutoff value associated with clinical risk; and 2) limited data to support that alteration of therapy based on platelet function measurements actually improves outcomes. This review provides a consensus opinion on the definition of high on-treatment platelet reactivity to ADP based on various methods reported in the literature and proposes how this measurement may be used in the future care of patients. Copyright © 2010 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

  12. Separation and purification of uridine diphosphate-glucuronosyltransferases by chromatofocusing on a high-performance liquid chromatograph.

    Science.gov (United States)

    Takanashi, H; Homma, H; Matsui, M

    1989-06-01

    A rapid method for the separation and purification of uridine diphosphate-glucuronosyltransferases (GT) was developed with the use of chromatofocusing on a high-performance liquid chromatograph. GT isoenzymes solubilized from hepatic microsomes of Wistar rats were separated on a Mono P column, a pre-packed column for chromatofocusing. Using 4-nitrophenol, testosterone and androsterone as substrates, four fractions with different GT activities were separated in a pH gradient from 9.5 to 7.0. Two isoenzymes, testosterone GT and androsterone GT were purified to apparent homogeneity. They were eluted at pH 8.9 and 8.0 and had subunit molecular weight values of 50000 and 52000, respectively. Approximately 10 mg of solubilized microsomal proteins was applied and the elution was completed within 2 h. Addition of N-nitrodiethylamine, an in vitro activator of GT activity, enhanced the GT activity toward 4-nitrophenol in the three fractions. This chromatographic analysis confirmed the absence of androsterone GT isoenzyme in LA Wistar rats, a mutant strain in terms of androsterone glucuronidation.

  13. Altered expression and activities of enzymes involved in thiamine diphosphate biosynthesis in Saccharomyces cerevisiae under oxidative and osmotic stress.

    Science.gov (United States)

    Kowalska, Ewa; Kujda, Marta; Wolak, Natalia; Kozik, Andrzej

    2012-08-01

    Thiamine diphosphate (TDP) serves as a cofactor for enzymes engaged in pivotal carbohydrate metabolic pathways, which are known to be modulated under stress conditions to ensure the cell survival. Recent reports have proven a protective role of thiamine (vitamin B(1)) in the response of plants to abiotic stress. This work aimed at verifying a hypothesis that also baker's yeast, which can synthesize thiamine de novo similarly to plants and bacteria, adjust thiamine metabolism to adverse environmental conditions. Our analyses on the gene expression and enzymatic activity levels generally showed an increased production of thiamine biosynthesis enzymes (THI4 and THI6/THI6), a TDP synthesizing enzyme (THI80/THI80) and a TDP-requiring enzyme, transketolase (TKL1/TKL) by yeast subjected to oxidative (1 mM hydrogen peroxide) and osmotic (1 M sorbitol) stress. However, these effects differed in magnitude, depending on yeast growth phase and presence of thiamine in growth medium. A mutant thi4Δ with increased sensitivity to oxidative stress exhibited enhanced TDP biosynthesis as compared with the wild-type strain. Similar tendencies were observed in mutants yap1Δ and hog1Δ defective in the signaling pathways of the defense against oxidative and osmotic stress, respectively, suggesting that thiamine metabolism can partly compensate damages of yeast general defense systems.

  14. Progress in the experimental observation of thiamin diphosphate-bound intermediates on enzymes and mechanistic information derived from these observations.

    Science.gov (United States)

    Jordan, Frank; Nemeria, Natalia S

    2014-12-01

    Thiamin diphosphate (ThDP), the vitamin B1 coenzyme is an excellent representative of coenzymes, which carry out electrophilic catalysis by forming a covalent complex with their substrates. The function of ThDP is to greatly increase the acidity of two carbon acids by stabilizing their conjugate bases, the ylide/carbene/C2-carbanion of the thiazolium ring and the C2α-carbanion/enamine, once the substrate binds to ThDP. In recent years, several ThDP-bound intermediates on such pathways have been characterized by both solution and solid-state methods. Prominent among these advances are X-ray crystallographic results identifying both oxidative and non-oxidative intermediates, rapid chemical quench followed by NMR detection of several intermediates which are stable under acidic conditions, solid-state NMR and circular dichroism detection of the states of ionization and tautomerization of the 4'-aminopyrimidine moiety of ThDP in some of the intermediates. These methods also enabled in some cases determination of the rate-limiting step in the complex series of steps. This review is an update of a review with the same title published by the authors in 2005 in this Journal. Much progress has been made in the intervening decade in the identification of the intermediates and their application to gain additional mechanistic insight.

  15. Simultaneous liquid chromatographic assessment of thiamine, thiamine monophosphate and thiamine diphosphate in human erythrocytes: a study on alcoholics.

    Science.gov (United States)

    Mancinelli, Rosanna; Ceccanti, Mauro; Guiducci, Maria Soccorsa; Sasso, Guido Francesco; Sebastiani, Gemma; Attilia, Maria Luisa; Allen, John Paul

    2003-06-15

    An isocratic HPLC procedure for the assessment of thiamine (T), thiamine monophosphate (TMP) and thiamine diphosphate (TDP) in human erythrocytes is described. Several aspects of the procedure make it suitable for both clinical and research purposes: limits of detection and quantification of 1 and 2.5 nmol/l, respectively, recovery of 102% on average (range 93-112%), intra- and inter-day precisions within 5 and 9%, respectively, total elution time 15 min. This analytical methodology was applied to a case-control study on erythrocyte samples from 103 healthy subjects and 36 alcohol-dependent patients at risk of thiamine deficiency. Mean control values obtained were: T=89.6+/-22.7 nmol/l, TMP=4.4+/-6.6 nmol/l and TDP=222.23+/-56.3 nmol/l. T and TDP mean values of alcoholics were significantly lower than those of control cases: T=69.4+/-35.9 nmol/l (Pthiamine was established in the study of alcohol related problems.

  16. Cooperation of divalent ions and thiamin diphosphate in regulation of the function of pig heart pyruvate dehydrogenase complex.

    Science.gov (United States)

    Czerniecki, J; Czygier, M

    2001-12-01

    The role of Mg2+, Ca2+, and Mn2+ in regulation of purified pig heart pyruvate dehydrogenase complex (PDC) containing endogenous thiamin diphosphate (TDP) was studied. It was found that the effects of the cations depended on the presence of exogenous TDP. In the absence of added TDP, the divalent cations led to a shortening of a lag phase of the PDC reaction and a strong reduction of the Km value for pyruvate. The relative efficiency of the three types of ions are presented as follows: Mn2+>Ca2+>Mg2+. The other sources claim that in the presence of exogenous TDP, which alone strongly increased the affinity of PDC for pyruvate, any significant additional effects of the cations were not observed. However, Mg2+, Ca2+, and Mn2+ decreased the Km value for CoA in both cases, the absence and presence of exogenous TDP, in approximately a similar extent (about twofold). The affinity of PDC for NAD+ seems to be not sensitive to the presence of the divalent cations. The data obtained suggest that Mg2+, Ca2+, and Mn2+ can cooperate with TDP as positive regulatory effectors of pig heart PDC on the level of pyruvate dehydrogenase and lipoamide acetyltransferase components of the complex.

  17. Preparation and in vitro evaluation of liposomal chloroquine diphosphate loaded by a transmembrane pH-gradient method.

    Science.gov (United States)

    Qiu, Liyan; Jing, Na; Jin, Yi

    2008-09-01

    This study developed an active loading method for encapsulating chloroquine diphosphate (CQ) into liposomes. The effects of different formulation factors on the encapsulation efficiency (EE) and the size of CQ liposomes were investigated. These factors included the internal phase of liposomes, the external phase of liposomes, the ratio of drug to soybean phosphatidylcholine (drug/SPC), the ratio of cholesterol to soybean phosphatidylcholine (Chol/SPC), and the incubation temperature and time. The EE (93%) was obtained when using drug/SPC (1:50 mass ratio), SPC/Chol (1:5 mass ratio) at 0.10 M citrate-sodium citrate buffer (pH 3.6). As 5 mol% methoxypoly(ethylene glycol)(2,000) cholesteryl succinate (CHS-PEG(2000)) or distearoyl phosphatidylethanolamine-poly (ethylene glycol)(2,000) (DSPE-PEG(2000)) was added, the size of particle was reduced and the EE was improved. Freeze-drying with 5% trehalose as a cryoprotectant was carried out to achieve long-term stability. The drug release studies were performed in vitro simulating the desired application conditions, such as physiological fluids (pH 7.4), tumor tissues (pH 6.5) and endosomal compartments (pH 5.5). The release of CQ from the liposomes prepared via remote loading showed the significant pH-sensitivity and retention properties, which favored the application of liposomal CQ at tumor tissues and endosomal compartments.

  18. Bioinformatic and molecular analysis of hydroxymethylbutenyl diphosphate synthase (GCPE) gene expression during carotenoid accumulation in ripening tomato fruit.

    Science.gov (United States)

    Rodríguez-Concepción, Manuel; Querol, Jordi; Lois, Luisa María; Imperial, Santiago; Boronat, Albert

    2003-07-01

    Carotenoids are plastidic isoprenoid pigments of great biological and biotechnological interest. The precursors for carotenoid production are synthesized through the recently elucidated methylerythritol phosphate (MEP) pathway. Here we have identified a tomato ( Lycopersicon esculentum Mill.) cDNA sequence encoding a full-length protein with homology to the MEP pathway enzyme hydroxymethylbutenyl 4-diphosphate synthase (HDS, also called GCPE). Comparison with other plant and bacterial HDS sequences showed that the plant enzymes contain a plastid-targeting N-terminal sequence and two highly conserved plant-specific domains in the mature protein with no homology to any other sequence in the databases. The ubiquitous distribution of HDS-encoding expressed sequence tags (ESTs) in the tomato collections suggests that the corresponding gene is likely expressed throughout the plant. The role of HDS in controlling the supply of precursors for carotenoid biosynthesis was estimated from the bioinformatic and molecular analysis of transcript abundance in different stages of fruit development. No significant changes in HDS gene expression were deduced from the statistical analysis of EST distribution during fruit ripening, when an active MEP pathway is required to support a massive accumulation of carotenoids. RNA blot experiments confirmed that similar transcript levels were present in both the wild-type and carotenoid-depleted yellow ripe ( r) mutant fruit independent of the stage of development and the carotenoid composition of the fruit. Together, our results are consistent with a non-limiting role for HDS in carotenoid biosynthesis during tomato fruit ripening.

  19. Effects of cumene hydroperoxide on adenosine diphosphate ribosyl transferase in mononuclear leukocytes of patients with adenomatous polyps in the colon.

    Science.gov (United States)

    Markowitz, M M; Johnson, D B; Pero, R W; Winawer, S J; Miller, D G

    1988-03-01

    We have studied the effects of plasma and of cumene hydroperoxide (CUM) on adenosine diphosphate ribosyl transferase (ADPRT) from mononuclear leukocytes (HML) of patients with colonic adenomatous polyps (n = 22), with colonic hyperplastic polyps (n = 5) and with neither type of polyp (controls) (n = 6). ADPRT was measured after incubation of HML with plasma alone (termed the plasma value), and with plasma plus CUM (50 microM) (the activated value); the difference elicited by CUM was termed the induced value. There was no significant difference in values between the control and hyperplastic polyp groups: these were combined for further analysis. The plasma (P = 0.038), activated (P = 0.009) and induced (P = 0.0024) values of the combined group all differed significantly from those of the adenoma group. At low exposures, CUM stimulated both ADPRT and unscheduled DNA synthesis and, at higher exposures, inactivated both. Pretreatment of HML with vitamin E protected against these effects of CUM, while pretreatment with diamide (which depletes GSH) accentuated the effects. This study demonstrates a differential reaction of ADPRT in patients harboring colonic adenomas and suggests that the origin of this difference may lie in cellular responses to oxidative stress.

  20. Changes in enzymic activities of nucleoside diphosphate sugar interconversions during differentiation of cambium to xylem in sycamore and poplar.

    Science.gov (United States)

    Dalessandro, G; Northcote, D H

    1977-02-15

    During the transition from primary wall formation to secondary thickening there is a marked shift in the synthesis of pectin, hemicellulose and cellulose. The activities of the enzymes [UDP-D-galactose 4-epimerase (EC 5.1.3.2)8 UDP-l-arabinose 4-epimerase (EC 5.1.3.5), UDP-D-glucose dehydrogenase (EC 1.1.1.22) and UDP-D--glucuronate decarboxylase (EC 4.1.1.35)] were measured in cambial cells, differentiating xylem cells and differentiated xylem cells isolated from sycamore and poplar trees, and phloem cells from poplar. At the final stage of the differentiation of cambium to xylem there was a decrease in activity of the enzymes directly involved in producing the soluble precursors of pectin (DUP-D-galactose 4-epimerase and UDP-L-arabinose 4-epimerase and an increase in those producing the precursors of hemicellulose (UDP-D-glucose dehydrogenase and UDP-D-glucuronate decarboxylase). These results strongly suggest ahat the changes were correlated with the differences observed in the chemical composition of the wall during development. The changes found in the catalytic activity of the enzymes of nucleoside diphosphate sugar interconversion exert a coarse control over the synthesis of pectin and hemicelluloses. The tissues at all stages of development contained the necessary enzyme activities to produce all the precursors of pectin and hemicellulose, even at the final stage of differentiation when no pectin was formed.

  1. Global warming, plant paraquat resistance, and light signal transduction through nucleoside diphosphate kinase as a paradigm for increasing food supply.

    Science.gov (United States)

    Hasunuma, Kohji; Yoshida, Yusuke; Haque, Mohamed Emdadul; Wang, Ni-yan; Fukamatsu, Yosuke; Miyoshi, Osamu; Lee, Bumkyu

    2011-10-01

    Light signal transduction was studied in extracts of mycelia of the fungus Neurospora crassa, and the third internodes of dark-grown Pisum sativum cv Alaska. Both processes increased the phosphorylation of nucleoside diphosphate kinase (NDPK). NDPK may function as a carrier of reduction equivalents, as it binds NADH, thereby providing electrons to transform singlet oxygen to superoxide by catalases (CAT). As the C-termini of NDPK interact with CAT which receive singlet oxygen, emitted from photoreceptors post light perception (which is transmitted to ambient triplet oxygen), we hypothesize that this may increase phospho-NDPK. Singlet oxygen, emitted from the photoreceptor, also reacts with unsaturated fatty acids in membranes thereby forming malonedialdehyde, which in turn could release ions from, e.g., the thylacoid membrane thereby reducing the rate of photosynthesis. A mutant of Alaska pea, which exhibited two mutations in chloroplast NDPK-2 and one mutation in mitochondrial localized NDPK-3, was resistant to reactive oxygen species including singlet oxygen and showed an increase in the production of carotenoids, anthocyanine, and thereby could reduce the concentration of singlet oxygen. The reduction of the concentration of singlet oxygen is predicted to increase the yield of crop plants, such as Alaska pea, soybean, rice, wheat, barley, and sugarcane. This approach to increase the yield of crop plants may contribute not only to enhance food supply, but also to reduce the concentration of CO(2) in the atmosphere.

  2. Arabidopsis GERANYLGERANYL DIPHOSPHATE SYNTHASE 11 is a hub isozyme required for the production of most photosynthesis-related isoprenoids.

    Science.gov (United States)

    Ruiz-Sola, M Águila; Coman, Diana; Beck, Gilles; Barja, M Victoria; Colinas, Maite; Graf, Alexander; Welsch, Ralf; Rütimann, Philipp; Bühlmann, Peter; Bigler, Laurent; Gruissem, Wilhelm; Rodríguez-Concepción, Manuel; Vranová, Eva

    2016-01-01

    Most plastid isoprenoids, including photosynthesis-related metabolites such as carotenoids and the side chain of chlorophylls, tocopherols (vitamin E), phylloquinones (vitamin K), and plastoquinones, derive from geranylgeranyl diphosphate (GGPP) synthesized by GGPP synthase (GGPPS) enzymes. Seven out of 10 functional GGPPS isozymes in Arabidopsis thaliana reside in plastids. We aimed to address the function of different GGPPS paralogues for plastid isoprenoid biosynthesis. We constructed a gene co-expression network (GCN) using GGPPS paralogues as guide genes and genes from the upstream and downstream pathways as query genes. Furthermore, knock-out and/or knock-down ggpps mutants were generated and their growth and metabolic phenotypes were analyzed. Also, interacting protein partners of GGPPS11 were searched for. Our data showed that GGPPS11, encoding the only plastid isozyme essential for plant development, functions as a hub gene among GGPPS paralogues and is required for the production of all major groups of plastid isoprenoids. Furthermore, we showed that the GGPPS11 protein physically interacts with enzymes that use GGPP for the production of carotenoids, chlorophylls, tocopherols, phylloquinone, and plastoquinone. GGPPS11 is a hub isozyme required for the production of most photosynthesis-related isoprenoids. Both gene co-expression and protein-protein interaction likely contribute to the channeling of GGPP by GGPPS11. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  3. PART I. ESCHERICHIA COLI

    Directory of Open Access Journals (Sweden)

    Sanaa Mahdi Oraibi

    2016-11-01

    Full Text Available The presence of Escherichia coli in the air of facilities involved in management and composting of post-slaughter poultry wastes in selected plants of West Western Pomerania region was studied. Measurements were made on four dates in a variety of weather conditions during the year. The study was conducted at 5 objects that differ in the type of waste and the degree of preparation for composting. These were: chemical treatment and preliminary processing plant, liquid wastes reservoir, platform for preparation of materials for composting, storage of biological sediments, and composting facility. Measurement of bacteria count was carried out in accordance with the applicable procedures on selective chromogenic TBX medium. The assays revealed the presence of E. coli at all test objects, but not always on all measurement dates. It has been shown that the presence of E. coli was from 20 to 3047 CFU∙m-3 of air, although the largest quantities were most frequently detected in the air of the building for post-slaughter waste pre-treatment in chemical treatment plant.

  4. Design and optimization of N-acylhydrazone pyrimidine derivatives as E. coli PDHc E1 inhibitors: Structure-activity relationship analysis, biological evaluation and molecular docking study.

    Science.gov (United States)

    He, Haifeng; Xia, Hongying; Xia, Qin; Ren, Yanliang; He, Hongwu

    2017-08-23

    By targeting the thiamin diphosphate (ThDP) binding site of Escherichia coli (E. coli) pyruvate dehydrogenase multienzyme complex E1 (PDHc E1), a series of novel 'open-chain' classes of ThDP analogs A, B, and C with N-acylhydrazone moieties was designed and synthesized to explore their activities against E. coli PHDc E1 in vitro and their inhibitory activity against microbial diseases were further evaluated in vivo. As a result, A1-23 exhibited moderate to potent inhibitory activities against E. coli PDHc E1 (IC50=0.15-23.55μM). The potent inhibitors A13, A14, A15, C2, had strong inhibitory activities with IC50 values of 0.60, 0.15, 0.39 and 0.34μM against E. coli PDHc E1 and with good enzyme-selective inhibition between microorganisms and mammals. Especially, the most powerful inhibitor A14 could 99.37% control Xanthimonas oryzae pv. Oryzae. Furthermore, the binding features of compound A14 within E. coli PDHc E1 were investigated to provide useful insights for the further construction of new inhibitor by molecular docking, site-directed mutagenesis, and enzymatic assays. The results indicated that A14 had most powerful inhibition against E. coli PDHc E1 due to the establishment of stronger interaction with Glu571, Met194, Glu522, Leu264 and Phe602 at active site of E.coli PDHc E1. It could be used as a lead compound for further optimization, and may have potential as a new microbicide. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. The thorium phosphate diphosphate as matrix for radioactive waste conditioning: radionuclide immobilization and behavior under irradiation; Le phosphate diphosphate de thorium, matrice pour le conditionnement des dechets radioactifs: immobilisation de radionucleides, comportement sous irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Pichot, Erwan [Inst. de Physique Nucleaire, Paris-11 Univ., 91 - Orsay (France)

    1999-04-13

    The aim of this work was to perform successively the decontamination of liquid solutions and the final immobilization of radionuclide storage using the same matrix. For this, thorium phosphate-diphosphate (TPD) of the formula Th{sub 4}P{sub 6}O{sub 23}, is proposed as a very resistant to water corrosion matrix. A new compound, thorium phosphate hydrogeno-phosphate (TPHP) of the formula Th{sub 2}(PO{sub 4}){sub 2}(HPO{sub 4}), nH{sub 2}O with n=3-7 was synthesized and characterized. Heated at 1100 deg.C it is transformed into the TDP. Ion exchange properties of TPHP were investigated. The exchange yields of imponderable caesium, strontium and americium ion onto TPHP (NaNO{sub 3} 0.1 M media at pH=6) are equal to 60% for the first one and 100% for the two others. The results interpreted in terms of ion-exchange led to determine selectivity coefficient values for each cation and suggested that only hydrated ions are exchanged. While the TPD is proposed for the high level nuclear waste storage, the irradiation effects, particularly structural modifications were studied using both {gamma} irradiation and charged particle irradiation. ESR and TL methods were carried out in order to identify radicals created during gamma radiation exposure. Correlation between ESR and TL experiments performed at room temperature clearly show three of PO{sub 3}{sup 2-} species and one POO{center_dot} species of free radicals. We have shown that Au-ion irradiation in the range of MeV energy involved TPD structure and chemical modifications. Important sputtering was interpreted in terms of local thermal chemical decomposition. We have shown, at room temperature, that the amorphization dose for heavy ion irradiation is between 0.1 to 0.4 dpa. (author) 146 refs., 46 figs., 21 tabs.

  6. Study of the irradiation effects on thorium phosphate diphosphate ({beta}-TPD): consequences on its chemical durability; Etude des effets d'irradiation sur le phosphate diphosphate de thorium ({beta}-PDT): consequences sur la durabilite chimique

    Energy Technology Data Exchange (ETDEWEB)

    Tamain, C

    2005-12-15

    Since Thorium Phosphate Diphosphate (beta-TPD) can be considered as a potential host matrix for long-term storage in underground repository, it is necessary to study the irradiation effects on the structure of this ceramics and the consequences on its chemical durability. Sintered samples of beta-TPD and of associated solid solutions of beta-TUPD were irradiated under ion beams and then altered in aqueous solutions. Depending on the electronic LET value, beta-TPD can be completely or partly amorphized. Furthermore, the ability of recrystallization of the amorphous material by thermal annealing was also demonstrated. Some leaching tests, realized on these irradiated samples, have shown a significant effect of the amorphous fraction on the normalized dissolution rate which was increased by a factor of 10 from the crystallized to the fully amorphized material. Correlatively, the amorphous fraction also modified the delay to reach the saturation conditions associated to the thermodynamic equilibria involved. On the other hand, it exhibited no influence neither on other kinetic parameters, such as activation energy of the dissolution process or partial order related to the proton concentration, nor on the nature of the neo-formed phase formed at the saturation of the leachate and identified as Thorium Phosphate Hydrogeno-Phosphate Hydrate (TPHPH). Beta-TUPD samples were also irradiated by gamma and alpha rays during leaching tests to study the effects of radiolysis in the leaching medium on the normalized leaching rate. It appeared that the radiolytic species occurring in the dissolution mechanism were unstable, disappearing quickly when stopping the irradiation. (author)

  7. The molecular origin of the thiamin diphosphate-induced spectral bands of ThDP-dependent enzymes

    NARCIS (Netherlands)

    Kovina, M.V.; Kok, A.; Sevostyanova, I.A.; Khailova, L.S.; Belkina, N.V.; Kochetov, G.A.

    2004-01-01

    New and previously published data on a variety of ThDP-dependent enzymes such as baker's yeast transketolase, yeast pyruvate decarboxylase and pyruvate dehydrogenase from pigeon breast muscle, bovine heart, bovine kidney, Neisseria meningitidis and E. coli show their spectral sensitivity to ThDP bin

  8. Modulation of guanosine nucleotides biosynthetic pathways enhanced GDP-L-fucose production in recombinant Escherichia coli.

    Science.gov (United States)

    Lee, Won-Heong; Shin, So-Yeon; Kim, Myoung-Dong; Han, Nam Soo; Seo, Jin-Ho

    2012-03-01

    Guanosine 5'-triphosphate (GTP) is the key substrate for biosynthesis of guanosine 5'-diphosphate (GDP)-L-fucose. In this study, improvement of GDP-L-fucose production was attempted by manipulating the biosynthetic pathway for guanosine nucleotides in recombinant Escherichia coli-producing GDP-L-fucose. The effects of overexpression of inosine 5'-monophosphate (IMP) dehydrogenase, guanosine 5'-monophosphate (GMP) synthetase (GuaB and GuaA), GMP reductase (GuaC) and guanosine-inosine kinase (Gsk) on GDP-L-fucose production were investigated in a series of fed-batch fermentations. Among the enzymes tested, overexpression of Gsk led to a significant improvement of GDP-L-fucose production. Maximum GDP-L-fucose concentration of 305.5 ± 5.3 mg l(-1) was obtained in the pH-stat fed-batch fermentation of recombinant E. coli-overexpressing Gsk, which corresponds to a 58% enhancement in the GDP-L-fucose production compared with the control strain overexpressing GDP-L-fucose biosynthetic enzymes. Such an enhancement of GDP-L-fucose production could be due to the increase in the intracellular level of GMP.

  9. Hemodynamic effects of fructose 1,6-diphosphate in patients with normal and impaired left ventricular function.

    Science.gov (United States)

    Markov, A K; Brumley, M A; Figueroa, A; Skelton, T N; Lehan, P H

    1997-05-01

    We compared the short-term hemodynamic effects of intravenous fructose 1,6-diphosphate (FDP) administration in patients with coronary artery disease. Hemodynamic measurements were performed before and after administration of FDP in two groups of patients: those with impaired left ventricular (LV) function, elevated LV end-diastolic pressures (LVEDP > or =12 mm Hg, n = 30), and those with normal LV function (LVEDP <12 mm Hg, n = 17). In those with impaired LV function, FDP induced a decrease in LVEDP from 22 +/- 1.31 to 16.73 +/- 1.46 mm Hg (p< 0.0001). The cardiac index increased (2.50 +/- 0.11 to 2.81 +/- 0.13 L/m2 [p < 0.0001]), as did the LV stroke work index (31.7 +/- 2.04 to 40.3 +/- 2.67 gm x m x m2 [p < 0.0001]). FDP induced no significant change in heart rate and mean aortic pressure. Pulmonary pressure and resistance declined (p<0.002 and p< 0.0001, respectively). Systemic vascular resistance decreased because of increased cardiac output and unchanged arterial pressure (p < 0.001). In those patients with normal baseline LVEDP (5.06 +/- 0.27 mm Hg), FDP decreased heart rate (p< 0.0001) and systemic and pulmonary resistance (p < 0.03 and p < 0.004, respectively), whereas LVEDP and mean aortic and pulmonary pressures remained unchanged. FDP moderately increased cardiac output (p < 0.05), stroke volume index, and LV stroke work index (p< 0.002 and p< 0.003, respectively). The observed improvement in LV function in those patients with elevated LV filling pressures is thought to be a result of an increased energy production by the Embden-Meyerhoff pathway and to act as a positive inotrope.

  10. Consensus and update on the definition of on-treatment platelet reactivity to adenosine diphosphate associated with ischemia and bleeding.

    Science.gov (United States)

    Tantry, Udaya S; Bonello, Laurent; Aradi, Daniel; Price, Matthew J; Jeong, Young-Hoon; Angiolillo, Dominick J; Stone, Gregg W; Curzen, Nick; Geisler, Tobias; Ten Berg, Jurrien; Kirtane, Ajay; Siller-Matula, Jolanta; Mahla, Elisabeth; Becker, Richard C; Bhatt, Deepak L; Waksman, Ron; Rao, Sunil V; Alexopoulos, Dimitrios; Marcucci, Rossella; Reny, Jean-Luc; Trenk, Dietmar; Sibbing, Dirk; Gurbel, Paul A

    2013-12-17

    Dual antiplatelet therapy with aspirin and a P2Y12 receptor blocker is a key strategy to reduce platelet reactivity and to prevent thrombotic events in patients treated with percutaneous coronary intervention. In an earlier consensus document, we proposed cutoff values for high on-treatment platelet reactivity to adenosine diphosphate (ADP) associated with post-percutaneous coronary intervention ischemic events for various platelet function tests (PFTs). Updated American and European practice guidelines have issued a Class IIb recommendation for PFT to facilitate the choice of P2Y12 receptor inhibitor in selected high-risk patients treated with percutaneous coronary intervention, although routine testing is not recommended (Class III). Accumulated data from large studies underscore the importance of high on-treatment platelet reactivity to ADP as a prognostic risk factor. Recent prospective randomized trials of PFT did not demonstrate clinical benefit, thus questioning whether treatment modification based on the results of current PFT platforms can actually influence outcomes. However, there are major limitations associated with these randomized trials. In addition, recent data suggest that low on-treatment platelet reactivity to ADP is associated with a higher risk of bleeding. Therefore, a therapeutic window concept has been proposed for P2Y12 inhibitor therapy. In this updated consensus document, we review the available evidence addressing the relation of platelet reactivity to thrombotic and bleeding events. In addition, we propose cutoff values for high and low on-treatment platelet reactivity to ADP that might be used in future investigations of personalized antiplatelet therapy.

  11. Mitochondria from cultured cells derived from normal and thiamine-responsive megaloblastic anemia individuals efficiently import thiamine diphosphate

    Directory of Open Access Journals (Sweden)

    Singleton Charles K

    2002-04-01

    Full Text Available Abstract Background Thiamine diphosphate (ThDP is the active form of thiamine, and it serves as a cofactor for several enzymes, both cytosolic and mitochondrial. Isolated mitochondria have been shown to take up thiamine yet thiamine diphosphokinase is cytosolic and not present in mitochondria. Previous reports indicate that ThDP can also be taken up by rat mitochondria, but the kinetic constants associated with such uptake seemed not to be physiologically relevant. Results Here we examine ThDP uptake by mitochondria from several human cell types, including cells from patients with thiamine-responsive megaloblastic anemia (TRMA that lack a functional thiamine transporter of the plasma membrane. Although mitochondria from normal lymphoblasts took up thiamine in the low micromolar range, surprisingly mitochondria from TRMA lymphoblasts lacked this uptake component. ThDP was taken up efficiently by mitochondria isolated from either normal or TRMA lymphoblasts. Uptake was saturable and biphasic with a high affinity component characterized by a Km of 0.4 to 0.6 μM. Mitochondria from other cell types possessed a similar high affinity uptake component with variation seen in uptake capacity as revealed by differences in Vmax values. Conclusions The results suggest a shared thiamine transporter for mitochondria and the plasma membrane. Additionally, a high affinity component of ThDP uptake by mitochondria was identified with the apparent affinity constant less than the estimates of the cytosolic concentration of free ThDP. This finding indicates that the high affinity uptake is physiologically significant and may represent the main mechanism for supplying phosphorylated thiamine for mitochondrial enzymes.

  12. Thiamine diphosphate in whole blood, thiamine and thiamine monophosphate in breast-milk in a refugee population.

    Directory of Open Access Journals (Sweden)

    Wolfgang Stuetz

    Full Text Available BACKGROUND: The provision of high doses of thiamine may prevent thiamine deficiency in the post-partum period of displaced persons. METHODOLOGY/PRINCIPAL FINDINGS: The study aimed to evaluate a supplementation regimen of thiamine mononitrate (100 mg daily at the antenatal clinics in Maela refugee camp. Women were enrolled during antenatal care and followed after delivery. Samples were collected at 12 weeks post partum. Thiamine diphosphate (TDP in whole blood and thiamine in breast-milk of 636 lactating women were measured. Thiamine in breast-milk consisted of thiamine monophosphate (TMP in addition to thiamine, with a mean TMP to total thiamine ratio of 63%. Mean whole blood TDP (130 nmol/L and total thiamine in breast-milk (755 nmol/L were within the upper range reported for well-nourished women. The prevalence of women with low whole blood TDP (<65 nmol/L was 5% and with deficient breast-milk total thiamine (<300 nmol/L was 4%. Whole blood TDP predicted both breast-milk thiamine and TMP (R(2 = 0.36 and 0.10, p<0.001. A ratio of TMP to total thiamine ≥63% was associated with a 7.5 and 4-fold higher risk of low whole blood TDP and deficient total breast-milk thiamine, respectively. Routine provision of daily 100 mg of thiamine mononitrate post-partum compared to the previous weekly 10 mg of thiamine hydrochloride resulted in significantly higher total thiamine in breast-milk. CONCLUSIONS/SIGNIFICANCE: Thiamine supplementation for lactating women in Maela refugee camp is effective and should be continued. TMP and its ratio to total thiamine in breast-milk, reported for the first time in this study, provided useful information on thiamine status and should be included in future studies of breast-milk thiamine.

  13. Conformational basis for substrate recognition and regulation of catalytic activity in Staphylococcus aureus nucleoside di-phosphate kinase.

    Science.gov (United States)

    Srivastava, Sandeep Kumar; Rajasree, Kalagiri; Gopal, B

    2011-10-01

    Nucleoside diphosphate kinases (NDK) are characterized by high catalytic turnover rates and diverse substrate specificity. These features make this enzyme an effective activator of a pro-drug-an application that has been actively pursued for a variety of therapeutic strategies. The catalytic mechanism of this enzyme is governed by a conserved histidine that coordinates a magnesium ion at the active site. Despite substantial structural and biochemical information on NDK, the mechanistic feature of the phospho-transfer that leads to auto-phosphorylation remains unclear. While the role of the histidine residue is well documented, the other active site residues, in particular the conserved serine remains poorly characterized. Studies on some homologues suggest no role for the serine residue at the active site, while others suggest a crucial role for this serine in the regulation and quaternary association of this enzyme in some species. Here we report the biochemical features of the Staphylococcus aureus NDK and the mutant enzymes. We also describe the crystal structures of the apo-NDK, as a transition state mimic with vanadate and in complex with different nucleotide substrates. These structures formed the basis for molecular dynamics simulations to understand the broad substrate specificity of this enzyme and the role of active site residues in the phospho-transfer mechanism and oligomerization. Put together, these data suggest that concerted changes in the conformation of specific residues facilitate the stabilization of nucleotide complexes thereby enabling the steps involved in the ping-pong reaction mechanism without large changes to the overall structure of this enzyme.

  14. Prognostic and clinicopathological value of poly (adenosine diphosphate-ribose) polymerase expression in breast cancer: A meta-analysis

    Science.gov (United States)

    Qiao, Weiqiang; Pan, Linlin; Kou, Changgui; Li, Ke; Yang, Ming

    2017-01-01

    Background Previous studies have shown that the poly (adenosine diphosphate-ribose) polymerase (PARP) level is a promising indicator of breast cancer. However, its prognostic value remains controversial. The present meta-analysis evaluated the prognostic value of PARP expression in breast cancer. Materials and methods Eligible studies were retrieved from the PubMed, Web of Science, Embase, and Cochrane Library databases through July 20, 2016. Studies investigating PARP expression as well as reporting survival data in breast cancer were included. Two independent reviewers carried out all literature searches. The pooled relative risk (RR) and hazard ratio (HR) with 95% confidence interval (95% CI) were applied to assess the association between PARP expression and the clinicopathological features and survival outcome in breast cancer. Results A total of 3506 patients from eight eligible studies were included. We found that higher PARP expression indicated a worse clinical outcome in early stage breast cancer, with a HR of 3.08 (95% CI, 1.14–8.29, P = 0.03) for disease-free survival and a HR of 1.82 (95% CI, 1.20–2.76; P = 0.005) for overall survival. Moreover, increased PARP expression was significantly associated with higher nuclear grade (RR, 1.51; 95% CI, 1.12–2.04; P = 0.008) in breast cancer. A similar correlation was detected in triple-negative breast cancer (TNBC; RR, 1.81; 95% CI, 1.04–3.17; P = 0.04). Conclusions Our findings indicated that elevated PARP expression correlated with worse prognosis in early stage breast cancer. Furthermore, high PARP expression was associated with higher nuclear grade and TNBC. PMID:28212434

  15. Mitochondria from cultured cells derived from normal and thiamine-responsive megaloblastic anemia individuals efficiently import thiamine diphosphate

    Science.gov (United States)

    Song, Qilin; Singleton, Charles K

    2002-01-01

    Background Thiamine diphosphate (ThDP) is the active form of thiamine, and it serves as a cofactor for several enzymes, both cytosolic and mitochondrial. Isolated mitochondria have been shown to take up thiamine yet thiamine diphosphokinase is cytosolic and not present in mitochondria. Previous reports indicate that ThDP can also be taken up by rat mitochondria, but the kinetic constants associated with such uptake seemed not to be physiologically relevant. Results Here we examine ThDP uptake by mitochondria from several human cell types, including cells from patients with thiamine-responsive megaloblastic anemia (TRMA) that lack a functional thiamine transporter of the plasma membrane. Although mitochondria from normal lymphoblasts took up thiamine in the low micromolar range, surprisingly mitochondria from TRMA lymphoblasts lacked this uptake component. ThDP was taken up efficiently by mitochondria isolated from either normal or TRMA lymphoblasts. Uptake was saturable and biphasic with a high affinity component characterized by a Km of 0.4 to 0.6 μM. Mitochondria from other cell types possessed a similar high affinity uptake component with variation seen in uptake capacity as revealed by differences in Vmax values. Conclusions The results suggest a shared thiamine transporter for mitochondria and the plasma membrane. Additionally, a high affinity component of ThDP uptake by mitochondria was identified with the apparent affinity constant less than the estimates of the cytosolic concentration of free ThDP. This finding indicates that the high affinity uptake is physiologically significant and may represent the main mechanism for supplying phosphorylated thiamine for mitochondrial enzymes. PMID:12014993

  16. Inhibition by cromoglycate and some flavonoids of nucleoside diphosphate kinase and of exocytosis from permeabilized mast cells.

    Science.gov (United States)

    Martin, M. W.; O'Sullivan, A. J.; Gomperts, B. D.

    1995-01-01

    1. The anti-allergic compound, cromoglycate, is reported to possess affinity for, and to suppress the autophosphorylation of a 72kDa protein having the sequence of nucleoside diphosphate kinase (NDPK). 2. We have tested the ability of cromoglycate, and a panel of ten structurally related flavonoids of plant origin, to inhibit the NDPK reaction and the exocytotic process of permeabilized mast cells. The conditions of permeabilization (use of an isotonic medium based on sodium glutamate) were selected to ensure that NDPK activity would be an essential component in the induction of Ca(2+)-induced exocytosis in which ATP is required for generation of GTP. For comparison, we also measured the inhibition of exocytosis induced by GTP-gamma-S; this proceeds in the absence of ATP and bypasses the need for NDPK activity. 3. We found that cromoglycate does not discriminate between Ca2+ and GTP-gamma-S-induced exocytosis and is a poor inhibitor of NDPK activity. Concentrations in the millimolar range are required for inhibition of all these functions. By comparison, many of the flavonoids are effective at concentrations in the micromolar range. 4. While we were unable to discern any systematic relationships between their ability to inhibit the three functions, two compounds, quercetin and genistein, inhibit Ca(2+)-induced, but not GTP-gamma-S-induced exocytosis. Inhibition of the late stages of the stimulus-response pathway in mast cells by these compounds is therefore likely to be due to inhibition of NDPK and the consequent failure to generate GTP. PMID:7582506

  17. A type-B response regulator drives the expression of the hydroxymethylbutenyl diphosphate synthase gene in periwinkle.

    Science.gov (United States)

    Ginis, Olivia; Oudin, Audrey; Guirimand, Grégory; Chebbi, Mouadh; Courdavault, Vincent; Glévarec, Gaëlle; Papon, Nicolas; Crèche, Joel; Courtois, Martine

    2012-10-15

    In plant cytokinin (CK) signaling, type-B response regulators (RRs) act as major players in orchestrating the transcriptome changes in response to CK. However, their direct targets are poorly known. The identification of putative type-ARR1 motifs located within the promoter of the CK-responsive hydroxyl methyl butenyl diphosphate synthase (HDS) gene from the methyl erythritol phosphate (MEP) pathway prompted us to investigate the ability of a previously isolated periwinkle type-B RR (CrRR5) that presents high homologies with ARR1 to interact with the promoter. Electrophoretic mobility shift assays (EMSAs) demonstrated that the CrRR5 DNA-binding domain binds specifically type-ARR1 motifs within the HDS promoter. We also established through yellow fluorescent protein (YFP) imaging the targeting of CrRR5 into cell nucleus in accordance with its putative function of transcription factor. In transient assays performed on periwinkle cells cultivated with CK, overexpression of the full-length CrRR5 or a truncated CrRR5 engineering a constitutive active form (35S:ΔDDK) did not affect the HDS promoter activity that reached a threshold. By contrast, in absence of CK, overexpression of CrRR5ΔDDK enhanced promoter activity up to the threshold level observed in cells grown with CK. Our results strongly suggest that CrRR5 directly transactivates the HDS promoter. CrRR5 is the first identified transcription factor mediating the CK signaling that targets a gene from the MEP pathway involved in isoprenoid metabolism. Moreover, CrRR5 could play a role in a regulatory mechanism controlling CK homeostasis in periwinkle cells.

  18. No association between cyclooxygenase-2 and uridine diphosphate glucuronosyltransferase 1A6 genetic polymorphisms and colon cancer risk

    Institute of Scientific and Technical Information of China (English)

    Cheryl L Thompson; Sarah J Plummer; Alona Merkulova; Iona Cheng; Thomas C Tucker; Graham Casey; Li Li

    2009-01-01

    AIM: To investigate the association of variations in the cyclooxygenase-2 (COX2) and uridine diphosphate glucuronosyltransferase 1A6 (UGT1A6) genes and nonsteroidal anti-inflammatory drugs (NSAIDs) use with risk of colon cancer. METHODS: NSAIDs, which are known to reduce the risk of colon cancer, act directly on COX2 and reduce its activity. Epidemiological studies have associated variations in the COX2 gene with colon cancer risk, but others were unable to replicate this finding. Similarly, enzymes in the UGT1A6 gene have been demonstrated to modify the therapeutic effect of NSAIDs on colon adenomas. Polymorphisms in the UGT1A6 gene have been statistically shown to interact with NSAID intake to influence risk of developing colon adenomas, but not colon cancer. Here we examined the association of tagging single nucleotide polymorphisms (SNPs) in the COX2 and UGT1A6 genes, and their interaction with NSAID consumption, on risk of colon cancer in a population of 422 colon cancer cases and 481 population controls. RESULTS: No SNP in either gene was individually statistically significantly associated with colon cancer, nor did they statistically significantly change the protective effect of NSAID consumption in our sample. Like others, we were unable to replicate the association of variants in the COX2 gene with colon cancer risk ( P > 0.05), and we did not observe that these variants modify the protective effect of NSAIDs ( P > 0.05). We were able to confirm the lack of association of variants in UGT1A6 with colon cancer risk, although further studies will have to be conducted to confirm the association of these variants with colon adenomas. CONCLUSION: Our study does not support a role of COX2 and UGT1A6 genetic variations in the development of colon cancer.

  19. Asymptomatic bacteriuria Escherichia coli strains

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Nielsen, E.M.; Klemm, Per

    2006-01-01

    Urinary tract infections (UTIs) affect millions of people each year. Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU) in humans. Persons affected by ABU may carry a particular E. coli strain for extended periods of time without any symptoms. In contrast...

  20. Asymptomatic bacteriuria Escherichia coli strains

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Nielsen, E.M.; Klemm, Per

    2006-01-01

    Urinary tract infections (UTIs) affect millions of people each year. Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU) in humans. Persons affected by ABU may carry a particular E. coli strain for extended periods of time without any symptoms. In contrast...

  1. Influence of the temperature in the uranium (Vi) sorption in zirconium diphosphate; Influencia de la temperatura en la sorcion de uranio (VI) en difosfato de circonio

    Energy Technology Data Exchange (ETDEWEB)

    Garcia G, N.; Solis, D. [Universidad Autonoma del Estado de Mexico, Facultad de Quimica, Paseo Colon y Paseo Tollocan, 50120 Toluca, Estado de Mexico (Mexico); Ordonez R, E., E-mail: nidgg@yahoo.com.mx [ININ, Departamento de Quimica, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (Mexico)

    2012-10-15

    In the present work was evaluated the uranium (Vi) sorption at 10, 20, 30, 40 and 60 C on the zirconium diphosphate (ZrP{sub 2}O{sub 7}). They were carried out kinetic and isotherms using the method by lots, these will allow to fix the sorption time (kinetic) and to explain the behavior of this sorption in different ph conditions and temperature (isotherm). The quantity of retained uranium in the surface was quantified by means of the fluorescence technique. (Author)

  2. Whole cell biosynthesis of a functional oligosaccharide, 2′-fucosyllactose, using engineered Escherichia coli

    Directory of Open Access Journals (Sweden)

    Lee Won-Heong

    2012-04-01

    Full Text Available Abstract Background 2'-Fucosyllactose (2-FL is a functional oligosaccharide present in human milk which protects against the infection of enteric pathogens. Because 2-FL can be synthesized through the enzymatic fucosylation of lactose with guanosine 5′-diphosphate (GDP-l-fucose by α-1,2-fucosyltransferase (FucT2, an 2-FL producing Escherichia coli can be constructed through overexpressing genes coding for endogenous GDP- l-fucose biosynthetic enzymes and heterologous fucosyltransferase. Results The gene for FucT2 from Helicobacter pylori was introduced to the GDP- l-fucose producing recombinant E. coli BL21 star(DE3 strain. However, only small amount of 2-FL was produced in a batch fermentation because the E. coli BL21star(DE3 strain assimilated lactose instead of converting to 2-FL. As an alternative host, the E. coli JM109(DE3 strain which is incapable of assimilating lactose was chosen as a 2-FL producer. Whole cell biosynthesis of 2-FL from lactose was investigated in a series of batch fermentations using various concentrations of lactose. The results of batch fermentations showed that lactose was slowly assimilated by the engineered E. coli JM109(DE3 strain and 2-FL was synthesized without supplementation of another auxiliary sugar for cell growth. A maximum 2-FL concentration of 1.23 g/l was obtained from a batch fermentation with 14.5 g/l lactose. The experimentally obtained yield (g 2-FL/g lactose corresponded to 20% of the theoretical maximum yield estimated by the elementary flux mode (EFM analysis. Conclusions The experimental 2-FL yield in this study corresponded to about 20% of the theoretical maximum yield, which suggests further modifications via metabolic engineering of a host strain or optimization of fermentation processes might be carried out for improving 2-FL yield. Improvement of microbial production of 2-FL from lactose by engineered E. coli would increase the feasibility of utilizing 2-FL as a prebiotic in various

  3. Engineering Escherichia coli to convert acetic acid to β-caryophyllene.

    Science.gov (United States)

    Yang, Jianming; Nie, Qingjuan

    2016-05-05

    Under aerobic conditions, acetic acid is the major byproduct produced by E. coli during the fermentation. And acetic acid is detrimental to cell growth as it destroys transmembrane pH gradients. Hence, how to reduce the production of acetic acid and how to utilize it as a feedstock are of intriguing interest. In this study, we provided an evidence to produce β-caryophyllene by the engineered E. coli using acetic acid as the only carbon source. Firstly, to construct the robust acetate-utilizing strain, acetyl-CoA synthases from three different sources were introduced and screened in the E. coli. Secondly, to establish the engineered strains converting acetic acid to β-caryophyllene, acetyl-CoA synthase (ACS), β-caryophyllene synthase (QHS1) and geranyl diphosphate synthase (GPPS2) were co-expressed in the E. coli cells. Thirdly, to further enhance β-caryophyllene production from acetic acid, the heterologous MVA pathway was introduced into the cells. What's more, acetoacetyl-CoA synthase (AACS) was also expressed in the cells to increase the precursor acetoacetyl-CoA and accordingly resulted in the increase of β-caryophyllene. The final genetically modified strain, YJM67, could accumulate the production of biomass and β-caryophyllene up to 12.6 and 1.05 g/L during 72 h, respectively, with a specific productivity of 1.15 mg h(-1) g(-1) dry cells, and the conversion efficiency of acetic acid to β-caryophyllene (gram to gram) reached 2.1%. The yield of β-caryophyllene on acetic acid of this strain also reached approximately 5.6% of the theoretical yield. In the present study, a novel biosynthetic pathway for β-caryophyllene has been investigated by means of conversion of acetic acid to β-caryophyllene using an engineered Escherichia coli. This was the first successful attempt in β-caryophyllene production by E. coli using acetic acid as the only carbon source. Therefore, we have provided a new metabolic engineering tool for β-caryophyllene synthesis.

  4. Comparison of cationic propyl gallate and adenosine diphosphate for the measurement of aspirin effectivity with optical aggregometry.

    Science.gov (United States)

    Motovska, Zuzana; Sujanova, Zdenka; Wimmerova, Sona; Ardo, Jan; Skrakova, Marcela; Widimsky, Petr

    2007-10-01

    To compare the newer inductor of platelet aggregation cationic propyl gallate (CPG) with adenosine diphosphate (ADP) for the examination of aspirin (ASA) effectivity with optical aggregometry. In total,116 patients were prospectively enrolled with a stable cardiovascular disease, taking ASA 100 mg/day for >or=1 month. The control group consisted of 62 healthy volunteers. A platelet aggregation was investigated by optical aggregometry (aggregometer LASER 4x; BIO ART, Sint-Katelijne-Waver, Belgium). CPG and ADP were added as aggregating agents. The measured parameters were CPG-slope (%/min) and ADP max (%). Using the CPG-slope values from the control group, the CPG-slope cut-off value was determined to define a laboratory ASA-noneffectively treated patient. The values from control group followed a normal distribution (Shapiro-Wilk test). We calculated the cut-off value using the 1-tailed 95% confidence interval. The CPG-slope cut-off value was 79 %/min for an ASA-effectively treated patient. We marked the patients as laboratory ASA-noneffective treated when the CPG-slope was >79%/min. In the same way we defined the cut-off value for ADP-max. We identified the aspirin treatment as ineffective when the value of ADP-max was >62%. The values of CPG-slope and ADP-max were in close correlation in the group of patients treated with aspirin with a highly significant correlation index (r=0.671, Pinductors, the proportion of ASA-noneffectively treated patients was 25%. Using both tests, 72.4% of patients were equally divided. ASA-noneffectively treated patients were commonly more obese (46.2%), had hypertension (94.9%) and hypercholesterolemia (73.7%), and were less commonly treated with statins (30.8%) than the aspirin effectively treated patients (42%, 88.2%, 59.2%, and 42.1%, respectively). The detected differences were not statistically significant. Cationic propyl gallate is an optimal inductor for optical aggregometry to monitor laboratory effectiveness of aspirin

  5. The nucleoside diphosphate kinase gene Nme3 acts as quantitative trait locus promoting non-Mendelian inheritance.

    Directory of Open Access Journals (Sweden)

    Hermann Bauer

    Full Text Available The t-haplotype, a variant form of the t-complex region on mouse chromosome 17, acts as selfish genetic element and is transmitted at high frequencies (> 95% from heterozygous (t/+ males to their offspring. This phenotype is termed transmission ratio distortion (TRD and is caused by the interaction of the t-complex responder (Tcr with several quantitative trait loci (QTL, the t-complex distorters (Tcd1 to Tcd4, all located within the t-haplotype region. Current data suggest that the distorters collectively impair motility of all sperm derived from t/+ males; t-sperm is rescued by the responder, whereas (+-sperm remains partially dysfunctional. Recently we have identified two distorters as regulators of RHO small G proteins. Here we show that the nucleoside diphosphate kinase gene Nme3 acts as a QTL on TRD. Reduction of the Nme3 dosage by gene targeting of the wild-type allele enhanced the transmission rate of the t-haplotype and phenocopied distorter function. Genetic and biochemical analysis showed that the t-allele of Nme3 harbors a mutation (P89S that compromises enzymatic activity of the protein and genetically acts as a hypomorph. Transgenic overexpression of the Nme3 t-allele reduced t-haplotype transmission, proving it to be a distorter. We propose that the NME3 protein interacts with RHO signaling cascades to impair sperm motility through hyperactivation of SMOK, the wild-type form of the responder. This deleterious effect of the distorters is counter-balanced by the responder, SMOK(Tcr, a dominant-negative protein kinase exclusively expressed in t-sperm, thus permitting selfish behaviour and preferential transmission of the t-haplotype. In addition, the previously reported association of NME family members with RHO signaling in somatic cell motility and metastasis, in conjunction with our data involving RHO signaling in sperm motility, suggests a functional conservation between mechanisms for motility control in somatic cells and

  6. Effects of nimodipine and fructose-1,6-diphosphate on cerebral damage in carbon monoxide poisoning mice

    Institute of Scientific and Technical Information of China (English)

    杨俊卿; 赵晓辉; 周岐新; 蒋青松

    2003-01-01

    Objective To study the dose- and time- dependent protective effects and the synergistic effects of nimodipine (NMDP) and fructose-1,6-diphosphate (FDP) against cerebral damage induced by acute carbon monoxide (CO) poisoning in mice.Methods Male mice were exposed to CO 170 mL/kg, i.p. After CO intraperitonealy exposure, mortality of mice, change in memory function estimated by passive avoidance test, the pathomorphologic observation of brain tissue slices, as well as changes of activities of monoamine oxidase (MAO)-B and Ca2+-Mg2+-ATPase in cerebral tissue were studied. In dose-dependent protective effect study, NMDP (10.6, 5.3, 2.7 mg/kg) and FDP (2.6, 1.3, 0.67 g/kg) was injected ip, respectively 15 min after CO exposure. To study the time-effect relationship of drugs, NMDP (5.3 mg/kg) and FDP (1.3 g/kg) were administered ip respectively 15 minutes, 45 minutes and 120 minutes after CO exposure. The combination of NMDP (2.7 mg/kg) and FDP (0.67 g/kg) was administered ip15 minutes, 45 min and 120 minutes after CO exposure to study the synergism of the two drugs.Results Either NMDP (10.6, 5.3 mg/kg) or FDP (2.6, 1.3 g/kg) administered ip within 15 minutes after CO exposure significantly decreased the impairment of memory function and mortality rate induced by CO, inhibited the decrease of Ca2+-Mg2+-ATPase activity, blunted the rising of MAO-B activity and prevented the delayed hippocampal neuronal death in poisoning mice. To our surprise, the combined use of NMDP (2.7 mg/kg) and FDP (0.67 g/kg) within 15 minutes after CO exposure had similar effects to that in NMDP (10.6, 5.3 mg/kg) and FDP (2.6, 1.3 g/kg).Conclusions These results suggest that the impairment of CO on brain can be attenuated if NMDP or FDP are administered sufficiently and quickly as soon as possible after CO exposure and there exists a synergism of FDP and NMDP against CO poisoning damage.

  7. Association of farnesyl diphosphate synthase polymorphisms and response to alendronate treatment in Chinese postmenopausal women with osteoporosis

    Institute of Scientific and Technical Information of China (English)

    Liu Yi; Liu Haijuan; Li Mei; Zhou Peiran; Xing Xiaoping; Xia Weibo; Zhang Zhenlin

    2014-01-01

    Background Genetic factors are important in the pathogenesis of osteoporosis,but less is known about the genetic determinants of osteoporosis treatment.We aimed to explore the association between the gene polymorphisms of key enzyme farnesyl diphosphate synthase (FDPS) in mevalonate signaling pathway of osteoclast and response to alendronate therapy in osteoporotic postmenopausal women in China.Methods The study group comprised 639 postmenopausal women aged (62.2±7.0) years with osteoporosis or osteopenia who had been randomly assigned to low dose group (70 mg/2w) or standard dose group (70 mg/w) of alendronate in this 1-year study.We identified allelic variant of the FDPS gene using the polymerase chain reaction and restriction enzyme Faul.Before and after treatment,serum levels of calcium,phosphate,alkaline phosphatase (ALP),cross linked C-telopeptide of type Ⅰ collagen (β-CTX) were detected.Bone mineral density (BMD) at lumbar spine and proximal femur was measured.The association was analyzed between the polymorphisms of FDPS gene and the changes of BMD,bone turnover biomarkers after the treatment.Results The FDPS rs2297480 polymorphisms were associated with baseline BMD at femoral neck,and patients with CC genotype had significantly higher baseline femoral neck BMD ((733.6±84.1) mg/cm2) than those with AC genotypes ((703.0±86.9) mg/cm2) and AA genotypes ((649.8±62.4) mg/cm2) (P <0.01).No significant difference in BMD at lumbar spine was observed among different genotypes of FDPS.The percentage change of serum ALP level was significantly lower in patients with CC genotype (-22.9%) than that in those with AC genotype (-24.1%) and AA genotype (-29.8%) of FDPS after 12 months of alendronate treatment (P <0.05).Neither percentage change of BMD nor β-CTX level after alendronate treatment had association with FDPS genotype.Conclusions FDPS gene was probably a candidate gene to predict femoral neck BMD at baseline.FDPS gene alleles could predict

  8. Biophysical Characterization and Activity of Lymphostatin, a Multifunctional Virulence Factor of Attaching and Effacing Escherichia coli.

    Science.gov (United States)

    Cassady-Cain, Robin L; Blackburn, Elizabeth A; Alsarraf, Husam; Dedic, Emil; Bease, Andrew G; Böttcher, Bettina; Jørgensen, René; Wear, Martin; Stevens, Mark P

    2016-03-11

    Attaching and effacing Escherichia coli cause diarrhea and typically produce lymphostatin (LifA), an inhibitor of mitogen-activated proliferation of lymphocytes and pro-inflammatory cytokine synthesis. A near-identical factor (Efa1) has been reported to mediate adherence of E. coli to epithelial cells. An amino-terminal region of LifA shares homology with the catalytic domain of the large clostridial toxins, which are retaining glycosyltransferases with a DXD motif involved in binding of a metal ion. Understanding the mode(s) of action of lymphostatin has been constrained by difficulties obtaining a stably transformed plasmid expression clone. We constructed a tightly inducible clone of enteropathogenic E. coli O127:H6 lifA for affinity purification of lymphostatin. The purified protein inhibited mitogen-activated proliferation of bovine T lymphocytes in the femtomolar range. It is a monomer in solution and the molecular envelope was determined using both transmission electron microscopy and small-angle x-ray scattering. Domain architecture was further studied by limited proteolysis. The largest proteolytic fragment containing the putative glycosyltransferase domain was tested in isolation for activity against T cells, and was not sufficient for activity. Tryptophan fluorescence studies indicated thatlymphostatin binds uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) but not UDP-glucose (UDP-Glc). Substitution of the predicted DXD glycosyltransferase motif with alanine residues abolished UDP-GlcNAc binding and lymphostatin activity, although other biophysical properties were unchanged. The data indicate that lymphostatin has UDP-sugar binding potential that is critical for activity, and is a major leap toward identifying the nature and consequences of modifications of host cell factors.

  9. Enhanced production of GDP-L-fucose by overexpression of NADPH regenerator in recombinant Escherichia coli.

    Science.gov (United States)

    Lee, Won-Heong; Chin, Young-Wook; Han, Nam Soo; Kim, Myoung-Dong; Seo, Jin-Ho

    2011-08-01

    Biosynthesis of guanosine 5'-diphosphate-L-fucose (GDP-L-fucose) requires NADPH as a reducing cofactor. In this study, endogenous NADPH regenerating enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (Icd), and NADP(+)-dependent malate dehydrogenase (MaeB) were overexpressed to increase GDP-L-fucose production in recombinant Escherichia coli. The effects of overexpression of each NADPH regenerating enzyme on GDP-L-fucose production were investigated in a series of batch and fed-batch fermentations. Batch fermentations showed that overexpression of G6PDH was the most effective for GDP-L-fucose production. However, GDP-L-fucose production was not enhanced by overexpression of G6PDH in the glucose-limited fed-batch fermentation. Hence, a glucose feeding strategy was optimized to enhance GDP-L-fucose production. Fed-batch fermentation with a pH-stat feeding mode for sufficient supply of glucose significantly enhanced GDP-L-fucose production compared with glucose-limited fed-batch fermentation. A maximum GDP-L-fucose concentration of 235.2 ± 3.3 mg l(-1), corresponding to a 21% enhancement in the GDP-L-fucose production compared with the control strain overexpressing GDP-L-fucose biosynthetic enzymes only, was achieved in the pH-stat fed-batch fermentation of the recombinant E. coli overexpressing G6PDH. It was concluded that sufficient glucose supply and efficient NADPH regeneration are crucial for NADPH-dependent GDP-L-fucose production in recombinant E. coli.

  10. Thiophene metabolism by E. coli

    Energy Technology Data Exchange (ETDEWEB)

    Clark, D.P.

    1990-01-01

    The objective of this project is to investigate the mechanism of degradation of sulfur containing heterocyclic molecules such as those found in coal, by mutants of Escherichia coli K-12. We previously isolated multiple mutants of E. coli which were selected for improved oxidation of furan and thiophene derivatives. We have focused on the thdA mutation in our subsequent research as it appears to be of central importance in thiophene oxidation. We hope that analysis of the thd genes of E. coli will lead to improvement of our thiophene metabolizing bacterial strains. 1 tab.

  11. Bacillus caldolyticus prs gene encoding phosphoribosyldiphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-l-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  12. Toward Network Biology in E. coli Cell.

    Science.gov (United States)

    Mori, Hirotada; Takeuchi, Rikiya; Otsuka, Yuta; Bowden, Steven; Yokoyama, Katsushi; Muto, Ai; Libourel, Igor; Wanner, Barry L

    2015-01-01

    E. coli has been a critically important model research organism for more than 50 years, particularly in molecular biology. In 1997, the E. coli draft genome sequence was published. Post-genomic techniques and resources were then developed that allowed E. coli to become a model organism for systems biology. Progress made since publication of the E. coli genome sequence will be summarized.

  13. Pulsed ELDOR spectroscopy measures the distance between the two tyrosyl dadicals in the R2 subunit of the E. coli ribonucleotide reductase.

    Science.gov (United States)

    Bennati, Marina; Weber, Axel; Antonic, Jelena; Perlstein, Deborah L; Robblee, John; Stubbe, JoAnne

    2003-12-10

    Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates (NDPs) to deoxynucleoside diphosphates (dNDPs). This RNR is composed of two homodimeric subunits: R1 and R2. R1 binds the NDPs in the active site, and R2 harbors the essential di-iron tyrosyl radical (Y*) cofactor. In this paper, we used PELDOR, a method that detects weak electron-electron dipolar coupling, to make the first direct measurement of the distance between the two Y*'s on each monomer of R2. In the crystal structure of R2, the Y*'s are reduced to tyrosines, and consequently R2 is inactive. In R2, where the Y*'s assume a well-defined geometry with respect to the protein backbone, the PELDOR method allows measurement of a distance of 33.1 +/- 0.2 A that compares favorably to the distance (32.4 A) between the center of mass of the spin density distribution of each Y* on each R2 monomer from the structure. The experiments provide the first direct experimental evidence for two Y*'s in a single R2 in solution.

  14. Analysis of an invariant cofactor-protein interaction in thiamin diphosphate-dependent enzymes by site-directed mutagenesis. Glutamic acid 418 in transketolase is essential for catalysis.

    Science.gov (United States)

    Wikner, C; Meshalkina, L; Nilsson, U; Nikkola, M; Lindqvist, Y; Sundström, M; Schneider, G

    1994-12-23

    A homologous expression system and a purification protocol for pure, highly active recombinant yeast transketolase have been developed. The invariant transketolase residue Glu418, which forms a hydrogen bond to the N-1' nitrogen atom of the pyrimidine ring of the cofactor thiamin diphosphate has been replaced by glutamine and alanine. Crystallographic analyses of the mutants show that these amino acid substitutions do not induce structural changes beyond the site of mutation. In both cases, the cofactor binds in a manner identical to the wild-type enzyme. Significant differences in the CD spectra of the mutant transketolases compared with the spectrum of wild-type enzyme indicate differences in the electron distribution of the aminopyrimidine ring of the cofactor. The E418Q mutant shows 2% and the E418A mutant shows about 0.1% of the catalytic activity of wild-type enzyme. The affinities of the mutant enzymes for thiamin diphosphate are comparable with wild-type transketolase. The hydrogen bond between the coenzyme and the side chain of Glu418 is thus not required for coenzyme binding but essential for catalytic activity. The results demonstrate the functional importance of this interaction and support the molecular model for cofactor deprotonation, the first step in enzymatic thiamin catalysis.

  15. Overexpression and Suppression of Artemisia annua 4-Hydroxy-3-Methylbut-2-enyl Diphosphate Reductase 1 Gene (AaHDR1) Differentially Regulate Artemisinin and Terpenoid Biosynthesis

    Science.gov (United States)

    Ma, Dongming; Li, Gui; Zhu, Yue; Xie, De-Yu

    2017-01-01

    4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) catalyzes the last step of the 2-C-methyl-D-erythritol 4- phosphate (MEP) pathway to synthesize isopentenyl pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP). To date, little is known regarding effects of an increase or a decrease of a HDR expression on terpenoid and other metabolite profiles in plants. In our study, an Artemisia annua HDR cDNA (namely AaHDR1) was cloned from leaves. Expression profiling showed that it was highly expressed in leaves, roots, stems, and flowers with different levels. Green florescence protein fusion and confocal microscope analyses showed that AaHDR1 was localized in chloroplasts. The overexpression of AaHDR1 increased contents of artemisinin, arteannuin B and other sesquiterpenes, and multiple monoterpenes. By contrast, the suppression of AaHDR1 by anti-sense led to opposite results. In addition, an untargeted metabolic profiling showed that the overexpression and suppression altered non-polar metabolite profiles. In conclusion, the overexpression and suppression of AaHDR1 protein level in plastids differentially affect artemisinin and other terpenoid biosynthesis, and alter non-polar metabolite profiles of A. annua. Particularly, its overexpression leading to the increase of artemisinin production is informative to future metabolic engineering of this antimalarial medicine. PMID:28197158

  16. Synthesis, Rietveld refinements, Infrared and Raman spectroscopy studies of the sodium diphosphate NaCryFe1-yP2O7 (0 ≤ y ≤ 1)

    Science.gov (United States)

    Bih, H.; Saadoune, I.; Bih, L.; Mansori, M.; ToufiK, H.; Fuess, H.; Ehrenberg, H.

    2016-01-01

    In the present study we report on the synthesis and crystal structure studies of NaCryFe1-yP2O7 sodium diphosphate solid solution (0 ≤ y ≤ 1). The X-ray diffraction shows that these compounds are isostructural with NaFeP2O7 and NaCrP2O7 (space group P21/c (C2h5) Z = 4). The Rietveld refinements based on the XRD patterns show the existence of a continuous solid solution over the whole composition range (0 ≤ y ≤ 1). A continuous evolution of the monoclinic unit cell parameters was obtained. The transition metal ions (Cr3+ and/or Fe3+) connect the diphosphate anions forming a three-dimensional network with cages filled by Na+ cations. IR and Raman spectra have been interpreted using factor group analysis. A small shift of the band frequencies is observed when Fe is substituted by Cr. The POP bridge angles are determined from Lazarev's relation and agree well with those deduced from the crystal structure refinement.

  17. Escherichia coli Uropathogenesis In Vitro

    DEFF Research Database (Denmark)

    Andersen, Thomas E; Khandige, Surabhi; Madelung, Michelle

    2012-01-01

    Uropathogenic Escherichia coli (UPEC) strains are capable of invading bladder epithelial cells (BECs) on the bladder luminal surface. Based primarily on studies in mouse models, invasion is proposed to trigger an intracellular uropathogenic cascade involving intracellular bacterial proliferation...

  18. Adenosine thiamine triphosphate accumulates in Escherichia coli cells in response to specific conditions of metabolic stress

    Directory of Open Access Journals (Sweden)

    Zorzi Willy

    2010-05-01

    Full Text Available Abstract Background E. coli cells are rich in thiamine, most of it in the form of the cofactor thiamine diphosphate (ThDP. Free ThDP is the precursor for two triphosphorylated derivatives, thiamine triphosphate (ThTP and the newly discovered adenosine thiamine triphosphate (AThTP. While, ThTP accumulation requires oxidation of a carbon source, AThTP slowly accumulates in response to carbon starvation, reaching ~15% of total thiamine. Here, we address the question whether AThTP accumulation in E. coli is triggered by the absence of a carbon source in the medium, the resulting drop in energy charge or other forms of metabolic stress. Results In minimal M9 medium, E. coli cells produce AThTP not only when energy substrates are lacking but also when their metabolization is inhibited. Thus AThTP accumulates in the presence of glucose, when glycolysis is blocked by iodoacetate, or in the presence lactate, when respiration is blocked by cyanide or anoxia. In both cases, ATP synthesis is impaired, but AThTP accumulation does not appear to be a direct consequence of reduced ATP levels. Indeed, in the CV2 E. coli strain (containing a thermolabile adenylate kinase, the ATP content is very low at 37°C, even in the presence of metabolizable substrates (glucose or lactate and under these conditions, the cells produce ThTP but not AThTP. Furthermore, we show that ThTP inhibits AThTP accumulation. Therefore, we conclude that a low energy charge is not sufficient to trigger AThTP accumulation and the latter can only accumulate under conditions where no ThTP is synthesized. We further show that AThTP production can also be induced by the uncoupler CCCP but, unexpectedly, this requires the presence of pyruvate or a substrate yielding pyruvate (such a D-glucose or L-lactate. Under the conditions described, AThTP production is not different when RelA or SpoT mutants are used. Conclusions In E. coli, AThTP accumulates in response to two different conditions of

  19. Diarrheagenic Escherichia coli Markers and Phenotypes among Fecal E. coli Isolates Collected from Nicaraguan Infants ▿

    OpenAIRE

    Reyes, Daniel; Vilchez, Samuel; Paniagua, Margarita; Colque-Navarro, Patricia; Weintraub, Andrej; Möllby, Roland; Kühn, Inger

    2010-01-01

    We analyzed the prevalence of diarrheagenic Escherichia coli (DEC) markers and common phenotypes in 2,164 E. coli isolates from 282 DEC-positive samples. Enteropathogenic E. coli (EPEC) and enteroaggregative E. coli (EAEC) were very diverse and were not correlated with diarrhea. Enterotoxigenic E. coli (ETEC) estA and enterohemorrhagic E. coli (EHEC) belonged to a few phenotypes and were significantly correlated with diarrhea.

  20. Diarrheagenic Escherichia coli Markers and Phenotypes among Fecal E. coli Isolates Collected from Nicaraguan Infants ▿

    OpenAIRE

    2010-01-01

    We analyzed the prevalence of diarrheagenic Escherichia coli (DEC) markers and common phenotypes in 2,164 E. coli isolates from 282 DEC-positive samples. Enteropathogenic E. coli (EPEC) and enteroaggregative E. coli (EAEC) were very diverse and were not correlated with diarrhea. Enterotoxigenic E. coli (ETEC) estA and enterohemorrhagic E. coli (EHEC) belonged to a few phenotypes and were significantly correlated with diarrhea.

  1. Modification of zirconium diphosphate with salicylic acid and its effect on the uranium (Vi) sorption; Modificacion del difosfato de circonio con acido salicilico y su efecto sobre la sorcion de uranio (VI)

    Energy Technology Data Exchange (ETDEWEB)

    Almazan T, M. G.; Garcia G, N. [ININ, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (Mexico); Simoni, E., E-mail: guadalupe.almazan@inin.gob.mx [Universidad Paris Sud, Instituto de Fisica Nuclear, Georges Clemenceau No. 15, Orsay (France)

    2014-10-15

    The surface of zirconium diphosphate (ZrP{sub 2}O{sub 7}) was modified with salicylic acid and its effect was evaluated on the uranium (Vi) sorption. The modified surface of the material was analyzed with different analytical techniques among which are included the atomic force microscopy, scanning electron microscopy and X-ray photoelectron spectroscopy. This analysis allowed showing that the salicylic acid is being held on the surface of the zirconium diphosphate. The reactivity of modified zirconium diphosphate compared with uranium (Vi) was investigated using the classical method of batch sorption. The analysis of sorption isotherms shows that the salicylic acid has an important effect in the uranium (Vi) sorption. According to the study conducted, the interaction among the uranium (Vi) and the surface of zirconium diphosphate modified with the salicylic acid most likely leads to the complexes formation of binary (U(Vi)/ZrP{sub 2}O{sub 7}) and ternary (U(Vi)/salicylate/ZrP{sub 2}O{sub 7}) surface. (Author)

  2. Safety, tolerability, and initial efficacy of AZD6140, the first reversible oral adenosine diphosphate receptor antagonist, compared with clopidogrel, in patients with non-ST-segment elevation acute coronary syndrome: primary results of the DISPERSE-2 trial

    DEFF Research Database (Denmark)

    Cannon, Christopher P; Husted, Steen; Harrington, Robert A;

    2007-01-01

    OBJECTIVES: Our goal was to compare the safety and initial efficacy of AZD6140, the first reversible oral adenosine diphosphate receptor antagonist, with clopidogrel in patients with non-ST-segment elevation acute coronary syndromes (NSTE-ACS). BACKGROUND: AZD6140 achieves higher mean levels of p...

  3. Crystallization and preliminary X-ray diffraction analysis of full-length and proteolytically activated pyruvate oxidase from Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Weidner, Annett; Neumann, Piotr; Wille, Georg; Stubbs, Milton T.; Tittmann, Kai, E-mail: kai.tittmann@biochemtech.uni-halle.de [Martin-Luther-Universität Halle-Wittenberg, Naturwissenschaftliche Fakultät I, Institut für Biochemie und Biotechnologie, Kurt-Mothes-Strasse 3, D-06120 Halle (Germany)

    2008-03-01

    The peripheral membrane flavoprotein pyruvate oxidase from E. coli has been crystallized in the full-length form and as a proteolytically activated truncation variant lacking the last 23 amino acids at the C-terminus. The thiamine diphosphate- and flavin-dependent peripheral membrane enzyme pyruvate oxidase from Escherichia coli (EcPOX) has been crystallized in the full-length form and as a proteolytically activated C-terminal truncation variant which lacks the last 23 amino acids (Δ23 EcPOX). Crystals were grown by the hanging-drop vapour-diffusion method using either protamine sulfate (full-length EcPOX) or 2-methyl-2,4-pentanediol (Δ23 EcPOX) as precipitants. Native data sets were collected at a X-ray home source to a resolution of 2.9 Å. The two forms of EcPOX crystallize in different space groups. Whereas full-length EcPOX crystallizes in the tetragonal space group P4{sub 3}2{sub 1}2 with two monomers per asymmetric unit, the crystals of Δ23 EcPOX belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1} and contain 12 monomers per asymmetric unit.

  4. Expression of Codon-Optimized Plant Glycosyltransferase UGT72B14 in Escherichia coli Enhances Salidroside Production

    Directory of Open Access Journals (Sweden)

    Feiyan Xue

    2016-01-01

    Full Text Available Salidroside, a plant secondary metabolite in Rhodiola, has been demonstrated to have several adaptogenic properties as a medicinal herb. Due to the limitation of plant source, microbial production of salidroside by expression of plant uridine diphosphate glycosyltransferase (UGT is promising. However, glycoside production usually remains hampered by poor expression of plant UGTs in microorganisms. Herein, we achieved salidroside production by expression of Rhodiola UGT72B14 in Escherichia coli (E. coli and codon optimization was accordingly applied. UGT72B14 expression was optimized by changing 278 nucleotides and decreasing the G+C content to 51.05% without altering the amino acid sequence. The effect of codon optimization on UGT72B14 catalysis for salidroside production was assessed both in vitro and in vivo. In vitro, salidroside production by codon-optimized UGT72B14 is enhanced because of a significantly improved protein yield (increased by 4.8-fold and an equivalently high activity as demonstrated by similar kinetic parameters (KM and Vmax, compared to that by wild-type protein. In vivo, both batch and fed-batch cultivation using the codon-optimized gene resulted in a significant increase in salidroside production, which was up to 6.7 mg/L increasing 3.2-fold over the wild-type UGT72B14.

  5. Expression of Codon-Optimized Plant Glycosyltransferase UGT72B14 in Escherichia coli Enhances Salidroside Production.

    Science.gov (United States)

    Xue, Feiyan; Guo, Huili; Hu, Yingying; Liu, Ran; Huang, Lina; Lv, Heshu; Liu, Chunmei; Yang, Mingfeng; Ma, Lanqing

    2016-01-01

    Salidroside, a plant secondary metabolite in Rhodiola, has been demonstrated to have several adaptogenic properties as a medicinal herb. Due to the limitation of plant source, microbial production of salidroside by expression of plant uridine diphosphate glycosyltransferase (UGT) is promising. However, glycoside production usually remains hampered by poor expression of plant UGTs in microorganisms. Herein, we achieved salidroside production by expression of Rhodiola UGT72B14 in Escherichia coli (E. coli) and codon optimization was accordingly applied. UGT72B14 expression was optimized by changing 278 nucleotides and decreasing the G+C content to 51.05% without altering the amino acid sequence. The effect of codon optimization on UGT72B14 catalysis for salidroside production was assessed both in vitro and in vivo. In vitro, salidroside production by codon-optimized UGT72B14 is enhanced because of a significantly improved protein yield (increased by 4.8-fold) and an equivalently high activity as demonstrated by similar kinetic parameters (K M and V max), compared to that by wild-type protein. In vivo, both batch and fed-batch cultivation using the codon-optimized gene resulted in a significant increase in salidroside production, which was up to 6.7 mg/L increasing 3.2-fold over the wild-type UGT72B14.

  6. Theoretical model of interactions between ligand-binding sites in a dimeric protein and its application for the analysis of thiamine diphosphate binding to yeast transketolase.

    Science.gov (United States)

    Ospanov, Ruslan; Kochetov, German; Kurganov, Boris

    2006-11-20

    The binding of thiamin diphosphate (ThDP) to yeast dimeric apotransketolase (apoTK) is accompanied by the appearance of a band in the absorption spectrum with maximum at 320 nm. The saturation function has been analyzed using a scheme that involves binding of ThDP to each subunit followed by the conformational transition of this subunit. It is assumed that the binding of ThDP to one subunit may affect the conformational transition of the other subunit. Rigorous mathematical expressions describing the dependence of the optical absorption on the total concentration of ThDP are first developed. Equilibrium constants and corresponding rate constants for the binding of ThDP to apoTK have been estimated. The negative cooperativity in the ThDP binding has been characterized by the function reflecting the dependence of the conformational change on the saturation of apoTK by ThDP.

  7. Crystal structure of the Bacillus anthracis nucleoside diphosphate kinase and its characterization reveals an enzyme adapted to perform under stress conditions.

    Science.gov (United States)

    Misra, Gauri; Aggarwal, Anita; Dube, Divya; Zaman, Mohd Saif; Singh, Yogendra; Ramachandran, Ravishankar

    2009-08-01

    Nucleoside diphosphate kinases (Ndks) play an important role in a plethora of regulatory and metabolic functions. Inhibition of the B. anthracis Ndk mRNA results in the formation of nonviable aberrant spores. We report the characterization and crystal structure of the enzyme from B. anthracis nucleoside diphosphate kinase (BaNdk), the first from sporulating bacteria. The enzyme, although from a mesophilic source, is active at extremes of pH (3.5-10.5), temperature (10-95 degrees C) and ionic strength (0.25-4.0M NaCl). It exists as a hexamer that is composed of two SDS-stable trimers interacting in a back-to-back association; mutational analysis confirms that the enzyme is a histidine kinase. The high-resolution crystal structure reported here reveals an unanticipated change in the conformation of residues between 43 and 63 that also regulates substrate entry in other Ndks. A comparative structural analysis involving Ndks from seven mesophiles and three thermophiles has resulted in the delineation of the structure into relatively rigid and flexible regions. The analysis suggests that the larger number of intramolecular hydrogen bonds and to a lesser extent ionic interactions in BaNdk contributes to its high thermostability. Mutational analysis and Molecular Dynamics simulations were used to probe the role of a highly conserved Gly19 (present at the oligomeric interface in most of the Ndks). The results suggest that the mutation leads to a rigidification of those residues that facilitate substrate entry and consequently leads to a large reduction in the kinase activity. Overall, the enzyme characterization helps to understand its apparent adaptation to perform under stress conditions.

  8. Modulation of hexa-acyl pyrophosphate lipid A population under Escherichia coli phosphate (Pho) regulon activation.

    Science.gov (United States)

    Lamarche, Martin G; Kim, Sang-Hyun; Crépin, Sébastien; Mourez, Michael; Bertrand, Nicolas; Bishop, Russell E; Dubreuil, J Daniel; Harel, Josée

    2008-08-01

    Environmental phosphate is an important signal for microorganism gene regulation, and it has recently been shown to trigger some key bacterial virulence mechanisms. In many bacteria, the Pho regulon is the major circuit involved in adaptation to phosphate limitation. The Pho regulon is controlled jointly by the two-component regulatory system PhoR/PhoB and by the phosphate-specific transport (Pst) system, which both belong to the Pho regulon. We showed that a pst mutation results in virulence attenuation in extraintestinal pathogenic Escherichia coli (ExPEC) strains. Our results indicate that the bacterial cell surface of the pst mutants is altered. In this study, we show that pst mutants of ExPEC strains display an increased sensitivity to different cationic antimicrobial peptides and vancomycin. Remarkably, the hexa-acylated 1-pyrophosphate form of lipid A is significantly less abundant in pst mutants. Among differentially expressed genes in the pst mutant, lpxT coding for an enzyme that transfers a phosphoryl group to lipid A, forming the 1-diphosphate species, was found to be downregulated. Our results strongly suggest that the Pho regulon is involved in lipid A modifications, which could contribute to bacterial surface perturbations. Since the Pho regulon and the Pst system are conserved in many bacteria, such a lipid A modification mechanism could be widely distributed among gram-negative bacterial species.

  9. Suppression of the E. coli SOS response by dNTP pool changes.

    Science.gov (United States)

    Maslowska, Katarzyna H; Makiela-Dzbenska, Karolina; Fijalkowska, Iwona J; Schaaper, Roel M

    2015-04-30

    The Escherichia coli SOS system is a well-established model for the cellular response to DNA damage. Control of SOS depends largely on the RecA protein. When RecA is activated by single-stranded DNA in the presence of a nucleotide triphosphate cofactor, it mediates cleavage of the LexA repressor, leading to expression of the 30(+)-member SOS regulon. RecA activation generally requires the introduction of DNA damage. However, certain recA mutants, like recA730, bypass this requirement and display constitutive SOS expression as well as a spontaneous (SOS) mutator effect. Presently, we investigated the possible interaction between SOS and the cellular deoxynucleoside triphosphate (dNTP) pools. We found that dNTP pool changes caused by deficiencies in the ndk or dcd genes, encoding nucleoside diphosphate kinase and dCTP deaminase, respectively, had a strongly suppressive effect on constitutive SOS expression in recA730 strains. The suppression of the recA730 mutator effect was alleviated in a lexA-deficient background. Overall, the findings suggest a model in which the dNTP alterations in the ndk and dcd strains interfere with the activation of RecA, thereby preventing LexA cleavage and SOS induction.

  10. Enhancing GDP-fucose production in recombinant Escherichia coli by metabolic pathway engineering.

    Science.gov (United States)

    Zhai, Yafei; Han, Donglei; Pan, Ying; Wang, Shuaishuai; Fang, Junqiang; Wang, Peng; Liu, Xian-wei

    2015-02-01

    Guanosine 5'-diphosphate (GDP)-fucose is the indispensible donor substrate for fucosyltransferase-catalyzed synthesis of fucose-containing biomolecules, which have been found involving in various biological functions. In this work, the salvage pathway for GDP-fucose biosynthesis from Bacterioides fragilis was introduced into Escherichia coli. Besides, the biosynthesis of guanosine 5'-triphosphate (GTP), an essential substrate for GDP-fucose biosynthesis, was enhanced via overexpression of enzymes involved in the salvage pathway of GTP biosynthesis. The production capacities of metabolically engineered strains bearing different combinations of recombinant enzymes were compared. The shake flask fermentation of the strain expressing Fkp, Gpt, Gmk and Ndk obtained the maximum GDP-fucose content of 4.6 ± 0.22 μmol/g (dry cell mass), which is 4.2 fold that of the strain only expressing Fkp. Through fed-batch fermentation, the GDP-fucose content further rose to 6.6 ± 0.14 μmol/g (dry cell mass). In addition to a better productivity than previous fermentation processes based on the de novo pathway for GDP-fucose biosynthesis, the established schemes in this work also have the advantage to be a potential avenue to GDP-fucose analogs encompassing chemical modification on the fucose residue. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Shiga Toxin Producing Escherichia coli.

    Science.gov (United States)

    Bryan, Allen; Youngster, Ilan; McAdam, Alexander J

    2015-06-01

    Shiga toxin-producing Escherichia coli (STEC) is among the common causes of foodborne gastroenteritis. STEC is defined by the production of specific toxins, but within this pathotype there is a diverse group of organisms. This diversity has important consequences for understanding the pathogenesis of the organism, as well as for selecting the optimum strategy for diagnostic testing in the clinical laboratory. This review includes discussions of the mechanisms of pathogenesis, the range of manifestations of infection, and the several different methods of laboratory detection of Shiga toxin-producing E coli.

  12. Experimental evolution of E. coli

    Science.gov (United States)

    Zhang, Mengshi

    The evolution from unicellular to multicellular behavior is an essential step in the history of life. Our aim is to investigate the emergence of collective behavior in the model organism Escherichia coli (E. coli) and its selection advantages, such as better utilization of public goods. Our preliminary results suggest that the evolution of collective behavior may be a natural response to stressed conditions. Mailing address: Room 306 Science Centre North Block, The Chinese University of Hong Kong, Shatin, N.T. Hong Kong SAR. Phone: +852-3943-6354. Fax: +852-2603-5204. E-mail: mengshi0928@gmail.com.

  13. Expression in E. coli systems

    DEFF Research Database (Denmark)

    Krogsdam, Anne-M; Kristiansen, Karsten; Nøhr, Jane

    2003-01-01

    intracellularly in soluble form. In E. coli, proteins containing disulfide bonds are best produced by secretion because the disulfide forming foldases reside in the periplasm. Likewise, a correct N-terminus is more likely to be obtained upon secretion. Moreover, potentially toxic proteins are more likely......Owing to cost advantage, speed of production, and often high product yield (up to 50% of total cell protein), expression in Escherichia coli is generally the first choice when attempting to express a recombinant protein. Expression systems exist to produce recombinant protein intracellularly...

  14. Pathogenomics of uropathogenic Escherichia coli

    Directory of Open Access Journals (Sweden)

    J Agarwal

    2012-01-01

    Full Text Available Subset of faecal E. coli that can enter, colonize urinary tract and cause infection are known as uropathogenic E. coli (UPEC. UPEC strains act as opportunistic intracellular pathogens taking advantage of host susceptibility using a diverse array of virulence factors. Presence of specific virulence associated genes on genomic/pathogenicity islands and involvement of horizontal gene transfer appears to account for evolution and diversity of UPEC. Recent success in large-scale genome sequencing and comparative genomics has helped in unravelling UPEC pathogenomics. Here we review recent findings regarding virulence characteristics of UPEC and mechanisms involved in pathogenesis of urinary tract infection.

  15. Infektionen mit darmpathogenen Escherichia coli.

    NARCIS (Netherlands)

    Friedrich, Alexander; Stein, Jürgen; Dignass, Axel

    2001-01-01

    E. coli ist ein wesentlicher Bestandteil der physiologischen Darmflora des Menschen. Die üblicherweise im Darm vorkommenden Kolibakterien sind apathogen und für den Menschen eher nützlich (Sonnenborn u. Greinwald 1990). Allerdings kennen wir bei dieser Bakterienspezies auch ein breites Spektrum von

  16. Expression in E. coli systems

    DEFF Research Database (Denmark)

    Krogsdam, Anne-M; Kristiansen, Karsten; Nøhr, Jane

    2003-01-01

    intracellularly in soluble form. In E. coli, proteins containing disulfide bonds are best produced by secretion because the disulfide forming foldases reside in the periplasm. Likewise, a correct N-terminus is more likely to be obtained upon secretion. Moreover, potentially toxic proteins are more likely...

  17. Facilitated geranylgeranylation of shrimp ras-encoded p25 fusion protein by the binding with guanosine diphosphate.

    Science.gov (United States)

    Huang, C F; Chuang, N N

    1999-05-01

    A cDNA was isolated from the shrimp Penaeus japonicus by homology cloning. Similar to the mammalian Ras proteins, this shrimp hepatopancreas cDNA encodes a 187-residue polypeptide whose predicted amino acid sequence shares 85% homology with mammalian KB-Ras proteins and demonstrates identity in the guanine nucleotide binding domains. Expression of the cDNA of shrimp in Escherichia coli yielded a 25-kDa polypeptide with positive reactivity toward the monoclonal antibodies against Ras of mammals. As judged by nitrocellulose filtration assay, the specific GTP binding activity of ras-encoded p25 fusion protein was approximately 30,000 units/mg of protein, whereas that of GDP was 5,000 units/mg of protein. In other words, the GTP bound form of ras-encoded p25 fusion protein prevails. Fluorography analysis demonstrated that the prenylation of both shrimp Ras-GDP and shrimp Ras-GTP by protein geranylgeranyltransferase I of shrimp Penaeus japonicus exceeded that of nucleotide-free form of Ras by 10-fold and four-fold, respectively. That is, the protein geranylgeranyl transferase I prefers to react with ras-encoded p25 fusion protein in the GDP bound form.

  18. The molecular origin of the thiamine diphosphate-induced spectral bands of ThDP-dependent enzymes.

    Science.gov (United States)

    Kovina, Marina V; De Kok, Aart; Sevostyanova, Irina A; Khailova, Ludmila S; Belkina, Natalya V; Kochetov, German A

    2004-08-01

    New and previously published data on a variety of ThDP-dependent enzymes such as baker's yeast transketolase, yeast pyruvate decarboxylase and pyruvate dehydrogenase from pigeon breast muscle, bovine heart, bovine kidney, Neisseria meningitidis and E. coli show their spectral sensitivity to ThDP binding. Although ThDP-induced spectral changes are different for different enzymes, their universal origin is suggested as being caused by the intrinsic absorption of the pyrimidine ring of ThDP, bound in different tautomeric forms with different enzymes. Non-enzymatic models with pyrimidine-like compounds indicate that the specific protein environment of the aminopyrimidine ring of ThDP determines its tautomeric form and therefore the changeable features of the inducible effect. A polar environment causes the prevalence of the aminopyrimidine tautomeric form (short wavelength region is affected). For stabilization of the iminopyrimidine tautomeric form (both short- and long-wavelength regions are affected) two factors appear essential: (i) a nonpolar environment and (ii) a conservative carboxyl group of a specific glutamate residue interacting with the N1' atom of the aminopyrimidine ring. The two types of optical effect depend in a different way upon the pH, in full accordance with the hypothesis tested. From these studies it is concluded that the inducible optical rotation results from interaction of the aminopyrimidine ring with its asymmetric environment and is defined by the protonation state of N1' and the 4'-nitrogen.

  19. Functional characterization of UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferases of Escherichia coli and Caulobacter crescentus.

    Science.gov (United States)

    Patel, Kinnari B; Toh, Evelyn; Fernandez, Ximena B; Hanuszkiewicz, Anna; Hardy, Gail G; Brun, Yves V; Bernards, Mark A; Valvano, Miguel A

    2012-05-01

    Escherichia coli K-12 WcaJ and the Caulobacter crescentus HfsE, PssY, and PssZ enzymes are predicted to initiate the synthesis of colanic acid (CA) capsule and holdfast polysaccharide, respectively. These proteins belong to a prokaryotic family of membrane enzymes that catalyze the formation of a phosphoanhydride bond joining a hexose-1-phosphate with undecaprenyl phosphate (Und-P). In this study, in vivo complementation assays of an E. coli K-12 wcaJ mutant demonstrated that WcaJ and PssY can complement CA synthesis. Furthermore, WcaJ can restore holdfast production in C. crescentus. In vitro transferase assays demonstrated that both WcaJ and PssY utilize UDP-glucose but not UDP-galactose. However, in a strain of Salmonella enterica serovar Typhimurium deficient in the WbaP O antigen initiating galactosyltransferase, complementation with WcaJ or PssY resulted in O-antigen production. Gas chromatography-mass spectrometry (GC-MS) analysis of the lipopolysaccharide (LPS) revealed the attachment of both CA and O-antigen molecules to lipid A-core oligosaccharide (OS). Therefore, while UDP-glucose is the preferred substrate of WcaJ and PssY, these enzymes can also utilize UDP-galactose. This unexpected feature of WcaJ and PssY may help to map specific residues responsible for the nucleotide diphosphate specificity of these or similar enzymes. Also, the reconstitution of O-antigen synthesis in Salmonella, CA capsule synthesis in E. coli, and holdfast synthesis provide biological assays of high sensitivity to examine the sugar-1-phosphate transferase specificity of heterologous proteins.

  20. ADPase activity of recombinantly expressed thermotolerant ATPases may be caused by copurification of adenylate kinase of Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Baoyu; Sysoeva, Tatyana A.; Chowdhury, Saikat; Guo, Liang; Nixon, B.Tracy; (IIT); (Penn)

    2009-10-06

    Except for apyrases, ATPases generally target only the {gamma}-phosphate of a nucleotide. Some non-apyrase ATPases from thermophilic microorganisms are reported to hydrolyze ADP as well as ATP, which has been described as a novel property of the ATPases from extreme thermophiles. Here, we describe an apparent ADP hydrolysis by highly purified preparations of the AAA+ ATPase NtrC1 from an extremely thermophilic bacterium, Aquifex aeolicus. This activity is actually a combination of the activities of the ATPase and contaminating adenylate kinase (AK) from Escherichia coli, which is present at 1/10 000 of the level of the ATPase. AK catalyzes conversion of two molecules of ADP into AMP and ATP, the latter being a substrate for the ATPase. We raise concern that the observed thermotolerance of E. coli AK and its copurification with thermostable proteins by commonly used methods may confound studies of enzymes that specifically catalyze hydrolysis of nucleoside diphosphates or triphosphates. For example, contamination with E. coli AK may be responsible for reported ADPase activities of the ATPase chaperonins from Pyrococcus furiosus, Pyrococcus horikoshii, Methanococcus jannaschii and Thermoplasma acidophilum; the ATP/ADP-dependent DNA ligases from Aeropyrum pernix K1 and Staphylothermus marinus; or the reported ATP-dependent activities of ADP-dependent phosphofructokinase of P. furiosus. Purification methods developed to separate NtrC1 ATPase from AK also revealed two distinct forms of the ATPase. One is tightly bound to ADP or GDP and able to bind to Q but not S ion exchange matrixes. The other is nucleotide-free and binds to both Q and S ion exchange matrixes.

  1. Regulation of resin acid synthesis in Pinus densiflora by differential transcription of genes encoding multiple 1-deoxy-D-xylulose 5-phosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase genes.

    Science.gov (United States)

    Kim, Yeon-Bok; Kim, Sang-Min; Kang, Min-Kyoung; Kuzuyama, Tomohisa; Lee, Jong Kyu; Park, Seung-Chan; Shin, Sang-Chul; Kim, Soo-Un

    2009-05-01

    Pinus densiflora Siebold et Zucc. is the major green canopy species in the mountainous area of Korea. To assess the response of resin acid biosynthetic genes to mechanical and chemical stimuli, we cloned cDNAs of genes encoding enzymes involved in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway (1-deoxy-d-xylulose 5-phosphate synthase (PdDXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (PdDXR) and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (PdHDR)) by the rapid amplification of cDNA ends (RACE) technique. In addition, we cloned the gene encoding abietadiene synthase (PdABS) as a marker for the site of pine resin biosynthesis. PdHDR and PdDXS occurred as two gene families. In the phylogenetic trees, PdDXSs, PdDXR and PdHDRs each formed a separate clade from their respective angiosperm homologs. PdDXS2, PdHDR2 and PdDXR were most actively transcribed in stem wood, whereas PdABS was specifically transcribed. The abundance of PdDXS2 transcripts in wood in the resting state was generally 50-fold higher than the abundance of PdDXS1 transcripts, and PdHDR2 transcripts were more abundant by an order of magnitude in wood than in other tissues, with the ratio of PdHDR2 to PdHDR1 transcripts in wood being about 1. Application of 1 mM methyl jasmonate (MeJA) selectively enhanced the transcript levels of PdDXS2 and PdHDR2 in wood. The ratios of PdDXS2 to PdDXS1 and PdHDR2 to PdHDR1 reached 900 and 20, respectively, on the second day after MeJA treatment, whereas the transcript level of PdABS increased twofold by 3 days after MeJA treatment. Wounding of the stem differentially enhanced the transcript ratios of PdDXS2 to PdDXS1 and PdHDR2 to PdHDR1 to 300 and 70, respectively. The increase in the transcript levels of the MEP pathway genes in response to wounding was accompanied by two orders of magnitude increase in PdABS transcripts. These observations indicated that resin acid biosynthesis activity, represented by PdABS transcription, was correlated

  2. Chromosomal features of Escherichia coli serotype O2:K2, an avian pathogenic E. coli

    DEFF Research Database (Denmark)

    Jørgensen, Steffen L; Kudirkiene, Egle; Li, Lili

    2017-01-01

    Escherichia coli causing infection outside the gastrointestinal system are referred to as extra-intestinal pathogenic E. coli. Avian pathogenic E. coli is a subgroup of extra-intestinal pathogenic E. coli and infections due to avian pathogenic E. coli have major impact on poultry production economy...... and welfare worldwide. An almost defining characteristic of avian pathogenic E. coli is the carriage of plasmids, which may encode virulence factors and antibiotic resistance determinates. For the same reason, plasmids of avian pathogenic E. coli have been intensively studied. However, genes encoded...... by the chromosome may also be important for disease manifestation and antimicrobial resistance. For the E. coli strain APEC_O2 the plasmids have been sequenced and analyzed in several studies, and E. coli APEC_O2 may therefore serve as a reference strain in future studies. Here we describe the chromosomal features...

  3. Thiophene metabolism by E. coli

    Energy Technology Data Exchange (ETDEWEB)

    Clark, D.P.

    1991-01-01

    The objective of this project is to investigate the mechanism of degradation of sulfur-containing heterocyclic molecules by mutant strains of Escherichia coli K-12. We have previously isolated multiple mutants of E. coli which had gained the capacity to oxidize thiophene compounds and their furan analogs. We have focused on the thdA mutation in our subsequent research, as this appears to be in a regulatory gene central to the thiophene/furan oxidation system. The thdF gene appears to be more directly involved in the oxidation reactions, whereas thdC and thdD are apparently required for increased protection against the toxic effects of thiophene and furan compounds. 4 tabs.

  4. Multiplex PCR Assay for Identification of Human Diarrheagenic Escherichia coli

    OpenAIRE

    Toma, Claudia; Lu, Yan; Higa, Naomi; Nakasone, Noboru; Isabel CHINEN; Baschkier, Ariela; Rivas, Marta; Iwanaga, Masaaki

    2003-01-01

    A multiplex PCR assay for the identification of human diarrheagenic Escherichia coli was developed. The targets selected for each category were eae for enteropathogenic E. coli, stx for Shiga toxin-producing E. coli, elt and est for enterotoxigenic E. coli, ipaH for enteroinvasive E. coli, and aggR for enteroaggregative E. coli. This assay allowed the categorization of a diarrheagenic E. coli strain in a single reaction tube.

  5. Multiplex PCR Assay for Identification of Human Diarrheagenic Escherichia coli

    OpenAIRE

    2003-01-01

    A multiplex PCR assay for the identification of human diarrheagenic Escherichia coli was developed. The targets selected for each category were eae for enteropathogenic E. coli, stx for Shiga toxin-producing E. coli, elt and est for enterotoxigenic E. coli, ipaH for enteroinvasive E. coli, and aggR for enteroaggregative E. coli. This assay allowed the categorization of a diarrheagenic E. coli strain in a single reaction tube.

  6. Strontium fructose 1,6-diphosphate prevents bone loss in a rat model of postmenopausal osteoporosis via the OPG/RANKL/RANK pathway

    Institute of Scientific and Technical Information of China (English)

    Bo MA; Qi ZHANG; Di WU; Yong-lu WANG; Ying-ying HU; Yan-ping CHENG; Zhen-dong YANG; Ya-ya ZHENG; Han-jie YING

    2012-01-01

    Aim:To evaluate the protective effects of strontium fructose 1,6-diphosphate (FDP-Sr),a novel strontium salt that combined fructose 1,6-diphosphate (FDP)with strontium,on bone in an ovariectomy-induced model of bone loss.Methods:Eighty female Sprague-Dawley rats were ovariectomized (OVX)or sham-operated.Three months later,the rats were assigned to six groups (10 for each):sham-operated,OVX control,OVX+FDP-Sr (110,220,or 440 mg/kg),or OVX+strontium ranelate (SR,180 mg/kg).Drugs were administered orally for 3 months.When the treatment was terminated,the following parameters were assessed:bone mineral density (BMD),the biomechanical properties of the femur and lumbar vertebrae,trabecular histomorphology,serum phosphorus,calcium,bone-specific alkaline phosphatase (B-ALP),tartrate-resistant acid phosphatase 5b (TRACP5b),N-telopeptide of type I collagen (NTx)and a series of markers for oxidative stress.Receptor activator of NF-kB ligand (RANKL)and osteoprotegerin (OPG)levels in serum were measured using ELISA and their gene expression levels in the bone were measured using R-T PCR.Results:Treatment with FDP-Sr (220 or 440 mg/kg)or SR (180 mg/kg)significantly increased the BMD and improved the bone microarchitecture and bone strength in OVX rats.The treatments also decreased in the levels of H202 and MDA,restored the CAT level in serum and bone marrow,increased the serum B-ALP and decreased NTx and TRACP 5b in OVX rats.Treatment with FDP-Sr decreased the RANKL level,and increased the OPG level in serum in a dose-dependent manner.It also significantly down-regulated the RANKL expression and ul-regulated OPG expression in bone marrow.Conclusion:FDP-Sr may be an effectve treatment for postmenopausal osteoporosis that acts,in part,via a decrease in osteoclastogenesis through the OPG\\RANKL\\RANK pathway.

  7. [The synthesis of P1-[11-(anthracen-9-ylmethoxy)undecyl]-P2(2-Acetamido-2-deoxy-α-D-glucopyranosyl) diphosphate and the study of its acceptor properties in the enzymic reaction catalyzed by D-rhamnosyltransferase from Pseudomonas aeruginosa].

    Science.gov (United States)

    Vinnikova, A N; Torgov, V I; Utkina, N S; Veselovsky, V V; Druzhinina, T N; Wang, S; Brockhausen; Danilov, L L

    2015-01-01

    P1-[11-(Anthracen-9-ylmethoxy)undecyl]-P2-(2-acetamido-2-deoxy-α-D-glucopyranosyl) diphosphate, a fluorescent derivative of undecyl diphosphate 2-acetamido-2-deoxyglucose, was chemically synthesized. The ability of the compound to serve as acceptor substrate of D-rhamnose residue in the enzymatic reaction catalyzed by D-rhamnosyltransferase from Pseudomonas aeruginosa PAO1 was demonstrated.

  8. Enterohemorrhagic E. coli (EHEC) pathogenesis

    OpenAIRE

    Vanessa eSperandio; Y eNguyen

    2012-01-01

    Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a human pathogen responsible for outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) worldwide. Conventional antimicrobials trigger an SOS response in EHEC that promotes the release of the potent Shiga toxin that is responsible for much of the morbidity and mortality associated with EHEC infection. Cattle are a natural reservoir of EHEC, and approximately 75% of EHEC outbreaks are linked to the consumption of contamina...

  9. Robust growth of Escherichia coli.

    Science.gov (United States)

    Wang, Ping; Robert, Lydia; Pelletier, James; Dang, Wei Lien; Taddei, Francois; Wright, Andrew; Jun, Suckjoon

    2010-06-22

    The quantitative study of the cell growth has led to many fundamental insights in our understanding of a wide range of subjects, from the cell cycle to senescence. Of particular importance is the growth rate, whose constancy represents a physiological steady state of an organism. Recent studies, however, suggest that the rate of elongation during exponential growth of bacterial cells decreases cumulatively with replicative age for both asymmetrically and symmetrically dividing organisms, implying that a "steady-state" population consists of individual cells that are never in a steady state of growth. To resolve this seeming paradoxical observation, we studied the long-term growth and division patterns of Escherichia coli cells by employing a microfluidic device designed to follow steady-state growth and division of a large number of cells at a defined reproductive age. Our analysis of approximately 10(5) individual cells reveals a remarkable stability of growth whereby the mother cell inherits the same pole for hundreds of generations. We further show that death of E. coli is not purely stochastic but is the result of accumulating damages. We conclude that E. coli, unlike all other aging model systems studied to date, has a robust mechanism of growth that is decoupled from cell death.

  10. 1,6-二磷酸果糖治疗小儿病毒性心肌炎临床疗效观察%Clinical effect of Fructose-1, 6-diphosphate on pedo-viral myocarditis

    Institute of Scientific and Technical Information of China (English)

    郭妍妍

    2010-01-01

    目的 观察1,6-二磷酸果糖治疗小儿病毒性心肌炎的临床效果.方法 将122例病毒性心肌炎的患儿随机抽签分治疗组60例及对照组62例,治疗组应用1,6-二磷酸果糖,对照组应用能量合剂.结果 治疗组总有效52例(86.7%),优于对照组42例(67.7%);治疗组合并早搏的患儿减少早搏总有效16例(88.9%),优于对照组19例(70.4%),2组差异有统计学意义(P<0.05).结论 1,6-二磷酸果糖治疗小儿病毒性心肌炎有明显疗效,值得临床应用.%Objectives To investigate the curative effect of Fructose-1, 6-diphosphate on pedo-viral myo-carditis. Methods Sixty patients in the treatment group had Fructose-1, 6-diphosphate and conventional therapy.62 patients in the control group were only treated with conventional therapy. Results The total effective rate of treatment group(86.7%) was higher than that of control group(67.7%). Fructose-1,6-diphosphate increased the effective rate, decreased the myo-creatase and improved the clinical symptoms. Conclusions Fructose-1, 6-diphosphate is effective for treating pedo-viral myocarditis.

  11. Structure of the ent -Copalyl Diphosphate Synthase PtmT2 from Streptomyces platensis CB00739, a Bacterial Type II Diterpene Synthase

    Energy Technology Data Exchange (ETDEWEB)

    Rudolf, Jeffrey D.; Dong, Liao-Bin; Cao, Hongnan; Hatzos-Skintges, Catherine; Osipiuk, Jerzy; Endres, Michael; Chang, Chin-Yuan; Ma, Ming; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N.; Shen, Ben

    2016-08-31

    Terpenoids are the largest and most structurally diverse family of natural products found in nature, yet their presence in bacteria is underappreciated. The carbon skeletons of terpenoids are generated through carbocation-dependent cyclization cascades catalyzed by terpene synthases (TSs). Type I and type II TSs initiate cyclization via diphosphate ionization and protonation, respectively, and protein structures of both types are known. Most plant diterpene synthases (DTSs) possess three alpha-helical domains (alpha beta gamma), which are thought to have arisen from the fusion of discrete, ancestral bacterial type I TSs (alpha) and type II TSs (beta gamma). Type II DTSs of bacterial origin, of which there are no structurally characterized members, are a missing piece in the structural evolution of TSs. Here, we report the first crystal structure of a type II DTS from bacteria. PtnaT2 from Streptomyces platensis CB00739 was verified as an ent-copalyl diphosphate synthase involved in the biosynthesis of platensimycin and platencin. The crystal structure of PtmT2 was solved at a resolution of 1.80 angstrom, and docking studies suggest the catalytically active conformation of geranylgeranyl diphosphate (GGPP). Site-directed mutagenesis confirmed residues involved in binding the diphosphate moiety of GGPP and identified DxxxxE as a potential Mg2+-binding motif for type II DTSs of bacterial origin. Finally, both the shape and physicochemical properties of the active sites are responsible for determining specific catalytic outcomes of TSs. The structure of PtmT2 fundamentally advances the knowledge of bacterial TSs, their mechanisms, and their role in the evolution of TSs.

  12. Utilizing the O-antigen lipopolysaccharide biosynthesis pathway in Escherichia coli to interrogate the substrate specificities of exogenous glycosyltransferase genes in a combinatorial approach.

    Science.gov (United States)

    Johansen, Eric B; Szoka, Francis C; Zaleski, Anthony; Apicella, Michael A; Gibson, Bradford W

    2010-06-01

    In previous work, our laboratory generated novel chimeric lipopolysaccharides (LPS) in Escherichia coli transformed with a plasmid containing exogenous lipooligosaccharide synthesis genes (lsg) from Haemophilus influenzae. Analysis of these novel oligosaccharide-LPS chimeras allowed characterization of the carbohydrate structures generated by several putative glycosyltransferase genes within the lsg locus. Here, we adapted this strategy to construct a modular approach to study the synthetic properties of individual glycosyltransferases expressed alone and in combinations. To this end, a set of expression vectors containing one to four putative glycosyltransferase genes from the lsg locus, lsgC-F, were transformed into E. coli K12 (XL-1) which is defective in LPS O-antigen biosynthesis. This strategy relied on the inclusion of the H. influenzae gene product lsgG in every plasmid construct, which partially rescues the E. coli LPS biosynthesis defect by priming uridine diphosphate-undecaprenyl in the WecA-dependent O-antigen synthetic pathway with N-acetyl-glucosamine (GlcNAc). This GlcNAc-undecaprenyl then served as an acceptor substrate for further carbohydrate extension by transformed glycosyltransferases. The resultant LPS-linked chimeric glycans were isolated from their E. coli constructs and characterized by mass spectrometry, methylation analysis and enzyme-linked immunosorbent assays. These structural data allowed the specificity of various glycosyltransferases to be unambiguously assigned to individual genes. LsgF was found to transfer a galactose (Gal) to terminal GlcNAc. LsgE was found to transfer GlcNAc to Gal-GlcNAc, and both LsgF and LsgD were found to transfer Gal to GlcNAc-Gal-GlcNAc but with differing linkage specificities. This method can be generalized and readily adapted to study the substrate specificity of other putative or uncharacterized glycosyltransferases.

  13. Involvement of nucleotide diphosphate kinase 2 in the reopening of the sensitive period of filial imprinting of domestic chicks (Gallus gallus domesticus).

    Science.gov (United States)

    Yamaguchi, Shinji; Aoki, Naoya; Takehara, Akihiko; Mori, Masaru; Kanai, Akio; Matsushima, Toshiya; Homma, Koichi J

    2016-01-26

    Filial imprinting is a behavior characterized by the sensitive or critical period restricted to the first few days after hatching. Once the sensitive period is closed, it is widely believed that chicks can never be imprinted under natural conditions. Previously, we showed that the exogenous injection of T3 reopened the sensitive period which was already closed. That study suggested that T3 functioned by way of a rapid non-genomic action; however, the molecular mechanism of how T3 reopens the sensitive period remains unknown. Here, we show that the phosphorylation level of nucleotide diphosphate kinase 2 (NDPK2) was upregulated following T3 injection. Pharmacological deprivation of the kinase activity of NDPK hampered the molecular process prerequisite for the reopening of the sensitive period of filial imprinting. Moreover, it is shown that the kinase activity of NDPK2 participates in the priming process by T3 signaling which endows the potential for learning. Our data indicate that NDPK2 plays a crucial role downstream of T3 action and that its phosphorylation is involved in the non-genomic signaling during imprinting. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  14. Effects of fructose-1,6-diphosphate on concentration of calcium and activities of sarcoplosnic Ca2+-ATPase in cardiomyocytes of Adriamycin-treated rats

    Institute of Scientific and Technical Information of China (English)

    CAI Wei; CHEN Jun-zhu; RUAN Li-ming; WANG Yi-na

    2005-01-01

    Objective: To observe the effects of fructose-1,6-diphosphate (FDP) on serum levels of cardiac troponin I (cTnI) and creatine kinase-MB (CK-MB), as well as the concentration of calcium in cardiomyocytes (Myo[Ca2+]) and activity of sarcoplosnic Ca2+-ATPase (SRCa2+-ATPase) in Adriamycin (ADR)-treated rats. Methods: Rats were intraperitoneally injected with ADR (2.5mg/kg every other day for 6 times) and then with different dosages of FDP (every other day for twenty-one times). Bi-antibodies sandwich Enzyme linked immune absorption assay (ELISA) was performed to detect serum level of cTnI. CK-MB was detected by monoclonal antibody, Myo[Ca2+] was detected by fluorescent spectrophotometry and the activity of SRCa2+-ATPase was detected by inorganic phosphate method. Results: FDP (300, 600, 1200 mg/kg) significantly reduced the serum levels of cTnI and CK-MB, while at the same time decreased calcium concentration and increased SRCa2+-ATPase activity in cardiomyocytes of ADR-treated rats (P<0.01). Conclusions: FDP might alleviate the cardiotoxic effects induced by ADR through decreasing calcium level as well as increasing SRCa2+-ATPase activity in cardiomyocytes.

  15. Adenosine diphosphate-induced aggregation of human platelets in flow through tubes. I. Measurement of concentration and size of single platelets and aggregates.

    Science.gov (United States)

    Bell, D N; Spain, S; Goldsmith, H L

    1989-11-01

    A double infusion flow system and particle sizing technique were developed to study the effect of time and shear rate on adenosine diphosphate-induced platelet aggregation in Poiseuille flow. Citrated platelet-rich plasma, PRP, and 2 microM ADP were simultaneously infused into a 40-microliters cylindrical mixing chamber at a fixed flow ratio, PRP/ADP = 9:1. After rapid mixing by a rotating magnetic stirbar, the platelet suspension flowed through 1.19 or 0.76 mm i.d. polyethylene tubing for mean transit times, t, from 0.1 to 86 s, over a range of mean tube shear rate, G, from 41.9 to 1,000 s-1. Known volumes of suspension were collected into 0.5% buffered glutaraldehyde, and all particles in the volume range 1-10(5) microns 3 were counted and sized using a model ZM particle counter (Coulter Electronics Inc., Hialeah, FL) and a logarithmic amplifier. The decrease in the single platelet concentration served as an overall index of aggregation. The decrease in the total particle concentration was used to calculate the collision capture efficiency during the early stages of aggregation, and aggregate growth was followed by changes in the volume fraction of particles of successively increasing size. Preliminary results demonstrate that both collision efficiency and particle volume fraction reveal important aspects of the aggregation process not indicated by changes in the single platelet concentration alone.

  16. Enzymatic synthesis of fructose 1,6-diphosphate with ATP regeneration in a batch reactor and a semibatch reactor using purified enzymes of Bacillus stearothermophilus.

    Science.gov (United States)

    Widjaja, A; Shiroshima, M; Yasuda, M; Ogino, H; Nakajima, H; Ishikawa, H

    1999-01-01

    The enzymatic synthesis of fructose 1,6-diphosphate (FDP), an important glycolytic intermediate whose applications in the field of medicine have generated a great deal of interest, was performed in a batch reactor and a semibatch reactor. Using the batch reactor, FDP was first synthesized from glucose by three enzymatic reactions and the ATP consumed was regenerated simultaneously using conjugated enzymes, all of which were purified from crude cell extract of thermophilic Bacillus stearothermophilus. The results of the experiments performed using several enzyme concentrations suggest the existence of an optimum concentration for each enzyme at which the maximum FDP yield can be attained. Since the thermal decomposition of acetyl phosphate reduced the yield of FDP in the batch reactor, the use of a semibatch reactor in which acetyl phosphate was fed continuously was examined. The yield of FDP was improved but the time required to complete the reaction was longer, resulting in a lower productivity of FDP. The yields observed in the two reactors using various enzyme and substrate concentrations were in good agreement with the theoretical predictions calculated based on differential equations derived for the system using the rate equations and the kinetic parameters determined previously. This means that these equations can be used for the analysis of the experimental results as well as for determining the optimum experimental conditions.

  17. Thiamine increases the resistance of baker's yeast Saccharomyces cerevisiae against oxidative, osmotic and thermal stress, through mechanisms partly independent of thiamine diphosphate-bound enzymes.

    Science.gov (United States)

    Wolak, Natalia; Kowalska, Ewa; Kozik, Andrzej; Rapala-Kozik, Maria

    2014-12-01

    Numerous recent studies have established a hypothesis that thiamine (vitamin B1 ) is involved in the responses of different organisms against stress, also suggesting that underlying mechanisms are not limited to the universal role of thiamine diphosphate (TDP) in the central cellular metabolism. The current work aimed at characterising the effect of exogenously added thiamine on the response of baker's yeast Saccharomyces cerevisiae to the oxidative (1 mM H2 O2 ), osmotic (1 M sorbitol) and thermal (42 °C) stress. As compared to the yeast culture in thiamine-free medium, in the presence of 1.4 μM external thiamine, (1) the relative mRNA levels of major TDP-dependent enzymes under stress conditions vs. unstressed control (the 'stress/control ratio') were moderately lower, (2) the stress/control ratio was strongly decreased for the transcript levels of several stress markers localised to the cytoplasm, peroxisomes, the cell wall and (with the strongest effect observed) the mitochondria (e.g. Mn-superoxide dismutase), (3) the production of reactive oxygen and nitrogen species under stress conditions was markedly decreased, with the significant alleviation of concomitant protein oxidation. The results obtained suggest the involvement of thiamine in the maintenance of redox balance in yeast cells under oxidative stress conditions, partly independent of the functions of TDP-dependent enzymes.

  18. Which stage of the process of apotransketolase interaction with thiamine diphosphate is affected by the regulatory activity of the donor substrate?

    Science.gov (United States)

    Esakova, O A; Meshalkina, L E; Golbik, R; Brauer, J; Hübner, G; Kochetov, G A

    2007-02-01

    The interaction of thiamine diphosphate (ThDP) with transketolase (TK) involves at least two stages: [formula: see text] During the first stage, an inactive intermediate complex (TK...ThDP) is formed, which is then transformed into a catalytically active holoenzyme (TK* - ThDP). The second stage is related to conformational changes of the protein. In the preceding publication (Esakova, O. A., Meshalkina, L. E., Golbik, R., Hübner, G., and Kochetov, G. A. Eur. J. Biochem. 2004, 271, 4189 - 4194) we reported that the affinity of ThDP for TK considerably increases in the presence of the donor substrate, which may be a mechanism whereby the activity of the enzyme is regulated under the conditions of the coenzyme deficiency. Here, we demonstrate that the substrate affects the stage of the reverse conformational transition, characterized by the constant k(-1): in the presence of the substrate, its value is decreased several fold, whereas K(d) and k(+1) remain unchanged.

  19. An activity, stability and selectivity comparison of propioin synthesis by thiamine diphosphate-dependent enzymes in a solid/gas bioreactor.

    Science.gov (United States)

    Mikolajek, Renaud; Spiess, Antje C; Pohl, Martina; Lamare, Sylvain; Büchs, Jochen

    2007-06-18

    Enzymatic carboligation in a solid/gas bioreactor represents a new challenge in biotechnology. In this paper, the continuous gas-phase production of propioin from two propanal molecules by using thiamine diphosphate-dependent enzymes was studied. Two enzymes were used, namely benzaldehyde lyase (BAL) from Pseudomonas fluorescens and benzoylformate decarboxylase (BFD) from Pseudomonas putida. The enzymes are homologous and catalyze carboligase and carbolyase reactions in which no external cofactor regeneration is needed. The influence of water and substrate activity on the initial reaction rate and biocatalyst stability was investigated. An increase in water activity raised the initial reaction rates to the maximal values of 250 and 80 U g(-1) for BAL and BFD, respectively. The half-life showed the same trend with maximal values of 50 and 78 min for BAL and BFD, respectively. The increase in the half-life by increasing water activity was unexpected. It was also observed that BFD is more stable than BAL in the presence of the substrate propanal. Both enzymes showed substrate inhibition in the kinetic studies, and BAL was also deactivated during the reaction. Unexpectedly, the stereoselectivity of both enzymes (ee of 19 % for BAL and racemic mixture for BFD) was significantly impaired in the gas phase compared to the liquid phase.

  20. Adenosine Diphosphate Ribosylation Factor-GTPaseActivating Protein Stimulates the Transport of AUX1Endosome, Which Relies on Actin Cytoskeletal Organization in Rice Root DevelopmentF

    Institute of Scientific and Technical Information of China (English)

    Cheng Du; Yunyuan XU; Yingdian Wang; Kang Chong

    2011-01-01

    Polar auxin transport,which depends on polarized subcellular distribution of AUXIN RESISTANT 1/LIKE AUX1 (AUX1/LAX) influx carriers and PIN-FORMED (PIN) efflux carriers,mediates various processes of plant growth and development.Endosomal recycling of PIN1 is mediated by an adenosine diphosphate (ADP)ribosylation factor (ARF)-GTPase exchange factor protein,GNOM.However,the mediation of auxin influx carrier recycling is poorly understood.Here,we report that overexpression of OsAGAP,an ARF-GTPase-activating protein in rice,stimulates vesicle transport from the plasma membrane to the Golgi apparatus in protoplasts and transgenic plants and induces the accumulation of early endosomes and AUX1.AUX1 endosomes could partially colocalize with FM4-64 labeled early endosome after actin disruption.Furthermore,OsAGAP is involved in actin cytoskeletal organization,and its overexpression tends to reduce the thickness and bundling of actin filaments.Fluorescence recovery after photobleaching analysis revealed exocytosis of the AUX1 recycling endosome was not affected in the OsAGAP overexpression cells,and was only slightly promoted when the actin filaments were completely disrupted by Lat B.Thus,we propose that AUX1 accumulation in the OsAGAP overexpression and actin disrupted cells may be due to the fact that endocytosis of the auxin influx carrier AUX1 early endosome was greatly promoted by actin cytoskeleton disruption.

  1. Effects of n-3 fatty acid, fructose-1,6-diphosphate and glutamine on mucosal cell proliferation and apoptosis of small bowel graft after transplantation in rats

    Institute of Scientific and Technical Information of China (English)

    Xiao-Ting Wu; Jie-Shou Li; Xiao-Fei Zhao; Ning Li; Yu-Kui Ma; Wen Zhuang; Yong Zhou; Gang Yang

    2003-01-01

    AIM: To evaluate the effects of n-3 fatty acids (n-3FA),fructose-1,6-diphosphate (FDP) and glutamine (GLN) on mucosal cell proliferation and apoptosis of small bowel graft. METHODS: One hundred and ninety-six inbred strain Wistar rats were grouped as donors and recipients, and underwent heterotopic small bowel transplantation (SBT). n-3FA, FDP and GLN were administered via gastric tube as well as venous infusion for 10 days before and after surgery, respectively.The proliferation and apoptosis of mucosal cells were analyzed with flow cytometry and in situ cell death detection kits. RESULTS: Apparent apoptosis and minor proliferation of mucosal cells of small bowel graft after transplantation were observed. A higher mucosal cell proliferative index and lower apoptotic index were found in all small bowel grafts after supplying with n-3FA, FDP and GLN. CONCLUSION: Nutritional support with n-3FA, FDP and GLN promotes mucosal cell proliferation significantly, and prevents mucosal cell from undergoing apoptosis with different degrees. These regulatory effects on the apoptosis alter the structure and absorption function of transplanted small bowel favorably.

  2. Structural Views along the Mycobacterium tuberculosis MenD Reaction Pathway Illuminate Key Aspects of Thiamin Diphosphate-Dependent Enzyme Mechanisms.

    Science.gov (United States)

    Jirgis, Ehab N M; Bashiri, Ghader; Bulloch, Esther M M; Johnston, Jodie M; Baker, Edward N

    2016-07-06

    Menaquinone (MQ) is an essential component of the respiratory chains of many pathogenic organisms, including Mycobacterium tuberculosis (Mtb). The first committed step in MQ biosynthesis is catalyzed by 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD), a thiamin diphosphate (ThDP)-dependent enzyme. Catalysis proceeds through two covalent intermediates as the substrates 2-oxoglutarate and isochorismate are successively added to the cofactor before final cleavage of the product. We have determined a series of crystal structures of Mtb-MenD that map the binding of both substrates, visualizing each step in the MenD catalytic cycle, including both intermediates. ThDP binding induces a marked asymmetry between the coupled active sites of each dimer, and possible mechanisms of communication can be identified. The crystal structures also reveal conformational features of the two intermediates that facilitate reaction but prevent premature product release. These data fully map chemical space to inform early-stage drug discovery targeting MenD.

  3. Cloning of a NaCl-induced fructose-1, 6-diphosphate aldolase cDNA from Dunaliella salina and its expression in tobacco

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Xiaoning; (张晓宁); LIN; Changfa; (林长发); CHEN; Huoying; (陈火英); WANG; Hao; (王; 昊); QU; Zhicai; (曲志才); ZHANG; Hongwei; (张宏伟); YAO; Jianhong; (姚剑虹); SHEN; Daleng; (沈大棱)

    2003-01-01

    Using Rapid Amplification of cDNA ends (RACE) technique, the full-length cDNA encoding a NaCl-induced fructose-1, 6- diphosphate aldolase (DsALDP) was obtained. It was shown that the DsALDP had a relatively high homology (66%-73%) to chloroplast fructose-1, 6-diphos- phate aldolase (AldP) in many plants according to their amino acid sequences. The phylogenetic analysis further confirmed that AldP in alga is the nearest to DsALDP. As to its expression pattern, DsALDP was de novo synthesized by NaCl induction. Its expression level was significantly changed with inducing time. After the selected DsALDP cDNA subcloned into a binary vector pBI121, the new construct was introduced into tobacco by Agrobacterium tumefaciens. The results of Southern blot and RT-PCR analysis of four transgenic T1 plants indicated that DsALDP was integrated into genome of these transgenic plants and effectively expressed. Aldolase activities have been detected in T1-1, T1-2 and T1-3 plants by bioassay under 100-200 mmol/L NaCl. It was also observed that proline contents in them were differentially increased.

  4. Chemical synthesis of guanosine diphosphate mannuronic acid (GDP-ManA) and its C-4-O-methyl and C-4-deoxy congeners.

    Science.gov (United States)

    Zhang, Qingju; Howell, P Lynne; Overkleeft, Herman S; Filippov, Dmitri V; van der Marel, Gijsbert A; Codée, Jeroen D C

    2017-10-10

    Described is the first synthesis of guanosine diphosphate mannuronic acid (GDP-ManA), the sugar donor used by algae and bacteria for the production of alginate, an anionic polysaccharide composed of β-d-mannuronic acid (ManA) and α-l-guluronic acid (GulA). Understanding the biosynthesis of these polyanionic polysaccharides on the molecular level, opens up avenues to use and modulate the biosynthesis machinery for biotechnological and therapeutic applications. The synthesis reported here delivers multi-milligram amounts of the GDP-ManA donor that can be used to study the polymerase (Alg8 in Pseudomonas aeruginosa) that generates the poly-ManA chain. Also reported is the assembly of two close analogues of GDP-ManA: the first bears a C-4-O-methyl group, while the second has been deoxygenated at this position. Both molecules may be used as "chain stoppers" in future enzymatic ManA polymerisation reactions. The crucial pyrophosphate linkage of the GDP-mannuronic acids has been constructed by the phosphorylation of the appropriate ManA-1-phosphates with a guanosine phosphoramidite. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Identification of Guanosine 5‧-diphosphate as Potential Iron Mobilizer: Preventing the Hepcidin-Ferroportin Interaction and Modulating the Interleukin-6/Stat-3 Pathway

    Science.gov (United States)

    Angmo, Stanzin; Tripathi, Neha; Abbat, Sheenu; Sharma, Shailesh; Singh, Shelley Sardul; Halder, Avishek; Yadav, Kamalendra; Shukla, Geeta; Sandhir, Rajat; Rishi, Vikas; Bharatam, Prasad V.; Yadav, Hariom; Singhal, Nitin Kumar

    2017-01-01

    Hepcidin, a peptide hormone, is a key regulator in mammalian iron homeostasis. Increased level of hepcidin due to inflammatory conditions stimulates the ferroportin (FPN) transporter internalization, impairing the iron absorption; clinically manifested as anemia of inflammation (AI). Inhibiting hepcidin-mediated FPN degradation is proposed as an important strategy to combat AI. A systematic approach involving in silico, in vitro, ex vivo and in vivo studies is employed to identify hepcidin-binding agents. The virtual screening of 68,752 natural compounds via molecular docking resulted into identification of guanosine 5‧-diphosphate (GDP) as a promising hepcidin-binding agent. The molecular dynamics simulations helped to identify the important hepcidin residues involved in stabilization of hepcidin-GDP complex. The results gave a preliminary indication that GDP may possibly inhibit the hepcidin-FPN interactions. The in vitro studies revealed that GDP caused FPN stabilization (FPN-GFP cell lines) and increased the FPN-mediated cellular iron efflux (HepG2 and Caco-2 cells). Interestingly, the co-administration of GDP and ferrous sulphate (FeSO4) ameliorated the turpentine-induced AI in mice (indicated by increased haemoglobin level, serum iron, FPN expression and decreased ferritin level). These results suggest that GDP a promising natural small-molecule inhibitor that targets Hepcidin-FPN complex may be incorporated with iron supplement regimens to ameliorate AI.

  6. Chromosomal features of Escherichia coli serotype O2:K2, an avian pathogenic E. coli.

    Science.gov (United States)

    Jørgensen, Steffen L; Kudirkiene, Egle; Li, Lili; Christensen, Jens P; Olsen, John E; Nolan, Lisa; Olsen, Rikke H

    2017-01-01

    Escherichia coli causing infection outside the gastrointestinal system are referred to as extra-intestinal pathogenic E. coli. Avian pathogenic E. coli is a subgroup of extra-intestinal pathogenic E. coli and infections due to avian pathogenic E. coli have major impact on poultry production economy and welfare worldwide. An almost defining characteristic of avian pathogenic E. coli is the carriage of plasmids, which may encode virulence factors and antibiotic resistance determinates. For the same reason, plasmids of avian pathogenic E. coli have been intensively studied. However, genes encoded by the chromosome may also be important for disease manifestation and antimicrobial resistance. For the E. coli strain APEC_O2 the plasmids have been sequenced and analyzed in several studies, and E. coli APEC_O2 may therefore serve as a reference strain in future studies. Here we describe the chromosomal features of E. coli APEC_O2. E. coli APEC_O2 is a sequence type ST135, has a chromosome of 4,908,820 bp (plasmid removed), comprising 4672 protein-coding genes, 110 RNA genes, and 156 pseudogenes, with an average G + C content of 50.69%. We identified 82 insertion sequences as well as 4672 protein coding sequences, 12 predicated genomic islands, three prophage-related sequences, and two clustered regularly interspaced short palindromic repeats regions on the chromosome, suggesting the possible occurrence of horizontal gene transfer in this strain. The wildtype strain of E. coli APEC_O2 is resistant towards multiple antimicrobials, however, no (complete) antibiotic resistance genes were present on the chromosome, but a number of genes associated with extra-intestinal disease were identified. Together, the information provided here on E. coli APEC_O2 will assist in future studies of avian pathogenic E. coli strains, in particular regarding strain of E. coli APEC_O2, and aid in the general understanding of the pathogenesis of avian pathogenic E. coli.

  7. Biochemical characterization of WbdN, a β1,3-glucosyltransferase involved in O-antigen synthesis in enterohemorrhagic Escherichia coli O157.

    Science.gov (United States)

    Gao, Yin; Liu, Bin; Strum, Scott; Schutzbach, John S; Druzhinina, Tatyana N; Utkina, Natalia S; Torgov, Vladimir I; Danilov, Leonid L; Veselovsky, Vladimir V; Vlahakis, Jason Z; Szarek, Walter A; Wang, Lei; Brockhausen, Inka

    2012-08-01

    The enterohemorrhagic O157 strain of Escherichia coli, which is one of the most well-known bacterial pathogens, has an O-antigen repeating unit structure with the sequence [-2-d-Rha4NAcα1-3-l-Fucα1-4-d-Glcβ1-3-d-GalNAcα1-]. The O-antigen gene cluster of E. coli O157 contains the genes responsible for the assembly of this repeating unit and includes wbdN. In spite of cloning many O-antigen genes, biochemical characterization has been done on very few enzymes involved in O-antigen synthesis. In this work, we expressed the wbdN gene in E. coli BL21, and the His-tagged protein was purified. WbdN activity was characterized using the donor substrate UDP-[(14)C]Glc and the synthetic acceptor substrate GalNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph. The enzyme product was isolated by high pressure liquid chromatography, and mass spectrometry showed that one Glc residue was transferred to the acceptor by WbdN. Nuclear magnetic resonance analysis of the product structure indicated that Glc was β1-3 linked to GalNAc. WbdN contains a conserved DxD motif and requires divalent metal ions for full activity. WbdN activity has an optimal pH between 7 and 8 and is highly specific for UDP-Glc as the donor substrate. GalNAcα derivatives lacking the diphosphate group were inactive as substrates, and the enzyme did not transfer Glc to GlcNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph. Our results illustrate that WbdN is a specific UDP-Glc:GalNAcα-diphosphate-lipid β1,3-Glc-transferase. The enzyme is a target for the development of inhibitors to block O157-antigen synthesis.

  8. Presence of Shiga toxin-producing Escherichia coli, Enteroinvasive E. coli, Enteropathogenic E. coli, and Enterotoxigenic E. coli on tomatoes from public markets in Mexico.

    Science.gov (United States)

    Gómez-Aldapa, Carlos A; Torres-Vitela, M Del Refugio; Acevedo-Sandoval, Otilio A; Rangel-Vargas, Esmeralda; Villarruel-López, Angélica; Castro-Rosas, Andjavier

    2013-09-01

    Diarrheagenic Escherichia coli pathotypes (DEP) are important foodborne pathogens in various countries, including Mexico. However, no data exist on the presence of DEP on fresh tomatoes (Solanum lycopericum) from Mexico. The frequency of fecal coliforms (FC), E. coli, and DEP were determined for two tomato varieties. One hundred samples of a saladette tomato variety and 100 samples of a red round tomato variety were collected from public markets in Pachuca, Mexico. Each tomato sample consisted of four whole tomatoes. For the 100 saladette samples, coliform bacterial, FC, E. coli, and DEP were identified in 100, 70, 60, and 10% of samples, respectively. For the 100 red round samples, coliform bacterial, FC, E. coli, and DEP were identified in 100, 75, 65, and 11% of samples, respectively. Identified DEP included Shiga toxin-producing E. coli (STEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), and enterotoxigenic E. coli (ETEC). STEC were isolated from 6% of saladette samples and 5% of red round samples. ETEC were isolated from 3% of saladette samples and 4% of red round samples. EPEC were isolated from 2% of saladette samples and 3% of red round samples, and EIEC were isolated from 1% of saladette samples. Both STEC and ETEC were identified in two saladette samples and 1 red round sample. E. coli O157:H7 was not detected in any STEC-positive samples.

  9. Escherichia coli in Europe: An Overview

    Directory of Open Access Journals (Sweden)

    Nerino Allocati

    2013-11-01

    Full Text Available Escherichia coli remains one of the most frequent causes of several common bacterial infections in humans and animals. E. coli is the prominent cause of enteritis, urinary tract infection, septicaemia and other clinical infections, such as neonatal meningitis. E. coli is also prominently associated with diarrhoea in pet and farm animals. The therapeutic treatment of E. coli infections is threatened by the emergence of antimicrobial resistance. The prevalence of multidrug-resistant E. coli strains is increasing worldwide principally due to the spread of mobile genetic elements, such as plasmids. The rise of multidrug-resistant strains of E. coli also occurs in Europe. Therefore, the spread of resistance in E. coli is an increasing public health concern in European countries. This paper summarizes the current status of E. coli strains clinically relevant in European countries. Furthermore, therapeutic interventions and strategies to prevent and control infections are presented and discussed. The article also provides an overview of the current knowledge concerning promising alternative therapies against E. coli diseases.

  10. Annual Surveillance Summary: Escherichia coli (E. coli) Infections in the Military Health System (MHS), 2016

    Science.gov (United States)

    2017-06-30

    Annual Surveillance Summary: Escherichia coli (E. coli) Infections in the Military Health System (MHS...and prevalence among all beneficiaries seeking care within the Military Health System (MHS). This report describes demographics, clinical...linked to assess descriptive and clinical factors related to E. coli. Health Level 7 (HL7)-formatted Composite Health Care System (CHCS) microbiology data

  11. neonatal infections caused by escherichia coli at the national ...

    African Journals Online (AJOL)

    boaz

    Knowledge about common infections caused by E.coli as well as its antibiotics susceptibility pattern will guide ... Keywords: Neonates, E.coli Infections, Antibiotics Resistance, Abuja. ..... Resistance in E.coli 0157 Strains and the Public Health.

  12. E. coli survival in waters: temperature dependence

    Science.gov (United States)

    Knowing the survival rates of water-borne Escherichia coli is important for evaluating microbial contamination and in making appropriate management decisions. E. coli survival rates are dependent on temperature; this dependency is routinely expressed using an analog of the Q10 model. This suggestion...

  13. Genes under positive selection in Escherichia coli

    DEFF Research Database (Denmark)

    Petersen, Lise; Bollback, J.P.; Dimmic, Matt

    2007-01-01

    We used a comparative genomics approach to identify genes that are under positive selection in six strains of Escherichia coli and Shigella flexneri, including five strains that are human pathogens. We find that positive selection targets a wide range of different functions in the E. coli genome...

  14. Third International E. coli genome meeting

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1994-12-31

    Proceedings of the Third E. Coli Genome Meeting are provided. Presentations were divided into sessions entitled (1) Large Scale Sequencing, Sequence Analysis; (2) Databases; (3) Sequence Analysis; (4) Sequence Divergence in E. coli Strains; (5) Repeated Sequences and Regulatory Motifs; (6) Mutations, Rearrangements and Stress Responses; and (7) Origins of New Genes. The document provides a collection of abstracts of oral and poster presentations.

  15. Fosfomycin Resistance in Escherichia coli, Pennsylvania, USA.

    Science.gov (United States)

    Alrowais, Hind; McElheny, Christi L; Spychala, Caressa N; Sastry, Sangeeta; Guo, Qinglan; Butt, Adeel A; Doi, Yohei

    2015-11-01

    Fosfomycin resistance in Escherichia coli is rare in the United States. An extended-spectrum β-lactamase-producing E. coli clinical strain identified in Pennsylvania, USA, showed high-level fosfomycin resistance caused by the fosA3 gene. The IncFII plasmid carrying this gene had a structure similar to those found in China, where fosfomycin resistance is commonly described.

  16. Fimbrial adhesins from extraintestinal Escherichia coli

    DEFF Research Database (Denmark)

    Klemm, Per; Hancock, Viktoria; Schembri, Mark A.

    2010-01-01

    Extraintestinal pathogenic Escherichia coli (ExPEC) represent an important subclass of E. coli that cause a wide spectrum of diseases in human and animal hosts. Fimbriae are key virulence factors of ExPEC strains. These long surface located rod-shaped organelles mediate receptor-specific attachment...

  17. Infectious endocarditis caused by Escherichia coli

    DEFF Research Database (Denmark)

    Lauridsen, Trine Kiilerich; Arpi, Magnus; Fritz-Hansen, Thomas

    2011-01-01

    Although Escherichia coli is among the most common causes of Gram-negative bacteraemia, infectious endocarditis (IE) due to this pathogen is rare. A 67-y-old male without a previous medical history presented with a new mitral regurgitation murmur and persisting E. coli bacteraemia in spite of broad...

  18. Survival of Escherichia coli in stormwater biofilters.

    Science.gov (United States)

    Chandrasena, G I; Deletic, A; McCarthy, D T

    2014-04-01

    Biofilters are widely adopted in Australia for stormwater treatment, but the reported removal of common faecal indicators (such as Escherichia coli (E. coli)) varies from net removal to net leaching. Currently, the underlying mechanisms that govern the faecal microbial removal in the biofilters are poorly understood. Therefore, it is important to study retention and subsequent survival of faecal microorganisms in the biofilters under different biofilter designs and operational characteristics. The current study investigates how E. coli survival is influenced by temperature, moisture content, sunlight exposure and presence of other microorganisms in filter media and top surface sediment. Soil samples were taken from two different biofilters to investigate E. coli survival under controlled laboratory conditions. Results revealed that the presence of other microorganisms and temperature are vital stressors which govern the survival of E. coli captured either in the top surface sediment or filter media, while sunlight exposure and moisture content are important for the survival of E. coli captured in the top surface sediment compared to that of the filter media. Moreover, increased survival was found in the filter media compared to the top sediment, and sand filter media was found be more hostile than loamy sand filter media towards E. coli survival. Results also suggest that the contribution from the tested environmental stressors on E. coli survival in biofilters will be greatly affected by the seasonality and may vary from one site to another.

  19. Genes under positive selection in Escherichia coli

    DEFF Research Database (Denmark)

    Petersen, Lise; Bollback, J.P.; Dimmic, Matt

    2007-01-01

    We used a comparative genomics approach to identify genes that are under positive selection in six strains of Escherichia coli and Shigella flexneri, including five strains that are human pathogens. We find that positive selection targets a wide range of different functions in the E. coli genome...

  20. Prevalence of Escherichia coli O157

    NARCIS (Netherlands)

    Abdissa, Rosa; Haile, Woynshet; Fite, Akafete Teklu; Beyi, Ashenafi Feyisa; Agga, Getahun E.; Edao, Bedaso Mammo; Tadesse, Fanos; Korsa, Mesula Geloye; Beyene, Takele; Beyene, Tariku Jibat; Zutter, De Lieven; Cox, Eric; Goddeeris, Bruno Maria

    2017-01-01

    Background: There is paucity of information regarding the epidemiology of Escherichia coli O157: H7 in developing countries. In this study, we investigated the occurrence of E. coli O157: H7 associated with beef cattle at processing plants and at retail shops in Ethiopia. Methods: Various samples

  1. Escherichia coli survival in waters: Temperature dependence

    Science.gov (United States)

    Knowing the survival rates of water-borne Escherichia coli is important in evaluating microbial contamination and making appropriate management decisions. E. coli survival rates are dependent on temperature, a dependency that is routinely expressed using an analogue of the Q10 mo...

  2. Strategies for Protein Overproduction in Escherichia coli.

    Science.gov (United States)

    Mott, John E.

    1984-01-01

    Examines heterologous expression in Escherichia coli and the role of regulatory sequences which control gene expression at transcription resulting in abundant production of messenger RNA and regulatory sequences in mRNA which promote efficient translation. Also examines the role of E. coli cells in stabilizing mRNA and protein that is…

  3. The unexhausted potential of E. coli.

    Science.gov (United States)

    Blount, Zachary D

    2015-03-25

    E. coli's hardiness, versatility, broad palate and ease of handling have made it the most intensively studied and best understood organism on the planet. However, research on E.coli has primarily examined it as a model organism, one that is abstracted from any natural history. But E. coli is far more than just a microbial lab rat. Rather, it is a highly diverse organism with a complex, multi-faceted niche in the wild. Recent studies of 'wild' E. coli have, for example, revealed a great deal about its presence in the environment, its diversity and genomic evolution, as well as its role in the human microbiome and disease. These findings have shed light on aspects of its biology and ecology that pose far-reaching questions and illustrate how an appreciation of E. coli's natural history can expand its value as a model organism.

  4. Guiding E.coli to nanosensors

    Science.gov (United States)

    Park, Dong-Won; So, Hye-Mi; Kim, Beom Soo; Kong, Ki-Jeong; Chang, Hyunju; Lee, Jeong-O.

    2009-03-01

    Electronic nanosensors based on nanomaterials such as carbon nanotubes and nanowires are expected to have ultimate sensitivity. However, as an inherent problem of nanosensors, they have extremely small sensor surface for reaction. Therefore, simple diffusion of target biomolecules is not enough for such nanosensors, and the problem is even more serious in the case of motile bacteria. Previously, we have shown that we could estimate the titer of E.coli with arrays of single-walled carbon nanotube field effect transistors (SWNT-FET) combined with statistical method. Still, sensitivity of our method is inferior compared with incubation method, due to the limited sensor surface area. In this work, we actively guide E.coli to the sensor surface using micro-fabricated channels. Arrow-shaped and funnel shaped microstructures were fabricated in the channel to guide E. coli to the sensor surface, and we used green fluorescent protein expressed E.coli to monitor the guiding of E.coli.

  5. Functional and structural variation of uridine diphosphate glycosyltransferase (UGT) gene of Stevia rebaudiana-UGTSr involved in the synthesis of rebaudioside A.

    Science.gov (United States)

    Madhav, Harish; Bhasker, Salini; Chinnamma, Mohankumar

    2013-02-01

    The sweetness of honey leaf plant Stevia rebaudiana is attributed to steviol glycosides or steviosides, accumulated in the leaves. Steviol glycosides are diterpenoids derived from steviol as the final step of glycosylation by the marker enzyme Uridine diphosphate glycosyltransferase (UGT). Out of the eight different steviol glycosides, rebaudioside A was detected as the sweetest glycoside with reduced bitter aftertaste. The pattern of glycosylation of steviol has a crucial role in maintaining the sweetness as well as the taste perception of stevioside. Within the 12 UGTs of S. rebaudiana so far elucidated, the functional genomics of three UGTs-UGT76G1, UGT74G1 & UGT85C2 in stevioside synthesis were studied. In the present study a UGT gene of S. rebaudiana named UGTSr showing resemblance with UGT76G1 was structurally analyzed and the functional role of the recombinant UGTSr in the synthesis of rebaudioside A was ascertained. The relative expression of UGTSr by qPCR showed a higher level of expression in mature leaves than in tender. Despite the similarity of nucleotide with UGT76G1, the gene UGTSr exhibits 48 SNPs and 39 associated amino acid substitutions with remarkable variation in the secondary and tertiary structure of the protein. The helical changes, the presence of a new amino acid, novel substitutions of amino acids and the hydrogen bond in the conserved histidine and aspartame residues observed in UGTSr support its functional stability and specificity from that of other UGTs of S. rebaudiana. Based on these features UGTSr exhibits a novel status from other UGTs of S. rebaudiana.

  6. Crystal structures of human sulfotransferases SULT1B1 and SULT1C1 complexed with the cofactor product adenosine-3'- 5'-diphosphate (PAP)

    Energy Technology Data Exchange (ETDEWEB)

    Dombrovski, Luidmila; Dong, Aiping; Bochkarev, Alexey; Plotnikov, Alexander N. (Toronto)

    2008-09-17

    Cytosolic sulfotransferases (SULTs), often referred as Phase II enzymes of chemical defense, are a superfamily of enzymes that catalyze the transfer of a sulfonate group from 3{prime}-phosphoadenosine 5{prime}-phosphosulfate (PAPS) to an acceptor group of substrates. This reaction modulates the activities of a large array of small endogenous and foreign chemicals including drugs, toxic compounds, steroid hormones, and neurotransmitters. In some cases, however, SULTs activate certain food and environmental compounds to mutagenenic and carcinogenic metabolites. Twelve human SULTs have been identified, which are partitioned into three families: SULT1, SULT2 and SULT4. The SULT1 family is further divided in four subfamilies, A, B, C, and E, and comprises eight members (1A1, 1A2, 1A3, 1B1, 1C1, 1C2, 1C3, and 1E1). Despite sequence and structural similarity among the SULTs, the family and subfamily members appear to have different biological function. SULT1 family shows substrate-binding specificity for simple phenols, estradiol, and thyroid hormones, as well as environmental xenobiotics and drugs. Human SULT1B1 is expressed in liver, colon, small intestine, and blood leukocytes, and shows substrate-binding specificity to thyroid hormones and benzylic alcohols. Human SULT1C1 is expressed in the adult stomach, kidney, and thyroid, as well as in fetal kidney and liver. SULT1C1 catalyzes the sulfonation of p-nitrophenol and N-hydroxy-2-acetylaminofluorene in vitro. However, the in vivo function of the enzyme remains unknown. We intend to solve the structures for all of the SULTs for which structural information is not yet available, and compare the structural and functional features of the entire SULT superfamily. Here we report the structures of two members of SULT1 family, SULT1B1 and SULT1C1, both in complex with the product of the PAPS cofactor, adenosine-3{prime}-5{prime}-diphosphate (PAP).

  7. Borate-aided anion exchange high-performance liquid chromatography of uridine diphosphate-sugars in brain, heart, adipose and liver tissues.

    Science.gov (United States)

    Oikari, Sanna; Venäläinen, Tuula; Tammi, Markku

    2014-01-03

    In this paper we describe a method optimized for the purification of uridine diphosphate (UDP)-sugars from liver, adipose tissue, brain, and heart, with highly reproducible up to 85% recoveries. Rapid tissue homogenization in cold ethanol, lipid removal by butanol extraction, and purification with a graphitized carbon column resulted in isolation of picomolar quantities of the UDP-sugars from 10 to 30mg of tissue. The UDP-sugars were baseline separated from each other, and from all major nucleotides using a CarboPac PA1 anion exchange column eluted with a gradient of acetate and borate buffers. The extraction and purification protocol produced samples with few unidentified peaks. UDP-N-acetylglucosamine was a dominant UDP-sugar in all the rat tissues studied. However, brain and adipose tissue showed high UDP-glucose levels, equal to that of UDP-N-acetylglucosamine. The UDP-N-acetylglucosamine showed 2.3-2.7 times higher levels than UDP-N-acetylgalactosamine in all tissues, and about the same ratio was found between UDP-glucose and UDP-galactose in adipose tissue and brain (2.6 and 2.8, respectively). Interestingly, the UDP-glucose/UDP-galactose ratio was markedly lower in liver (1.1) and heart (1.7). The UDP-N-acetylglucosamine/UDP-glucuronic acid ratio was also constant, between 9.7 and 7.7, except in liver with the ratio as low as 1.8. The distinct UDP-glucose/galactose ratio, and the abundance of UDP-glucuronic acid may reflect the specific role of liver in glycogen synthesis, and metabolism of hormones and xenobiotics, respectively, using these UDP-sugars as substrates.

  8. Functional characterization of wheat copalyl diphosphate synthases sheds light on the early evolution of labdane-related diterpenoid metabolism in the cereals.

    Science.gov (United States)

    Wu, Yisheng; Zhou, Ke; Toyomasu, Tomonobu; Sugawara, Chizu; Oku, Madoka; Abe, Shiho; Usui, Masami; Mitsuhashi, Wataru; Chono, Makiko; Chandler, Peter M; Peters, Reuben J

    2012-12-01

    Two of the most agriculturally important cereal crop plants are wheat (Triticum aestivum) and rice (Oryza sativa). Rice has been shown to produce a number of diterpenoid natural products as phytoalexins and/or allelochemicals--specifically, labdane-related diterpenoids, whose biosynthesis proceeds via formation of an eponymous labdadienyl/copalyl diphosphate (CPP) intermediate (e.g., the ent-CPP of gibberellin phytohormone biosynthesis). Similar to rice, wheat encodes a number of CPP synthases (CPS), and the three CPS characterized to date (TaCPS1-3) all have been suggested to produce ent-CPP. However, several of the downstream diterpene synthases will only react with CPP intermediate of normal or syn, but not ent, stereochemistry, as described in the accompanying report. Investigation of additional CPS did not resolve this issue, as the only other functional synthase (TaCPS4) also produced ent-CPP. Chiral product characterization of all the TaCPS then established that TaCPS2 uniquely produces normal, rather than ent-, CPP, thus, providing a suitable substrate source for the downstream diterpene synthases. Notably, TaCPS2 is most homologous to the similarly stereochemically differentiated syn-CPP synthase from rice (OsCPS4), while the non-inducible TaCPS3 and TaCPS4 cluster with the rice OsCPS1 required for gibberellin phytohormone biosynthesis, as well as with a barley (Hordeum vulgare) CPS (HvCPS1) that also is characterized here as similarly producing ent-CPP. These results suggest that diversification of labdane-related diterpenoid metabolism beyond the ancestral gibberellins occurred early in cereal evolution, and included the type of stereochemical variation demonstrated here.

  9. Fructose-1,6-diphosphate protects against epileptogenesis by modifying cation-chloride co-transporters in a model of amygdaloid-kindling temporal epilepticus.

    Science.gov (United States)

    Ding, Yao; Wang, Shan; Jiang, Yan; Yang, Yi; Zhang, Manman; Guo, Yi; Wang, Shuang; Ding, Mei-ping

    2013-11-20

    Fructose-1,6-diphosphate (FDP) shifts the metabolism of glucose from glycolysis to the pentose phosphate pathway and has anticonvulsant activity in several acute seizure animal models. In the present study, we investigated the anti-epileptogenic effects of FDP in an amygdaloid-kindling seizure model, which is an animal model of the most common form of human temporal lobe epilepsy. We found that 1.0 g/kg FDP slowed seizure progression and shortened the corresponding after-discharge duration (ADD). FDP increased the number of stimulations needed to reach seizure stages 2-5 and prolonged the cumulative ADD prior to reaching stages 3-5. It also shortened staying days and cumulative ADD in stages 4-5. However, it demonstrated no significant protective effect when administered after the animals were fully kindled. In hippocampal neurons, cation-chloride co-transporters (CCCs) are suggested to play interesting roles in epilepsy by modulating γ-aminobutyric acid (GABA)ergic activity through controlling GABAA receptor-mediated reversal potential. We examined the potential link between FDP and the hippocampal expression of two main members of the CCCs: the neuron-specific K(+)-Cl(-)co-transporter 2 (KCC2) and Na(+)-K(+)-Cl(-)co-transporter 1 (NKCC1). FDP inhibited the kindling-induced downregulation of KCC2 expression and decreased NKCC1 expression during the kindling session. Taken together, our data reveal that FDP may have protective activity against epileptogenesis, from partial to generalized tonic-clonic seizures. Furthermore, our findings suggest that the FDP-induced imbalance between KCC2 and NKCC1 expression may be involved in the neuroprotective effect.

  10. Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A.

    Science.gov (United States)

    Borthwick, Lee A; Kerbiriou, Mathieu; Taylor, Christopher J; Cozza, Giorgio; Lascu, Ioan; Postel, Edith H; Cassidy, Diane; Trouvé, Pascal; Mehta, Anil; Robson, Louise; Muimo, Richmond

    2016-01-01

    Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent protein kinase A (PKA) and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2) forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent) disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36-54 from NDPK-B or NDPK-A). Overlay (Far-Western) and Surface Plasmon Resonance (SPR) analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351-727). Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive) showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent) reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A) peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia.

  11. A novel dynamic layer-by-layer assembled nano-scale biointerface: functionality tests with platelet adhesion and aggregate morphology influenced by adenosine diphosphate.

    Science.gov (United States)

    Watson, Melanie G; Lopez, Juan M; Paun, Mihaela; Jones, Steven A

    2013-11-01

    An improved biointerface was developed, dynamic layer-by-layer self-assembly surface (d-LbL), and utilized as a biologically-active substrate for platelet adhesion and aggregation. Possible clinical applications for this research include improved anti-coagulation surfaces. This work demonstrated the functionality of d-LbL biointerfaces in the presence of platelet-rich-plasma (PRP) with the addition of 20 μM adenosine diphosphate (ADP), a thrombus activator. The surface morphology of the experimental control, plain PRP, was compared to PRP containing additional ADP (PRP + ADP) and resulted in an expected increase of platelet adhesions along the fibrinogen d-LbL substrate. The d-LbL process was used to coat glass slides with fibrinogen, Poly (sodium 4-styrene-sulfonate), and Poly (diallydimethlyammonium chloride). Slides were exposed to PRP under flow and static conditions with and without 20 μM of ADP. Fluorescence microscopy (FM), phase contrast microscopy (PCM), atomic force microscopy (AFM), and field emission-scanning electron microscopy (FE-SEM) were used to evaluate platelet adhesions under the influence of varied shear conditions. PCM images illustrated differences between the standard LbL and d-LbL substrates. FM images provided percent surface coverage values. For high-shear conditions, percent surface coverage values increased when using ADP whereas plain PRP exposure displayed no significant increase. AFM scans also displayed higher mean peak height values and unique surface characteristics for PRP + ADP as opposed to plain PRP. FE-SEM images revealed platelet adhesions along the biointerface and unique characteristics of the d-LbL surface. In conclusion, PRP + ADP was more effective at increasing platelet aggregation, especially under high shear conditions, providing further validation of the improved biointerface.

  12. Crystal structure of a ring-cleaving cyclohexane-1,2-dione hydrolase, a novel member of the thiamine diphosphate enzyme family.

    Science.gov (United States)

    Steinbach, Alma; Fraas, Sonja; Harder, Jens; Warkentin, Eberhard; Kroneck, Peter M H; Ermler, Ulrich

    2012-04-01

    The thiamine diphosphate (ThDP) dependent flavoenzyme cyclohexane-1,2-dione hydrolase (CDH) (EC 3.7.1.11) catalyses a key step of a novel anaerobic degradation pathway for alicyclic alcohols by converting cyclohexane-1,2-dione (CDO) to 6-oxohexanoate and further to adipate using NAD(+) as electron acceptor. To gain insights into the molecular basis of these reactions CDH from denitrifying anaerobe Azoarcus sp. strain 22Lin was structurally characterized at 1.26 Å resolution. Notably, the active site funnel is rearranged in an unprecedented manner providing the structural basis for the specific binding and cleavage of an alicyclic compound. Crucial features include a decreased and displaced funnel entrance, a semi-circularly shaped loop segment preceding the C-terminal arm and the attachment of the C-terminal arm to other subunits of the CDH tetramer. Its structural scaffold and the ThDP activation is related to that observed for other members of the ThDP enzyme family. The selective binding of the competitive inhibitor 2-methyl-2,4-pentane-diol (MPD) to the open funnel of CDH reveals an asymmetry of the two active sites found also in the dimer of several other ThDP dependent enzymes. The substrate binding site is characterized by polar and non-polar moieties reflected in the structures of MPD and CDO and by three prominent histidine residues (His28, His31 and His76) that most probably play a crucial role in substrate activation. The NAD(+) dependent oxidation of 6-oxohexanoate remains enigmatic as the redox-active cofactor FAD seems not to participate in catalysis, and no obvious NAD(+) binding site is found. Based on the structural data both reactions are discussed.

  13. Enzymes that control the thiamine diphosphate pool in plant tissues. Properties of thiamine pyrophosphokinase and thiamine-(di)phosphate phosphatase purified from Zea mays seedlings.

    Science.gov (United States)

    Rapala-Kozik, Maria; Gołda, Anna; Kujda, Marta

    2009-04-01

    The pool of thiamine diphosphate (TDP), available for TDP-dependent enzymes involved in the major carbohydrate metabolic pathways, is controlled by two enzyme systems that act in the opposite directions. The thiamine pyrophosphokinase (TPK) activates thiamine into TDP and the numerous phosphatases perform the reverse two-step dephosphorylation of TDP to thiamine monophosphate (TMP) and then to free thiamine. Properties and a possible cooperation of those enzymes in higher plants have not been extensively studied. In this work, we characterize highly purified preparations of TPK and a TDP/TMP phosphatase isolated from 6-day Zea mays seedlings. TPK was the 29-kDa monomeric protein, with the optimal activity at pH 9.0, the K(m) values of 12.4microM and 4.7mM for thiamine and ATP, respectively, and the V(max) value of 360pmol TDPmin(-1)mg(-1) protein. The enzyme required magnesium ions, and the best phosphate donor was GTP. The purified phosphatase was the dimer of 24kDa subunits, showed the optimal activity at pH 5.0 and had a rather broad substrate specificity, although TDP, but not TMP, was one of the preferable substrates. The K(m) values for TDP and TMP were 36microM and 49microM, respectively, and the V(max) value for TDP was significantly higher than for TMP (164 versus 60nmolesmin(-1)mg(-1) protein). The total activities of TPK and TDP phosphatases were similarly decreased when the seedlings were grown under the illumination, suggesting a coordinated regulation of both enzymes to stabilize the pool of the essential coenzyme.

  14. Uridine diphosphate glucuronide transferase 1A1FNx0128 gene polymorphism and the toxicity of irinotecan in recurrent and refractory small cell lung cancer

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    Fan Yun

    2014-01-01

    Full Text Available Objective: The aim was to investigate the association between uridine diphosphate glucuronide transferase 1A1 (UGT1A1 gene promoter region polymorphism and irinotecan-related adverse effects and efficacy on recurrent and refractory small cell lung cancer (SCLC. Materials and Methods: A total of 31 patients with recurrent and refractory SCLC were enrolled in this study from June 2012 to August 2013 and received at least two cycles of single-agent irinotecan chemotherapy. The efficacy and adverse effects of irinotecan were evaluated. DNA was extracted from peripheral blood and direct sequencing method was employed to test UGT1A1FNx0128 polymorphism, thus analyzing the correlation between UGT1A1FNx0128 polymorphism and irinotecan-related side-effects and efficacy. Results: A total of 25 cases (80.6% were UGT1A1FNx0128 wild-type (TA 6 /(TA 6 ; 6 cases (19.4% were heterozygous mutant (TA 6 /(TA 7 , no homozygous mutant genotype (TA 7 /(TA 7 was found. The incidences of grade 3/4 neutropenia, diarrhea and thrombocytopenia were 35.5%, 25.8% and 22.6% in all the patients, respectively. The incidence of 3/4 adverse effects in patients with genotype (TA 6 /(TA 6 and heterozygous (TA 6 /(TA 7 had no statistical difference (P > 0.05 for all. The overall response rate (ORR was 32.3%. Median progression free survival (PFS and overall survival (OS were 4 months and 7.5 months in all patients, respectively. There was no statistical difference in ORR, PFS and OS between genotype (TA 6 /(TA 6 patients and heterozygous (TA 6 /(TA 7 patients. Conclusion: Irinotecan showed efficacy in patients with recurrent and refractory SCLC; UGT1A1 FNx01 28 polymorphism failed to predict the incidence of serious adverse effects and efficacy of irinotecan.

  15. Co-targeting Deoxyribonucleic Acid–Dependent Protein Kinase and Poly(Adenosine Diphosphate-Ribose) Polymerase-1 Promotes Accelerated Senescence of Irradiated Cancer Cells

    Energy Technology Data Exchange (ETDEWEB)

    Azad, Arun, E-mail: arun.azad@bccancer.bc.ca [Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Department of Pathology, St. Vincent' s Hospital, University of Melbourne, Parkville, Victoria (Australia); Bukczynska, Patricia; Jackson, Susan [Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Haput, Ygal; Cullinane, Carleen [Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria (Australia); McArthur, Grant A.; Solomon, Benjamin [Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Division of Cancer Medicine, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Department of Medicine, St. Vincent' s Hospital, University of Melbourne, Parkville, Victoria (Australia); Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria (Australia)

    2014-02-01

    Purpose: To examine the effects of combined blockade of DNA-dependent protein kinase (DNA-PK) and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) on accelerated senescence in irradiated H460 and A549 non-small cell lung cancer cells. Methods and Materials: The effects of KU5788 and AG014699 (inhibitors of DNA-PK and PARP-1, respectively) on clonogenic survival, DNA double-strand breaks (DSBs), apoptosis, mitotic catastrophe, and accelerated senescence in irradiated cells were examined in vitro. For in vivo experiments, H460 xenografts established in athymic nude mice were treated with BEZ235 (a DNA-PK, ATM, and phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor) and AG014699 to determine effects on proliferation, DNA DSBs, and accelerated senescence after radiation. Results: Compared with either inhibitor alone, combination treatment with KU57788 and AG014699 reduced postradiation clonogenic survival and significantly increased persistence of Gamma-H2AX (γH2AX) foci in irradiated H460 and A549 cells. Notably, these effects coincided with the induction of accelerated senescence in irradiated cells as reflected by positive β-galactosidase staining, G2-M cell-cycle arrest, enlarged and flattened cellular morphology, increased p21 expression, and senescence-associated cytokine secretion. In irradiated H460 xenografts, concurrent therapy with BEZ235 and AG014699 resulted in sustained Gamma-H2AX (γH2AX) staining and prominent β-galactosidase activity. Conclusion: Combined DNA-PK and PARP-1 blockade increased tumor cell radiosensitivity and enhanced the prosenescent properties of ionizing radiation in vitro and in vivo. These data provide a rationale for further preclinical and clinical testing of this therapeutic combination.

  16. Metabonomic profiling in studying anti-osteoporosis effects of strontium fructose 1,6-diphosphate on estrogen deficiency-induced osteoporosis in rats by GC/TOF-MS.

    Science.gov (United States)

    Ma, Bo; Li, Xiaotian; Zhang, Qi; Wu, Di; Wang, Guangji; A, Jiye; Sun, Jianguo; Li, Jing; Liu, Yinhui; Wang, Yonglu; Ying, Hanjie

    2013-10-15

    A novel strontium salt compound strontium fructose 1, 6-diphosphate (FDP-Sr) has been proved to have highly effective for bone loss via dual effects of stimulating bone formation and suppressing bone absorption. In the present study, metabolomic approach was used to identify and study potential biomarkers associated with the effect and safety of FDP-Sr. The metabolomic profiles of bone loss induced by estrogen deficiency in a rat model was described to attain a system-level map of the shift on the metabolic response in plasma using GC/TOF-MS, after FDP-Sr was orally administered at the dose of 110 mg/kg/day for the prevention and 220 mg/kg/day for the treatment. Meanwhile, bone turnover biomarkers and bone mineral density were investigated to identify the specific changes of potential anti-osteoporosis effects of FDP-Sr. The differences in metabolic profiles between osteoporosis rats and FDP-Sr treated rats were well observed by the partial least squares-discriminant analysis (PLS-DA) to the MS spectra. Some metabolites including homocysteine, arachidonic acid, alanine, and hydroxyproline, which significantly changed during osteoporosis progression could be effectively reversed after FDP-Sr therapy. Of course some metabolites such as uric acid, glyceric acid, octadecadienoic acid, docosahexaenoic acid, oleic acid, and hexadecanoic acid were not found to reverse significantly after FDP-Sr administration. These results delineated the FDP-Sr effects-related metabolic alterations in the bone loss rats, suggesting that metabonomic analysis could provide helpful information on the new potential biomarkers relating to the mechanism of anti-osteoporosis action and side effects of FDP-Sr against estrogen deficiency induced bone loss.

  17. Extracellular Adenosine Diphosphate Ribose Mobilizes Intracellular Ca2+ via Purinergic-Dependent Ca2+ Pathways in Rat Pulmonary Artery Smooth Muscle Cells

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    Chun Huang

    2015-11-01

    Full Text Available Background/Aims: Adenosine diphosphate ribose (ADPR, a product of β-NAD+ metabolism generated by the multifunctional enzyme CD38, is recognized as a novel signaling molecule. The catalytic site of CD38 orients extracellularly or intracellularly, capable of generating ADPR outside and inside the cells. CD38-dependent pathways have been characterized in pulmonary artery smooth muscle cells (PASMCs; however the physiological function of extracellular ADPR is unclear. Methods: Ca2+ mobilizing and proliferative effects of extracellular ADPR were characterized and compared with the ATP-induced responses in rat PASMCs; and the expression of purinergic receptor (P2X and P2Y subtypes were examined in pulmonary arteries. Results: ADPR elicited concentration-dependent increase in [Ca2+]i with a fast transient and a sustained phase in PASMCs. The sustained phase was abolished by Ca2+ removal and inhibited by the non-selective cation channel blocker SKF-96365, but was unaffected by TRPM2 antagonists or nifedipine. The purinergic receptor (P2X antagonist pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonate inhibited partially the transient and the sustained Ca2+ response, while the P2(XY inhibitor suramin and the phospholipase C inhibitor U73122 abolished the sustained Ca2+ influx. The P2Y1 antagonist MRS2179 had no effect on the response. By contrast, ATP and ADP activated Ca2+ response exhibited a high and a low affinity component, and the pharmacological profile of ATP-induced Ca2+ response was distinctive from that of ADPR. BrdU incorporation assay showed that ADPR caused significant inhibition whereas ATP caused slight stimulation of PASMC proliferation. RT-PCR analysis found that almost all P2X and P2Y subtypes are expressed in PAs. Conclusion: ADPR and ATP activate Ca2+ responses through different combinations of multiple purinergic receptor subtypes; and extracellular ADPR may exert an autocrine/paracrine action via purinergic receptors on PASMCs.

  18. Use of tailored loading-dose clopidogrel in patients undergoing selected percutaneous coronary intervention based on adenosine diphosphate-mediated platelet aggregation

    Institute of Scientific and Technical Information of China (English)

    MENG Kang; L(U) Shu-zheng; ZHU Hua-gang; CHEN Xin; GE Chang-jiang; SONG Xian-tao

    2010-01-01

    Background Adenosine phosphate-mediated platelet aggregation is a prognostic factor for major adverse cardiac events in patients who have undergone selective percutaneous coronary interventions. This study aimed to assess whether an adjusted loading dose of clopidogrel could more effectively inhibit platelet aggregation in patients undergoing selected percutaneous coronary intervention.Methods A total of 205 patients undergoing selected percutaneous coronary intervention were enrolled in this multicenter, prospective, randomized study. Patients receiving domestic clopidogrel (n=104) served as the Talcom (Taijia)group; others (n=101) received Plavix, the Plavix group, Patients received up to 3 additional 300-mg loading doses of clopidogrel to decrease the adenosine phosphate-mediated platelet aggregation index by more than 50% (the primary endpoint) compared with the baseline. The secondary endpoint was major adverse cardiovascular events at 12 months.Results Compared with the rational loading dosage, the tailored loading dosage better inhibited platelet aggregation based on a >50% decrease in adenosine phosphate-mediated platelet aggregation (rational loading dosage vs. tailored loading dosage, 48% vs. 73%, P=0.028). There was no significant difference in the eligible index between the Talcom and Plavix groups (47% vs. 49% at 300 mg; 62% vs. 59% at 600 mg; 74% vs. 72% at 900 mg; P >0.05) based on a standard adenosine diphosphate-mediated platelet aggregation decrease of >50%. After 12 months of follow-up, there were no significant differences in major adverse cardiac events (2.5% vs. 2.9%, P=5.43). No acute or subacute stent thrombosis events occurred.Conclusion An adjusted loading dose of clopidogrel could have significant effects on antiplatelet aggregation compared with a rational dose, decreasing 1-year major adverse cardiac events in patients undergoing percutaneous coronary interventions based on adenosine phosphate-mediated platelet aggregation with no

  19. Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A.

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    Lee A Borthwick

    Full Text Available Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR, a cAMP-dependent protein kinase A (PKA and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2 forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36-54 from NDPK-B or NDPK-A. Overlay (Far-Western and Surface Plasmon Resonance (SPR analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351-727. Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia.

  20. The Effect of Fructose-1,6-diphosphate and HTK Solution on Protecting Primary Cardiac Muscle Cells of Rat with Cold Preservation

    Institute of Scientific and Technical Information of China (English)

    SHI Xiaofeng; CHENG Jun; XIA Suisheng

    2005-01-01

    Summary: In this study we tried to investigate the effect of fructose-1,6-diphosphate and HTK solution on protecting primary cardiac muscle cells of rat with cold preservation. The primary cardiac muscle cells of rat were cultured in vitro with four preservation solutions respectively: 0.9 % sodium chloride solution (group A), FDP (group B), HTK solution (group C) and a mixture of FDP and HTK solution (group D). The cells were preserved for 6, 8 and 10 h at 0-4 ℃. The values of AST and LDH-L and the Na+-K+ ATPase activity in cardiac muscle cells were detected, and the survival rate of cardiac muscle cells was detected with trypan blue staining. The values of AST and LDH-L in group C and group D were remarkable lower those in group A and group B (P<0.001), while the Na+-K+ ATPase activity and the survival rate of cells in group C and group D were much higher than those in group A and group B (P<0.001). The values of AST and LDH-L after 6 hours in group D decreased much more than those in group C (P<0.01), while the Na+-K+ ATPase activity and the survival rate of cells in group D improved more than those in group C (P<0.01). Both of the HTK solution and the mixture of HTK and FDP solution have an evident effect on protecting the primary cardiac muscle cells of rat in vitro with cold preservation, Compared with the HTK solution, the mixture solution has a better short-term protective effect.

  1. Functional Analysis of Ribonucleotide Reductase from Cordyceps militaris Expressed in Escherichia coli.

    Science.gov (United States)

    Kato, Tatsuya; Ahmad, Suparmin; Park, Enoch Y

    2017-08-01

    Cordyceps militaris produces cordycepin (3'-deoxyadenosine), which has various activities, including anti-oxidant, anti-tumoral, anti-viral, and anti-inflammatory. Ribonucleotide reductase (RNR) seems to be a candidate to produce cordycepin in C. militaris because RNR catalyzes the reduction of nucleotides to 2'-deoxynucleotides, whose structures are similar to that of cordycepin. However, the role of RNR has not been confirmed yet. In this study, complementary DNAs (cDNAs) of C. militaris RNR (CmRNR) large and small subunits (CmR1 and CmR2) were cloned from C. militaris NBRC9787 to investigate the function of CmRNR for its cordycepin production. C. militaris NBRC9787 began to produce cordycepin when grown in a liquid surface culture in medium composed of glucose and yeast extract for 15 days. CmR1 cDNA and CmR2 cDNA were obtained from its genomic DNA and from total RNA extracted from its mycelia after cultivation for 21 days, respectively. Recombinant CmR1 and CmR2 were expressed individually in Escherichia coli and purified. Purified recombinant CmR1 and CmR2 showed RNR activity toward adenosine diphosphate (ADP) only when two subunits were mixed but only show the reduction of ADP to 2'-deoxyADP. These results indicate that the pathway from ADP to 3'deoxyADP via CmRNR does not exist in C. militaris and cordycepin production in C. militaris may be mediated by other enzymes.

  2. Escherichia coli transcriptional regulatory network

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    Agustino Martinez-Antonio

    2011-06-01

    Full Text Available Escherichia coli is the most well-know bacterial model about the function of its molecular components. In this review are presented several structural and functional aspects of their transcriptional regulatory network constituted by transcription factors and target genes. The network discussed here represent to 1531 genes and 3421 regulatory interactions. This network shows a power-law distribution with a few global regulators and most of genes poorly connected. 176 of genes in the network correspond to transcription factors, which form a sub-network of seven hierarchical layers where global regulators tend to be set in superior layers while local regulators are located in the lower ones. There is a small set of proteins know as nucleoid-associated proteins, which are in a high cellular concentrations and reshape the nucleoid structure to influence the running of global transcriptional programs, to this mode of regulation is named analog regulation. Specific signal effectors assist the activity of most of transcription factors in E. coli. These effectors switch and tune the activity of transcription factors. To this type of regulation, depending of environmental signals is named the digital-precise-regulation. The integration of regulatory programs have place in the promoter region of transcription units where it is common to observe co-regulation among global and local TFs as well as of TFs sensing exogenous and endogenous conditions. The mechanistic logic to understand the harmonious operation of regulatory programs in the network should consider the globalism of TFs, their signal perceived, coregulation, genome position, and cellular concentration. Finally, duplicated TFs and their horizontal transfer influence the evolvability of members of the network. The most duplicated and transferred TFs are located in the network periphery.

  3. Structure of Escherichia coli tryptophanase.

    Science.gov (United States)

    Ku, Shao Yang; Yip, Patrick; Howell, P Lynne

    2006-07-01

    Pyridoxal 5'-phosphate (PLP) dependent tryptophanase has been isolated from Escherichia coli and its crystal structure has been determined. The structure shares the same fold with and has similar quaternary structure to Proteus vulgaris tryptophanase and tyrosine-phenol lyase, but is found in a closed conformation when compared with these two enzymes. The tryptophanase structure, solved in its apo form, does not have covalent PLP bound in the active site, but two sulfate ions. The sulfate ions occupy the phosphoryl-binding site of PLP and the binding site of the alpha-carboxyl of the natural substrate tryptophan. One of the sulfate ions makes extensive interactions with both the transferase and PLP-binding domains of the protein and appears to be responsible for holding the enzyme in its closed conformation. Based on the sulfate density and the structure of the P. vulgaris enzyme, PLP and the substrate tryptophan were modeled into the active site. The resulting model is consistent with the roles of Arg419 in orienting the substrate to PLP and acidifying the alpha-proton of the substrate for beta-elimination, Lys269 in the formation and decomposition of the PLP quinonoid intermediate, Arg230 in orienting the substrate-PLP intermediates in the optimal conformation for catalysis, and His463 and Tyr74 in determining substrate specificity and suggests that the closed conformation observed in the structure could be induced by substrate binding and that significant conformational changes occur during catalysis. A catalytic mechanism for tryptophanase is proposed. Since E. coli tryptophanase has resisted forming diffraction-quality crystals for many years, the molecular surface of tryptophanase has been analyzed in various crystal forms and it was rationalized that strong crystal contacts occur on the flat surface of the protein and that the size of crystal contact surface seems to correlate with the diffraction quality of the crystal.

  4. Structure of Escherichia Coli Tryptophanase

    Energy Technology Data Exchange (ETDEWEB)

    Ku,S.; Yip, P.; Howell, P.

    2006-01-01

    Pyridoxal 5'-phosphate (PLP) dependent tryptophanase has been isolated from Escherichia coli and its crystal structure has been determined. The structure shares the same fold with and has similar quaternary structure to Proteus vulgaris tryptophanase and tyrosine-phenol lyase, but is found in a closed conformation when compared with these two enzymes. The tryptophanase structure, solved in its apo form, does not have covalent PLP bound in the active site, but two sulfate ions. The sulfate ions occupy the phosphoryl-binding site of PLP and the binding site of the {alpha}-carboxyl of the natural substrate tryptophan. One of the sulfate ions makes extensive interactions with both the transferase and PLP-binding domains of the protein and appears to be responsible for holding the enzyme in its closed conformation. Based on the sulfate density and the structure of the P. vulgaris enzyme, PLP and the substrate tryptophan were modeled into the active site. The resulting model is consistent with the roles of Arg419 in orienting the substrate to PLP and acidifying the {alpha}-proton of the substrate for {beta}-elimination, Lys269 in the formation and decomposition of the PLP quinonoid intermediate, Arg230 in orienting the substrate-PLP intermediates in the optimal conformation for catalysis, and His463 and Tyr74 in determining substrate specificity and suggests that the closed conformation observed in the structure could be induced by substrate binding and that significant conformational changes occur during catalysis. A catalytic mechanism for tryptophanase is proposed. Since E. coli tryptophanase has resisted forming diffraction-quality crystals for many years, the molecular surface of tryptophanase has been analyzed in various crystal forms and it was rationalized that strong crystal contacts occur on the flat surface of the protein and that the size of crystal contact surface seems to correlate with the diffraction quality of the crystal.

  5. The evolution of the Escherichia coli phylogeny.

    Science.gov (United States)

    Chaudhuri, Roy R; Henderson, Ian R

    2012-03-01

    Escherichia coli is familiar to biologists as a classical model system, ubiquitous in molecular biology laboratories around the world. Outside of the laboratory, E. coli strains exist as an almost universal component of the lower-gut flora of humans and animals. Although usually a commensal, E. coli has an alter ego as a pathogen, and is associated with diarrhoeal disease and extra-intestinal infections. The study of E. coli diversity predates the availability of molecular data, with strains initially distinguished by serotyping and metabolic profiling, and genomic diversity illustrated by DNA hybridisation. The quantitative study of E. coli diversity began with the application of multi-locus enzyme electrophoresis (MLEE), and has progressed with the accumulation of nucleotide sequence data, from single genes through multi-locus sequence typing (MLST) to whole genome sequencing. Phylogenetic methods have shed light on the processes of genomic evolution in this extraordinarily diverse species, and revealed the origins of pathogenic E. coli strains, including members of the phylogenetically indistinguishable "genus"Shigella. In May and June 2011, an outbreak of haemorrhagic uraemic syndrome in Germany was linked to a strain of enterohaemorrhagic E. coli (EHEC) O104:H4. Application of high-throughput sequencing technologies allowed the genome and origins of the outbreak strain to be characterised in real time as the outbreak was in progress.

  6. Effect of bile on growth, peritoneal absorption, and blood clearance of Escherichia coli in E coli peritonitis

    Energy Technology Data Exchange (ETDEWEB)

    Andersson, R.; Schalen, C.; Tranberg, K.G. (Department of Surgery, Lund University, Lund (Sweden))

    1991-06-01

    The effect of intraperitoneal bile on growth, peritoneal absorption, and clearance of Escherichia coli was determined in E coli peritonitis in the rat. In E coli peritonitis, intraperitoneal bacterial counts gradually decreased, whereas they increased (after 2 hours) with subsequent development of bacteremia in E coli plus bile peritonitis. After an intraperitoneal injection of labeled bacteria, blood radioactivity was only initially lower in E coli plus bile peritonitis compared with E coli peritonitis. Clearance from blood was lower in E coli plus bile peritonitis than in E coli peritonitis. Organ localization was similar in E coli peritonitis and E coli plus bile peritonitis with decreased splenic, increased pulmonary, and unchanged hepatic uptakes compared with controls. Impaired peritoneal absorption of bacteria, together with impaired local host defense, is likely to enhance the noxious effect of bile in E coli peritonitis.

  7. Identification and functional characterization of monofunctional ent-copalyl diphosphate and ent-kaurene synthases in white spruce reveal different patterns for diterpene synthase evolution for primary and secondary metabolism in gymnosperms.

    Science.gov (United States)

    Keeling, Christopher I; Dullat, Harpreet K; Yuen, Mack; Ralph, Steven G; Jancsik, Sharon; Bohlmann, Jörg

    2010-03-01

    The biosynthesis of the tetracyclic diterpene ent-kaurene is a critical step in the general (primary) metabolism of gibberellin hormones. ent-Kaurene is formed by a two-step cyclization of geranylgeranyl diphosphate via the intermediate ent-copalyl diphosphate. In a lower land plant, the moss Physcomitrella patens, a single bifunctional diterpene synthase (diTPS) catalyzes both steps. In contrast, in angiosperms, the two consecutive cyclizations are catalyzed by two distinct monofunctional enzymes, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). The enzyme, or enzymes, responsible for ent-kaurene biosynthesis in gymnosperms has been elusive. However, several bifunctional diTPS of specialized (secondary) metabolism have previously been characterized in gymnosperms, and all known diTPSs for resin acid biosynthesis in conifers are bifunctional. To further understand the evolution of ent-kaurene biosynthesis as well as the evolution of general and specialized diterpenoid metabolisms in gymnosperms, we set out to determine whether conifers use a single bifunctional diTPS or two monofunctional diTPSs in the ent-kaurene pathway. Using a combination of expressed sequence tag, full-length cDNA, genomic DNA, and targeted bacterial artificial chromosome sequencing, we identified two candidate CPS and KS genes from white spruce (Picea glauca) and their orthologs in Sitka spruce (Picea sitchensis). Functional characterization of the recombinant enzymes established that ent-kaurene biosynthesis in white spruce is catalyzed by two monofunctional diTPSs, PgCPS and PgKS. Comparative analysis of gene structures and enzyme functions highlights the molecular evolution of these diTPSs as conserved between gymnosperms and angiosperms. In contrast, diTPSs for specialized metabolism have evolved differently in angiosperms and gymnosperms.

  8. Gas-phase chemistry of diphosphate anions as a tool to investigate the intrinsic requirements of phosphate ester enzymatic reactions: the [M1M2HP2O7]- ions.

    Science.gov (United States)

    Pepi, Federico; Barone, Vincenzo; Cimino, Paola; Ricci, Andreina

    2007-01-01

    Experimental studies on gaseous inorganic phosphate ions are practically nonexistent, yet they can prove helpful for a better understanding of the mechanisms of phosphate ester enzymatic processes. The present contribution extends our previous investigations on the gas-phase ion chemistry of diphosphate species to the [M(1)M(2)HP(2)O(7)](-) ions where M(1) and M(2) are the same or different and correspond to the Li, Na, K, Cs, and Rb cations. The diphosphate ions are formed by electrospray ionization of 10(-4) M solutions of Na(5)P(3)O(10) in CH(3)CN/H(2)O (1/1) and MOH bases or M salts as a source of M(+) cations. The joint application of mass spectrometric techniques and quantum-mechanical calculations makes it possible to characterize the gaseous [M(1)M(2)HP(2)O(7)](-) ions as a mixed ionic population formed by two isomeric species: linear diphosphate anion coordinated to two M(+) cations (group I) and [PO(3)M(1)M(2)HPO(4)](-) clusters (group II). The relative gas-phase stabilities and activation barriers for the isomerization I-->II, which depend on the nature of the M(+) cations, highlight the electronic susceptibility of P-O-P bond breaking in the active site of enzymes. The previously unexplored gas-phase reactivity of [M(1)M(2)HP(2)O(7)](-) ions towards alcohols of different acidity was investigated by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS). The reaction proceeds by addition of the alcohol molecule followed by elimination of a water molecule.

  9. Identification and Functional Characterization of Monofunctional ent-Copalyl Diphosphate and ent-Kaurene Synthases in White Spruce Reveal Different Patterns for Diterpene Synthase Evolution for Primary and Secondary Metabolism in Gymnosperms1[W][OA

    Science.gov (United States)

    Keeling, Christopher I.; Dullat, Harpreet K.; Yuen, Mack; Ralph, Steven G.; Jancsik, Sharon; Bohlmann, Jörg

    2010-01-01

    The biosynthesis of the tetracyclic diterpene ent-kaurene is a critical step in the general (primary) metabolism of gibberellin hormones. ent-Kaurene is formed by a two-step cyclization of geranylgeranyl diphosphate via the intermediate ent-copalyl diphosphate. In a lower land plant, the moss Physcomitrella patens, a single bifunctional diterpene synthase (diTPS) catalyzes both steps. In contrast, in angiosperms, the two consecutive cyclizations are catalyzed by two distinct monofunctional enzymes, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). The enzyme, or enzymes, responsible for ent-kaurene biosynthesis in gymnosperms has been elusive. However, several bifunctional diTPS of specialized (secondary) metabolism have previously been characterized in gymnosperms, and all known diTPSs for resin acid biosynthesis in conifers are bifunctional. To further understand the evolution of ent-kaurene biosynthesis as well as the evolution of general and specialized diterpenoid metabolisms in gymnosperms, we set out to determine whether conifers use a single bifunctional diTPS or two monofunctional diTPSs in the ent-kaurene pathway. Using a combination of expressed sequence tag, full-length cDNA, genomic DNA, and targeted bacterial artificial chromosome sequencing, we identified two candidate CPS and KS genes from white spruce (Picea glauca) and their orthologs in Sitka spruce (Picea sitchensis). Functional characterization of the recombinant enzymes established that ent-kaurene biosynthesis in white spruce is catalyzed by two monofunctional diTPSs, PgCPS and PgKS. Comparative analysis of gene structures and enzyme functions highlights the molecular evolution of these diTPSs as conserved between gymnosperms and angiosperms. In contrast, diTPSs for specialized metabolism have evolved differently in angiosperms and gymnosperms. PMID:20044448

  10. Biosynthesis of the Common Polysaccharide Antigen of Pseudomonas aeruginosa PAO1: Characterization and Role of GDP-D-Rhamnose:GlcNAc/GalNAc-Diphosphate-Lipid α1,3-D-Rhamnosyltransferase WbpZ.

    Science.gov (United States)

    Wang, Shuo; Hao, Youai; Lam, Joseph S; Vlahakis, Jason Z; Szarek, Walter A; Vinnikova, Anna; Veselovsky, Vladimir V; Brockhausen, Inka

    2015-06-15

    The opportunistic pathogen Pseudomonas aeruginosa produces two major cell surface lipopolysaccharides, characterized by distinct O antigens, called common polysaccharide antigen (CPA) and O-specific antigen (OSA). CPA contains a polymer of D-rhamnose (D-Rha) in α1-2 and α1-3 linkages. Three putative glycosyltransferase genes, wbpX, wbpY, and wbpZ, are part of the CPA biosynthesis cluster. To characterize the enzymatic function of the wbpZ gene product, we chemically synthesized the donor substrate GDP-D-Rha and enzymatically synthesized GDP-D-[(3)H]Rha. Using nuclear magnetic resonance (NMR) spectroscopy, we showed that WbpZ transferred one D-Rha residue from GDP-D-Rha in α1-3 linkage to both GlcNAc- and GalNAc-diphosphate-lipid acceptor substrates. WbpZ is also capable of transferring D-mannose (D-Man) to these acceptors. Therefore, WbpZ has a relaxed specificity with respect to both acceptor and donor substrates. The diphosphate group of the acceptor, however, is required for activity. WbpZ does not require divalent metal ion for activity and exhibits an unusually high pH optimum of 9. WbpZ from PAO1 is therefore a GDP-D-Rha:GlcNAc/GalNAc-diphosphate-lipid α1,3-D-rhamnosyltransferase that has significant activity of GDP-D-Man:GlcNAc/GalNAc-diphosphate-lipid α1,3-D-mannosyltransferase. We used site-directed mutagenesis to replace the Asp residues of the two DXD motifs with Ala. Neither of the mutant constructs of wbpZ (D172A or D254A) could be used to rescue CPA biosynthesis in the ΔwbpZ knockout mutant in a complementation assay. This suggested that D172 and D254 are essential for WbpZ function. This work is the first detailed characterization study of a D-Rha-transferase and a critical step in the development of CPA synthesis inhibitors. This is the first characterization of a D-rhamnosyltransferase and shows that it is essential in Pseudomonas aeruginosa for the synthesis of the common polysaccharide antigen. Copyright © 2015, American Society for

  11. Advances in genoserotyping and subtyping of Escherichia coli

    Science.gov (United States)

    E. coli plays an important role as a member of the gut microbiota; however, pathogenic strains also exist, including various diarrheagenic E. coli pathotypes and extraintestinal pathogenic E. coli that cause illness outside of the GI-tract. E. coli have traditionally been serotyped using antisera a...

  12. 21 CFR 866.3255 - Escherichia coli serological reagents.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Escherichia coli serological reagents. 866.3255... coli serological reagents. (a) Identification. Escherichia coli serological reagents are devices that consist of antigens and antisera used in serological tests to identify Escherichia coli from...

  13. Protective effects of indigenous Escherichia coli against a pathogenic E. coli challenge strain in pigs.

    Science.gov (United States)

    Vahjen, W; Cuisiniere, T; Zentek, J

    2017-10-03

    To investigate the inhibitory effect of indigenous enterobacteria on pathogenic Escherichia coli, a challenge trial with postweaning pigs was conducted. A pathogenic E. coli strain was administered to all animals and their health was closely monitored thereafter. Faecal samples were taken from three healthy and three diarrhoeic animals. Samples were cultivated on MacConkey agar and isolates were subcultured. A soft agar overlay assay was used to determine the inhibitory activity of the isolates. A total of 1,173 enterobacterial isolates were screened for their ability to inhibit the E. coli challenge strain. Colony forming units of enterobacteria on MacConkey agar were not different between healthy and diarrhoeic animals in the original samples. Furthermore, numbers of isolates per animal were also not significantly different between healthy (482 isolates) and diarrhoeic animals (691 isolates). A total of 43 isolates (3.7%) with inhibitory activity against the pathogenic E. coli challenge strain were detected. All inhibitory isolates were identified as E. coli via MALDI-TOF. The isolates belonged to the phylotypes A, C and E. Many isolates (67.4%) were commensal E. coli without relevant porcine pathogenic factors, but toxin- and fimbrial genes (stx2e, fae, estIb, elt1a, fas, fan) were detected in 14 inhibitory isolates. Healthy animals showed significantly (P=0.003) more inhibitory isolates (36 of 482 isolates; 7.5%) than diseased animals (7 of 691 isolates; 1.0%). There were no significant correlations regarding phylotype or pathogenic factors between healthy and diseased animals. This study has shown that a small proportion of indigenous E. coli is able to inhibit in vitro growth of a pathogenic E. coli strain in pigs. Furthermore, healthy animals possess significantly more inhibitory E. coli strains than diarrhoeic animals. The inhibition of pathogenic E. coli by specific indigenous E. coli strains may be an underlying principle for the containment of pathogenic

  14. Differentiation between Shigella, enteroinvasive Escherichia coli (EIEC) and noninvasive Escherichia coli.

    Science.gov (United States)

    van den Beld, M J C; Reubsaet, F A G

    2012-06-01

    Shigella causes bacillary dysentery and is classified into four species based on their antigen characteristics. This classification does not reflect genetic relatedness; in fact, Shigella species are so related to Escherichia coli , they should be classified as one distinctive species in the genus Escherichia. The differentiation of Shigella and E. coli is even more complicated with the description of enteroinvasive E. coli (EIEC). EIEC are strains that possess some of the biochemical characteristics of E. coli and have the ability to cause dysentery using the same method of invasion as Shigella does. Sequencing of multiple housekeeping genes indicates that EIEC is more related to Shigella than to non-invasive E. coli. Shigella and EIEC evolved from the same ancestor and form a single pathovar within E. coli. Shigella and EIEC could be separated from other E. coli by a PCR targeting the ipaH-gene; this is a multicopy gene exclusively found in all Shigella and EIEC. It is possible to differentiate Shigella and all E. coli, including EIEC, by using multiple tests, including ipaH-gene PCR, physiological and biochemical typing and serological typing. Based on literature study, a key is designed for daily use in diagnostic laboratories to identify Shigella and all E. coli.

  15. Biofuels from E. Coli: Engineering E. coli as an Electrofuels Chassis for Isooctane Production

    Energy Technology Data Exchange (ETDEWEB)

    None

    2010-07-16

    Electrofuels Project: Ginkgo Bioworks is bypassing photosynthesis and engineering E. coli to directly use carbon dioxide (CO2) to produce biofuels. E. coli doesn’t naturally metabolize CO2, but Ginkgo Bioworks is manipulating and incorporating the genes responsible for CO2 metabolism into the microorganism. By genetically modifying E. coli, Ginkgo Bioworks will enhance its rate of CO2 consumption and liquid fuel production. Ginkgo Bioworks is delivering CO2 to E. coli as formic acid, a simple industrial chemical that provides energy and CO2 to the bacterial system.

  16. Adenosine diphosphate-decorated chitosan nanoparticles shorten blood clotting times, influencing the structures and varying the mechanical properties of the clots.

    Science.gov (United States)

    Chung, Tze-Wen; Lin, Pei-Yi; Wang, Shoei-Shen; Chen, Yen-Fung

    2014-01-01

    Chitosan nanoparticles (NPs) decorated with adenosine diphosphate (ADP) (ANPs) or fibrinogen (FNPs) were used to fabricate hemostatic NPs that can shorten blood clotting time and prevent severe local hemorrhage. The structure and mechanical properties of the blood clot induced with ANP (clot/ANP) or FNP (clot/FNP) were also investigated. The NPs, ANPs, and FNPs, which had particle sizes of 245.1 ± 14.0, 251.0 ± 9.8, and 326.5 ± 14.5 nm and zeta potentials of 24.1 ± 0.5, 20.6 ± 1.9, and 15.3 ± 1.5 mV (n=4), respectively, were fabricated by ionic gelation and then decorated with ADP and fibrinogen. The zeta potentials and Fourier transform infrared (FTIR) spectroscopy of the NPs confirmed that their surfaces were successfully coated with ADP and fibrinogen. The scanning electron microscope (SEM) micrographs of the structure of the clot induced with "undecorated" chitosan NPs (clot/NP), clot/ANP, and clot/FNP (at 0.05 wt%) were different, after citrated bloods had been recalcified by a calcium chloride solution containing NPs, ANPs, or FNPs. This indicated that many NPs adhered on the membrane surfaces of red blood cells, that ANPs induced many platelet aggregates, and that FNPs were incorporated into the fibrin network in the clots. Measurements of the blood clotting times (Tc) of blood clot/NPs, clot/ANPs, and clot/FNPs, based on 90% of ultimate frequency shifts measured on a quartz crystal microbalance (QCM), were significantly (P<0.05) (n=4) shorter than that of a clot induced by a phosphate-buffered solution (PBS) (clot/PBS) (63.6% ± 3.1%, 48.3% ± 6.2%, and 63.2% ± 4.7%, respectively). The ΔF2 values in the spectra of frequency shifts associated with the propagation of fibrin networks in the clot/ANPs and clot/FNPs were significantly lower than those of clot/PBS. Interestingly, texture profile analysis of the compressional properties showed significantly lower hardness and compressibility in clot/NPs and clot/ANPs (P<0.05 or better) (n=4) compared with

  17. Cyclohexane-1,2-dione hydrolase from denitrifying Azoarcus sp. strain 22Lin, a novel member of the thiamine diphosphate enzyme family.

    Science.gov (United States)

    Steinbach, Alma K; Fraas, Sonja; Harder, Jens; Tabbert, Anja; Brinkmann, Henner; Meyer, Axel; Ermler, Ulrich; Kroneck, Peter M H

    2011-12-01

    Alicyclic compounds with hydroxyl groups represent common structures in numerous natural compounds, such as terpenes and steroids. Their degradation by microorganisms in the absence of dioxygen may involve a C-C bond ring cleavage to form an aliphatic intermediate that can be further oxidized. The cyclohexane-1,2-dione hydrolase (CDH) (EC 3.7.1.11) from denitrifying Azoarcus sp. strain 22Lin, grown on cyclohexane-1,2-diol as a sole electron donor and carbon source, is the first thiamine diphosphate (ThDP)-dependent enzyme characterized to date that cleaves a cyclic aliphatic compound. The degradation of cyclohexane-1,2-dione (CDO) to 6-oxohexanoate comprises the cleavage of a C-C bond adjacent to a carbonyl group, a typical feature of reactions catalyzed by ThDP-dependent enzymes. In the subsequent NAD(+)-dependent reaction, 6-oxohexanoate is oxidized to adipate. CDH has been purified to homogeneity by the criteria of gel electrophoresis (a single band at ∼59 kDa; calculated molecular mass, 64.5 kDa); in solution, the enzyme is a homodimer (∼105 kDa; gel filtration). As isolated, CDH contains 0.8 ± 0.05 ThDP, 1.0 ± 0.02 Mg(2+), and 1.0 ± 0.015 flavin adenine dinucleotide (FAD) per monomer as a second organic cofactor, the role of which remains unclear. Strong reductants, Ti(III)-citrate, Na(+)-dithionite, and the photochemical 5-deazaflavin/oxalate system, led to a partial reduction of the FAD chromophore. The cleavage product of CDO, 6-oxohexanoate, was also a substrate; the corresponding cyclic 1,3- and 1,4-diones did not react with CDH, nor did the cis- and trans-cyclohexane diols. The enzymes acetohydroxyacid synthase (AHAS) from Saccharomyces cerevisiae, pyruvate oxidase (POX) from Lactobacillus plantarum, benzoylformate decarboxylase from Pseudomonas putida, and pyruvate decarboxylase from Zymomonas mobilis were identified as the closest relatives of CDH by comparative amino acid sequence analysis, and a ThDP binding motif and a 2-fold Rossmann fold

  18. Altered expression profile of glycolytic enzymes during testicular ischemia reperfusion injury is associated with the p53/TIGAR pathway: effect of fructose 1,6-diphosphate

    Directory of Open Access Journals (Sweden)

    May Al-Maghrebi

    2016-07-01

    Full Text Available Background. Testicular ischemia reperfusion injury (tIRI is considered the mechanism underlying the pathology of testicular torsion and detorsion. Left untreated, tIRI can induce testis dysfunction, damage to spermatogenesis and possible infertility. In this study, we aimed to assess the activities and expression of glycolytic enzymes (GEs in the testis and their possible modulation during tIRI. The effect of fructose 1,6-diphosphate (FDP, a glycolytic intermediate, on tIRI was also investigated. Methods. Male Sprague-Dawley rats were divided into three groups: sham, unilateral tIRI, and tIRI + FDP (2 mg/kg. tIRI was induced by occlusion of the testicular artery for 1 h followed by 4 h of reperfusion. FDP was injected peritoneally 30 min prior to reperfusion. Histological and biochemical analyses were used to assess damage to spermatogenesis, activities of major GEs, and energy and oxidative stress markers. The relative mRNA expression of GEs was evaluated by real-time PCR. ELISA and immunohistochemistry were used to evaluate the expression of p53 and TP53-induced glycolysis and apoptosis regulator (TIGAR. Results. Histological analysis revealed tIRI-induced spermatogenic damage as represented by a significant decrease in the Johnsen biopsy score. In addition, tIRI reduced the activities of hexokinase 1, phosphofructokinase-1, glyceraldehyde 3-phosphate dehydrogenase, and lactate dehydrogenase C. However, mRNA expression downregulation was detected only for hexokinase 1, phosphoglycerate kinase 2, and lactate dehydrogenase C. ATP and NADPH depletion was also induced by tIRI and was accompanied by an increased Malondialdehyde concentration, reduced glutathione level, and reduced superoxide dismutase and catalase enzyme activities. The immunoexpression of p53 and TIGAR was markedly increased after tIRI. The above tIRI-induced alterations were attenuated by FDP treatment. Discussion. Our findings indicate that tIRI-induced spermatogenic damage is

  19. Altered expression profile of glycolytic enzymes during testicular ischemia reperfusion injury is associated with the p53/TIGAR pathway: effect of fructose 1,6-diphosphate.

    Science.gov (United States)

    Al-Maghrebi, May; Renno, Waleed M

    2016-01-01

    Background. Testicular ischemia reperfusion injury (tIRI) is considered the mechanism underlying the pathology of testicular torsion and detorsion. Left untreated, tIRI can induce testis dysfunction, damage to spermatogenesis and possible infertility. In this study, we aimed to assess the activities and expression of glycolytic enzymes (GEs) in the testis and their possible modulation during tIRI. The effect of fructose 1,6-diphosphate (FDP), a glycolytic intermediate, on tIRI was also investigated. Methods. Male Sprague-Dawley rats were divided into three groups: sham, unilateral tIRI, and tIRI + FDP (2 mg/kg). tIRI was induced by occlusion of the testicular artery for 1 h followed by 4 h of reperfusion. FDP was injected peritoneally 30 min prior to reperfusion. Histological and biochemical analyses were used to assess damage to spermatogenesis, activities of major GEs, and energy and oxidative stress markers. The relative mRNA expression of GEs was evaluated by real-time PCR. ELISA and immunohistochemistry were used to evaluate the expression of p53 and TP53-induced glycolysis and apoptosis regulator (TIGAR). Results. Histological analysis revealed tIRI-induced spermatogenic damage as represented by a significant decrease in the Johnsen biopsy score. In addition, tIRI reduced the activities of hexokinase 1, phosphofructokinase-1, glyceraldehyde 3-phosphate dehydrogenase, and lactate dehydrogenase C. However, mRNA expression downregulation was detected only for hexokinase 1, phosphoglycerate kinase 2, and lactate dehydrogenase C. ATP and NADPH depletion was also induced by tIRI and was accompanied by an increased Malondialdehyde concentration, reduced glutathione level, and reduced superoxide dismutase and catalase enzyme activities. The immunoexpression of p53 and TIGAR was markedly increased after tIRI. The above tIRI-induced alterations were attenuated by FDP treatment. Discussion. Our findings indicate that tIRI-induced spermatogenic damage is associated with

  20. antimicrobial susceptibility and plasmids from escherichia coli ...

    African Journals Online (AJOL)

    2001-10-10

    Oct 10, 2001 ... transmission to humans of E. coli containing antibiotic resistance plasmids ... resistant micro-organisms, which may in turn transfer resistance to .... cells were washed with sterile normal saline to remove leached. Я-lactamase ...

  1. ANTIMICIROBIAL SUSCEPTIBILITY PATTERNS OF Escherichia coli ...

    African Journals Online (AJOL)

    DR. AMINU

    aetiological agents of diarrhoea diseases of humans in developing ... Pathogenic organisms have developed a number of elaborate .... reported that E.coli isolated from animals haboured plasmids .... coil 0157:H7 as a model of entry of a new.

  2. First international E. coli genome meeting

    Energy Technology Data Exchange (ETDEWEB)

    1992-12-31

    This volume is a collection of abstracts of oral presentations and poster sessions of studies reported at the First International E. Coli Genome Meeting, held September 10-14, 1992 at the University of Wisconsin.

  3. First international E. coli genome meeting

    Energy Technology Data Exchange (ETDEWEB)

    1992-01-01

    This volume is a collection of abstracts of oral presentations and poster sessions of studies reported at the First International E. Coli Genome Meeting, held September 10-14, 1992 at the University of Wisconsin.

  4. Experimental induced avian E. coli salpingitis

    DEFF Research Database (Denmark)

    Olsen, Rikke Heidemann; Thøfner, Ida; Pors, Susanne Elisabeth

    2016-01-01

    Several types of Escherichia coli have been associated with extra-intestinal infections in poultry, however, they may vary significantly in their virulence potential. The aim of the present study was to investigate the virulence of five strains of E. coli obtained from different disease......) had a distinct ability to cause disease. Results of the study shows major differences in virulence of different strains of E. coli in ascending infections; however, there was no indication of tissue-specific adaptation, since strains obtained from lesions unrelated to the reproductive system were...... fully capable of causing experimental infection. In conclusion, the study provides evidence for the clinical outcome of infection with E. coli in poultry is largely influenced by the specific strain as well as individual host factors....

  5. Prevalence of diarrheagenic Escherichia coli in suckling rabbits

    OpenAIRE

    2016-01-01

    Diarrheagenic Escherichia coli (E. coli) in suckling rabbit causes collibacillosis, which is characterized by sever yellow diarrhea, poor growth and high mortalities. This study was undertaken to investigate the prevalence of diarrheagenic E. coli in suckling rabbits in Egypt. Additionally, expression of some virulence-associated genes in the isolated E. coli serotypes were examined using the polymerase chain reaction. Finally, antibiogram of the identified E. coli serotypes was also investig...

  6. Methane production from kitchen waste using Escherichia coli.

    Science.gov (United States)

    Jayalakshmi, S; Joseph, Kurian; Sukumaran, V

    2007-04-01

    Escherichia coli (E. coli) strain isolated from biogas plant sludge was examined for its ability to enhance biogas from kitchen waste during solid phase anaerobic digestion. The laboratory experiments were conducted for total solid concentrations of 20% and 22%. Kitchen waste was characterized for physico-chemical parameters and laboratory experiments were conducted with and without E. coli strain. It was found that the reactor with E. coli produced 17% more biogas than the reactors that are operated without E. coli strain.

  7. Synergistic effects in mixed Escherichia coli biofilms

    DEFF Research Database (Denmark)

    Reisner, A.; Holler, B.M.; Molin, Søren

    2006-01-01

    the strongest effects was most often linked to conjugative transmission of natural plasmids carried by the E. coli isolates (70%). Thus, the capacity of an isolate to promote the biofilm through cocultivation was (i) transferable to the K-12 strain, (ii) was linked with the acquisition of conjugation genes...... promotion in this system is not dependent on the laboratory strain and that the described model system could provide relevant insights on mechanisms of biofilm development in natural E. coli populations....

  8. Native valve Escherichia coli endocarditis following urosepsis.

    Science.gov (United States)

    Rangarajan, D; Ramakrishnan, S; Patro, K C; Devaraj, S; Krishnamurthy, V; Kothari, Y; Satyaki, N

    2013-05-01

    Gram-negative organisms are a rare cause of infective endocarditis. Escherichia coli, the most common cause of urinary tract infection and gram-negative septicemia involves endocardium rarely. In this case report, we describe infection of native mitral valve by E. coli following septicemia of urinary tract origin in a diabetic male; subsequently, he required prosthetic tissue valve replacement indicated by persistent sepsis and congestive cardiac failure.

  9. Detection of attaching and effacing virulence gene of E. coli

    Directory of Open Access Journals (Sweden)

    Maratu Soleha

    2013-07-01

    Full Text Available AbstrakLatar belakang: Bakteri Escherichia coli (E. coli ada yang telah bermutasi menjadi patogen yang menimbulkan berbagai penyakit seperti hemorrhagic colitis (HC, hemolytic uremic syndrome (HUS, sepsis, pnemonia, neonatal meningitis, dan infeksi saluran kemih. Mutasi terjadi karena bakteri ini menerima transfer gen yang virulen dari bakteri lain yang hidup di sekitarnya. E. coli yang biasanya hidup normal di dalam usus manusia telah beradaptasi sehingga bisa hidup di tanah, makanan, dan saluran kemih. Penelitian ini mendeteksi gene yang virulen pada DNA isolat E. coli. Metode: Untuk deteksi E. coli yang virulen pada penelitian ini digunakan metode Real-time PCR dengan mencocokkan hasil sekuensing dengan sekuens E. coli virulen yang telah di publikasikan sebagai rujukan. Hasil: Sekuens RT PCR menggambarkan DNA gen eae pada BLAST mempunyai kesesuaian dengan rujukan segmen E. coli yang virulen. Dari sampel yang berasal dari E. coli di sekitar perairan lingkungan didapatkan gen Eae sebagai gen yang menyebabkan E. coli menjadi virulen sebesar 7,3%. Kesimpulan: E. coli yang virulen ditemukan pada sampel E. coli yang berasal dari perairan lingkungan dengan metode realtime PCR. (Health Science Indones 2013;1:41-6 Kata kunci: gen virulen E. coli, real-time PCR, perairan lingkunganAbstractBackground: Escherichia coli(E. coli bacteria have developed into pathogenic bacteria that caused diseases such as hemorrhagic colitis (HC, hemolytic uremic syndrome (HUS, sepsis, pneumonia, neonatal meningitis, and urinary tract infections. Pathogenic E. coli have acquired pathogenic/virulence genes from other bacteria in their environment. E. coli that normally lived in the human gut had adapted to other niches such as soil, food and the urinary tract. This study investigated the presence of pathogenic E. coli from water samples by examining E. coli virulence genes present in E. coli genomes of water sourced isolates. Methods:This study used Real-time PCR to detect

  10. Detection of attaching and effacing virulence gene of E. coli

    Directory of Open Access Journals (Sweden)

    Maratu Soleha

    2013-07-01

    Full Text Available AbstrakLatar belakang: Bakteri Escherichia coli (E. coli ada yang telah bermutasi menjadi patogen yang menimbulkan berbagai penyakit seperti hemorrhagic colitis (HC, hemolytic uremic syndrome (HUS, sepsis, pnemonia, neonatal meningitis, dan infeksi saluran kemih. Mutasi terjadi karena bakteri ini menerima transfer gen yang virulen dari bakteri lain yang hidup di sekitarnya. E. coli yang biasanya hidup normal di dalam usus manusia telah beradaptasi sehingga bisa hidup di tanah, makanan, dan saluran kemih. Penelitian ini mendeteksi gene yang virulen pada DNA isolat E. coli. Metode: Untuk deteksi E. coli yang virulen pada penelitian ini digunakan metode Real-time PCR dengan mencocokkan hasil sekuensing dengan sekuens E. coli virulen yang telah di publikasikan sebagai rujukan. Hasil: Sekuens RT PCR menggambarkan DNA gen eae pada BLAST mempunyai kesesuaian dengan rujukan segmen E. coli yang virulen. Dari sampel yang berasal dari E. coli di sekitar perairan lingkungan didapatkan gen Eae sebagai gen yang menyebabkan E. coli menjadi virulen sebesar 7,3%. Kesimpulan: E. coli yang virulen ditemukan pada sampel E. coli yang berasal dari perairan lingkungan dengan metode realtime PCR. (Health Science Indones 2013;1:41-6 Kata kunci: gen virulen E. coli, real-time PCR, perairan lingkunganAbstractBackground: Escherichia coli(E. coli bacteria have developed into pathogenic bacteria that caused diseases such as hemorrhagic colitis (HC, hemolytic uremic syndrome (HUS, sepsis, pneumonia, neonatal meningitis, and urinary tract infections. Pathogenic E. coli have acquired pathogenic/virulence genes from other bacteria in their environment. E. coli that normally lived in the human gut had adapted to other niches such as soil, food and the urinary tract. This study investigated the presence of pathogenic E. coli from water samples by examining E. coli virulence genes present in E. coli genomes of water sourced isolates. Methods:This study used Real-time PCR to detect

  11. Siderophore production by uropathogenic Escherichia coli

    Directory of Open Access Journals (Sweden)

    Vagrali Manjula

    2009-01-01

    Full Text Available Urinary tract infection (UTI is one of the most frequently encountered problems in ambulatory medicine. The present study was designed to determine siderophore production as the urovirulence factor of Escherichia coli isolated from the patients of UTI. A total of 160 strains of E. coli isolated from urine of patients with clinically diagnosed UTI were included in the study and 50 fecal isolates of E. coli, siderophore production was seen in 156 (97.5%. In 50 fecal isolates, siderophore production was seen in 2 (4%. Siderophore production has been shown to be more frequent in E. coli from patients with UTI, than in fecal isolates. The results suggest that siderophore production positive strains can be considered as UPEC. Thus, although a great deal has been learned regarding E. coli virulence mechanisms in UTI, much remains to be learned and the practical application of our growing understanding of E. coli virulence factors to the prevention and treatment of UTI has to be continued.

  12. E. coli on the move

    Science.gov (United States)

    Calne, S.

    2012-04-01

    Lynn Grove High School in Great Yarmouth, UK has been awarded a Royal Society partnership grant. Lynn Grove pupils aged between 11 and 16 years will carry out an investigation collaborating with scientists at the John Innes Centre in Norwich, UK to investigate the distribution of E.coli and other coliform bacteria within a school. The information will be used as an evidence base to educate pupils about the transmission of microbes and about methods of control. Through this work pupils will gain an appreciation of the diversity of microbial biochemistry and the chemistry behind chromogenic detection methodologies for specific bacterial enzymes. Inferences from the use of diagnostic selective media will be confirmed by carrying out DNA isolation and PCR to identify the genes responsible for the biochemical reactions. PCR will also be used to identify species of coliforms by reference to genomic sequence databases. These techniques will allow pupils to look into an unseen world in a way which has direct relevance to their everyday lives. Furthermore this partnership study will demonstrate to pupils that solving scientific questions requires the integration of a variety of scientific disciplines. The project will run from January 2012 until June 2012. We will present our preliminary results from the investigation and outline our future plans.

  13. Enterohemorrhagic E. coli (EHEC pathogenesis

    Directory of Open Access Journals (Sweden)

    Vanessa eSperandio

    2012-07-01

    Full Text Available Enterohemorrhagic Escherichia coli (EHEC serotype O157:H7 is a human pathogen responsible for outbreaks of bloody diarrhea and hemolytic uremic syndrome worldwide. Conventional antimicrobials trigger an SOS response in EHEC that promotes the release of the potent Shiga toxin that is responsible for much of the morbidity and mortality associated with EHEC infection. Cattle are a natural reservoir of EHEC, and approximately 75% of EHEC outbreaks are linked to the consumption of contaminated bovine-derived products. This review will discuss how EHEC causes disease in humans but is asymptomatic in adult ruminants. It will also analyze factors utilized by EHEC as it travels through the bovine gastrointestinal tract that allow for its survival through the acidic environment of the distal stomachs, and for its ultimate colonization in the recto-anal junction. Understanding the factors crucial for EHEC survival and colonization in cattle will aid in the development of alternative strategies to prevent EHEC shedding into the environment and consequent human infection.

  14. The pangenome structure of Escherichia coli: comparative genomic analysis of E. coli commensal and pathogenic isolates.

    Science.gov (United States)

    Rasko, David A; Rosovitz, M J; Myers, Garry S A; Mongodin, Emmanuel F; Fricke, W Florian; Gajer, Pawel; Crabtree, Jonathan; Sebaihia, Mohammed; Thomson, Nicholas R; Chaudhuri, Roy; Henderson, Ian R; Sperandio, Vanessa; Ravel, Jacques

    2008-10-01

    Whole-genome sequencing has been skewed toward bacterial pathogens as a consequence of the prioritization of medical and veterinary diseases. However, it is becoming clear that in order to accurately measure genetic variation within and between pathogenic groups, multiple isolates, as well as commensal species, must be sequenced. This study examined the pangenomic content of Escherichia coli. Six distinct E. coli pathovars can be distinguished using molecular or phenotypic markers, but only two of the six pathovars have been subjected to any genome sequencing previously. Thus, this report provides a seminal description of the genomic contents and unique features of three unsequenced pathovars, enterotoxigenic E. coli, enteropathogenic E. coli, and enteroaggregative E. coli. We also determined the first genome sequence of a human commensal E. coli isolate, E. coli HS, which will undoubtedly provide a new baseline from which workers can examine the evolution of pathogenic E. coli. Comparison of 17 E. coli genomes, 8 of which are new, resulted in identification of approximately 2,200 genes conserved in all isolates. We were also able to identify genes that were isolate and pathovar specific. Fewer pathovar-specific genes were identified than anticipated, suggesting that each isolate may have independently developed virulence capabilities. Pangenome calculations indicate that E. coli genomic diversity represents an open pangenome model containing a reservoir of more than 13,000 genes, many of which may be uncharacterized but important virulence factors. This comparative study of the species E. coli, while descriptive, should provide the basis for future functional work on this important group of pathogens.

  15. The asymptomatic bacteriuria Escherichia coli strain 83972 outcompetes uropathogenic E. coli strains in human urine

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Ulett, G.C.; Schembri, M.A.

    2006-01-01

    Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU). In contrast to uropathogenic E. coli (UPEC), which causes symptomatic urinary tract infections (UTI), very little is known about the mechanisms by which these strains colonize the human urinary tract...

  16. Identification and Prevalence of Escherichia coli and Escherichia coli O157: H7 in Foods

    Directory of Open Access Journals (Sweden)

    Ancuta Mihaela Rotar

    2013-11-01

    Full Text Available The objective of this study is to investigate the incidence of Escherichia coli in animal and non-animal foods, and mainly the incidence of the serotype O157: H7 producing verotoxin. The presence of common Escherichia coli and Escherichia coli O157: H7 in various foods (of animal and non animal origin was performed in Transylvania area. We analyzed a total of one hundred forty-one samples of minced meat, one hundred twenty-six samples of meat , twenty six samples of meat products, five samples of alcoholic beverages, three samples of seafood, one hundred samples of cheese from pasteurized milk, seventeen samples of butter, four samples of vegetables and one sample of milk powder, using the standard cultural method and Vidas Eco method for E. coli O157: H7 strains. E. coli was identified in 50 samples of minced meat, 55 samples of meat prepared, 4 samples of meat products, 2 samples of alcoholic beverages, 25 samples of cheese from pasteurized milk, 6 samples of butter and 1 sample of vegetables. In this study were not been identified any foods contaminated with the E. coli O157: H7 serotype. The results of this reasearch have demostrated that E. coli wich represents a hygienic indicator of recent food contamination, can be destroyed with heat treatment and hygienic handling of foods. Our country over the years has been among the few countries where the incidence of the E. coli O157: H7 serotype has been minimal.

  17. The asymptomatic bacteriuria Escherichia coli strain 83972 outcompetes uropathogenic E. coli strains in human urine

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Ulett, G.C.; Schembri, M.A.

    2006-01-01

    Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU). In contrast to uropathogenic E. coli (UPEC), which causes symptomatic urinary tract infections (UTI), very little is known about the mechanisms by which these strains colonize the human urinary tract....... The prototype ABU E. coli strain 83972 was originally isolated from a girl who had carried it asymptomatically for 3 years. Deliberate colonization of UTI-susceptible individuals with E. coli 83972 has been used successfully as an alternative approach for the treatment of patients who are refractory...... to conventional therapy. Colonization with strain 83972 appears to prevent infection with UPEC strains in such patients despite the fact that this strain is unable to express the primary adhesins involved in UTI, viz. P and type 1 fimbriae. Here we investigated the growth characteristics of E. coli 83972 in human...

  18. Purification and characterization of recombinant sugarcane sucrose phosphate synthase expressed in E. coli and insect Sf9 cells: an importance of the N-terminal domain for an allosteric regulatory property.

    Science.gov (United States)

    Sawitri, Widhi Dyah; Narita, Hirotaka; Ishizaka-Ikeda, Etsuko; Sugiharto, Bambang; Hase, Toshiharu; Nakagawa, Atsushi

    2016-06-01

    Sucrose phosphate synthase (SPS) catalyses the transfer of glycosyl group of uridine diphosphate glucose to fructose-6-phosphate to form sucrose-6-phosphate. Plant SPS plays a key role in photosynthetic carbon metabolisms, which activity is modulated by an allosteric activator glucose-6-phosphate (G6P). We produced recombinant sugarcane SPS using Escherichia coli and Sf9 insect cells to investigate its structure-function relationship. When expressed in E. coli, two forms of SPS with different sizes appeared; the larger was comparable in size with the authentic plant enzyme and the shorter was trimmed the N-terminal 20 kDa region off. In the insect cells, only enzyme with the authentic size was produced. We purified the trimmed SPS and the full size enzyme from insect cells and found their enzymatic properties differed significantly; the full size enzyme was activated allosterically by G6P, while the trimmed one showed a high activity even without G6P. We further introduced a series of N-terminal truncations up to 171 residue and found G6P-independent activity was enhanced by the truncation. These combined results indicated that the N-terminal region of sugarcane SPS is crucial for the allosteric regulation by G6P and may function like a suppressor domain for the enzyme activity.

  19. Transmission OF Campylobacter coli in chicken embryos

    Science.gov (United States)

    Rossi, Daise Aparecida; Fonseca, Belchiolina Beatriz; de Melo, Roberta Torres; Felipe, Gutembergue da Silva; da Silva, Paulo Lourenço; Mendonça, Eliane Pereira; Filgueiras, Ana Luzia Lauria; Beletti, Marcelo Emilio

    2012-01-01

    Campylobacter coli is an important species involved in human cases of enteritis, and chickens are carriers of the pathogen mainly in developing country. The current study aimed to evaluate the transmission of C. coli and its pathogenic effects in chicken embryos. Breeder hens were inoculated intra-esophageally with C. coli isolated from chickens, and their eggs and embryos were analyzed for the presence of bacteria using real-time PCR and plate culture. The viability of embryos was verified. In parallel, SPF eggs were inoculated with C. coli in the air sac; after incubation, the embryos were submitted to the same analysis as the embryos from breeder hens. In embryos and fertile eggs from breeder hens, the bacterium was only identified by molecular methods; in the SPF eggs, however, the bacterium was detected by both techniques. The results showed no relationship between embryo mortality and positivity for C. coli in the embryos from breeder hens. However, the presence of bacteria is a cause of precocious mortality for SPF embryos. This study revealed that although the vertical transmission is a possible event, the bacteria can not grow in embryonic field samples. PMID:24031861

  20. Behavior of non-O157 Shiga toxin-producing Escherichia coli, enteroinvasive E. coli, enteropathogenic E. coli, and enterotoxigenic E. coli strains on alfalfa sprouts.

    Science.gov (United States)

    Gómez-Aldapa, Carlos A; Rangel-Vargas, Esmeralda; Torres-Vitela, M Del Refugio; Villarruel-López, Angélica; Castro-Rosas, Javier

    2013-08-01

    Data about the behavior of non-O157 Shiga toxin-producing Escherichia coli (non-O157 STEC), enteroinvasive E. coli (EIEC), enterotoxigenic E. coli (ETEC), and enteropathogenic E. coli (EPEC) on seeds and alfalfa sprouts are not available. The behavior of STEC, EIEC, ETEC, and EPEC was determined during germination and sprouting of alfalfa seeds at 20 ± 2°C and 30 ± 2°C and on alfalfa sprouts at 3 ± 2°C. When alfalfa seeds were inoculated with STEC, EIEC, ETEC, or EPEC strains, all these diarrheagenic E. coli pathotypes (DEPs) grew during germination and sprouting of seeds, reaching counts of approximately 5 and 6 log CFU/g after 1 day at 20 ± 2°C and 30 ± 2°C, respectively. However, when the sprouts were inoculated after 1 day of seed germination and stored at 20 ± 2°C or 30 ± 2°C, no growth was observed for any DEP during sprouting at 20 ± 2°C or 30 ± 2°C for 9 days. Refrigeration reduced significantly (P < 0.0.5) the number of viable DEPs on sprouts after 20 days in storage; nevertheless, these decreases have no practical significance for the safety of the sprouts.

  1. Transmission of Campylobacter coli in chicken embryos

    Directory of Open Access Journals (Sweden)

    Daise Aparecida Rossi

    2012-06-01

    Full Text Available Campylobacter coli is an important species involved in human cases of enteritis, and chickens are carriers of the pathogen mainly in developing country. The current study aimed to evaluate the transmission of C. coli and its pathogenic effects in chicken embryos. Breeder hens were inoculated intra-esophageally with C. coli isolated from chickens, and their eggs and embryos were analyzed for the presence of bacteria using real-time PCR and plate culture. The viability of embryos was verified. In parallel, SPF eggs were inoculated with C. coli in the air sac; after incubation, the embryos were submitted to the same analysis as the embryos from breeder hens. In embryos and fertile eggs from breeder hens, the bacterium was only identified by molecular methods; in the SPF eggs, however, the bacterium was detected by both techniques. The results showed no relationship between embryo mortality and positivity for C. coli in the embryos from breeder hens. However, the presence of bacteria is a cause of precocious mortality for SPF embryos. This study revealed that although the vertical transmission is a possible event, the bacteria can not grow in embryonic field samples.

  2. Sensitivity Trend of E.coli

    Directory of Open Access Journals (Sweden)

    Molaabaszadeh H

    2013-03-01

    Full Text Available ABSTRACT Background and objective: The resistance to antibiotics among of pathogen bacteria is one of the main concerns of doctors all around the world, with consideration to different reports about E.coli bacteria’s sensitivity, this study was done to examine the pattern of sensitivity and antibiotic resistance of E.coli strains collected from clinical samples of patients hospitalized in Tehran’s Araad hospital. Materials and methods: In this descriptive examination, after extracting E.coli derivations from clinical samples (urine, catheter, phlegm, wound, abscess, vagina and blood, their sensitivity was measured using standard Kirby-Bauer test, in contract with following antibiotics Amikacin, Ciprofloxacin, Gentamicin, Imipenem, Sulfametoxazole Trimetoprime, Ceftriaxone and Cefotaxime. Results: Most of E. coli strains isolated were from urine samples every four years and the lowest of E. coli strains from vaginal samples. The most amount of sensibility to Imipenem and Amikacin and the most amount of resistant were seen to Sulfametoxazole-Trimetoprime and Ciprofloxacin. Conclusion: The results of this study are indicating that E.coli’s strains resistance has increased against Trimethoprim and Ciprofloxacin; presumably it is due to excessive consumption of these antibiotics. It is obvious that, with regard to increasing consumption of antibiotics, and consequently, augmentation of antibacterial resistance, control of this resistance factor is necessary and inevitable, so it is recommended to avoid unnecessary usage of antibiotics.

  3. Adenosine diphosphate-decorated chitosan nanoparticles shorten blood clotting times, influencing the structures and varying the mechanical properties of the clots

    Directory of Open Access Journals (Sweden)

    Chung TW

    2014-03-01

    Full Text Available Tze-Wen Chung,1,3 Pei-Yi Lin,2 Shoei-Shen Wang,2 Yen-Fung Chen31Department of Biomedical Engineering, National Yang-Ming University, 2Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan, Republic of China; 3Department of Chemical and Materials Engineering, National Yunlin University of Science and Technology, Yunlin, Taiwan, Republic of ChinaAbstract: Chitosan nanoparticles (NPs decorated with adenosine diphosphate (ADP (ANPs or fibrinogen (FNPs were used to fabricate hemostatic NPs that can shorten blood clotting time and prevent severe local hemorrhage. The structure and mechanical properties of the blood clot induced with ANP (clot/ANP or FNP (clot/FNP were also investigated. The NPs, ANPs, and FNPs, which had particle sizes of 245.1±14.0, 251.0±9.8, and 326.5±14.5 nm and zeta potentials of 24.1±0.5, 20.6±1.9, and 15.3±1.5 mV (n=4, respectively, were fabricated by ionic gelation and then decorated with ADP and fibrinogen. The zeta potentials and Fourier transform infrared (FTIR spectroscopy of the NPs confirmed that their surfaces were successfully coated with ADP and fibrinogen. The scanning electron microscope (SEM micrographs of the structure of the clot induced with "undecorated" chitosan NPs (clot/NP, clot/ANP, and clot/FNP (at 0.05 wt% were different, after citrated bloods had been recalcified by a calcium chloride solution containing NPs, ANPs, or FNPs. This indicated that many NPs adhered on the membrane surfaces of red blood cells, that ANPs induced many platelet aggregates, and that FNPs were incorporated into the fibrin network in the clots. Measurements of the blood clotting times (Tc of blood clot/NPs, clot/ANPs, and clot/FNPs, based on 90% of ultimate frequency shifts measured on a quartz crystal microbalance (QCM, were significantly (P<0.05 (n=4 shorter than that of a clot induced by a phosphate-buffered solution (PBS (clot/PBS (63.6%±3.1%, 48.3%±6.2%, and 63.2%±4.7%, respectively. The ∆F2

  4. Cellular chain formation in Escherichia coli biofilms

    DEFF Research Database (Denmark)

    Vejborg, Rebecca Munk; Klemm, Per

    2009-01-01

    In this study we report on a novel structural phenotype in Escherichia coli biofilms: cellular chain formation. Biofilm chaining in E. coli K-12 was found to occur primarily by clonal expansion, but was not due to filamentous growth. Rather, chain formation was the result of intercellular......; type I fimbriae expression significantly reduced cellular chain formation, presumably by steric hindrance. Cellular chain formation did not appear to be specific to E coli K-12. Although many urinary tract infection (UTI) isolates were found to form rather homogeneous, flat biofilms, three isolates......, including the prototypic asymptomatic bacteriuria strain, 83972, formed highly elaborate cellular chains during biofilm growth in human urine. Combined, these results illustrate the diversity of biofilm architectures that can be observed even within a single microbial species....

  5. Interaction between Escherichia coli and lunar fines

    Science.gov (United States)

    Johansson, K. R.

    1983-01-01

    A sample of mature lunar fines (10084.151) was solubilized to a high degree (about 17 percent) by the chelating agent salicylic acid (0.01. M). The neutralized (pH adjusted to 7.0) leachate was found to inhibit the growth of Escherichia coli (ATCC 259922) in a minimial mineral salts glucose medium; however, the inhibition was somewhat less than that caused by neutralized salicylic acid alone. The presence of lunar fines in the minimal medium was highly stimulatory to growth of E. coli following an early inhibitory response. The bacterium survived less well in the lunar leachate than in distilled water, no doubt because of the salicylate. It was concluded that the sample of lunar soil tested has nutritional value to E. coli and that certain products of fermentation helped to solubilize the lunar soil.

  6. Intramammary challenge with Escherichia coli following immunization with a curli-producing Escherichia coli.

    Science.gov (United States)

    Todhunter, D A; Smith, K L; Hogan, J S; Nelson, L

    1991-03-01

    Holstein and Jersey cattle were immunized with a curli-producing strain of Escherichia coli (pCRL65/A012) or a noncurli-producing strain (pUC18/HB101) to determine differences in resistance to establishment of experimental intramammary infection. Cows (n = 6 per group) were immunized at 14 d prior to drying off, 7 d of involution, and at calving with 3 x 10(10) E. coli in Freund's Incomplete Adjuvant. At 30 d of lactation, one mammary quarter of each cow was infused with a wild strain of E. coli (727). Escherichia coli 727 was isolated from a naturally occurring intramammary infection and produced curli. All challenged quarters became infected, and all cows developed acute clinical mastitis. Geometric mean duration of intramammary infections was 6 d for both immunization groups. All infections were spontaneously eliminated within 10 d. No differences occurred between immunization groups in blood selenium and glutathione peroxidase activity, plasma selenium, number of E. coli 727 isolated from secretion after challenge, rectal temperature and SCC response, clinical status of mammary quarters, or DMI. Reduction in milk production after challenge was greater for cows immunized with E. coli pCRL65/A012. Immunization of dairy cattle with a curli-producing strain of E. coli did not protect against experimental intramammary challenge during lactation.

  7. 胞二磷胆碱联合治疗急性脑卒中临床观察%Efficacy of Cytidine Diphosphate Choline Combination Therapy in Acute Cerebral Infarction

    Institute of Scientific and Technical Information of China (English)

    陈瑞红

    2015-01-01

    目的:评价疏血通联合胞二磷胆碱注射液治疗急性脑卒中的临床疗效。方法选择急性脑卒中且不宜溶栓患者42例,随机分为治疗组和对照组,在常规抗血小板聚集、降血压、降血脂等治疗基础上,治疗组以疏血通和胞二磷胆碱注射液静滴14 d;对照组予以丹参注射液和吡拉西坦氯化钠注射液静滴l4 d;观察两组的临床疗效及Glasgow评分、MMSE评分等变化。结果治疗组总有效率95.4%,高于对照组80.0%(P<0.05)。结论疏血通联合胞二磷胆碱注射液治疗急性脑卒中临床疗效肯定,能改善患者意识、智能及预后。%Objective To evaluate the clinical curative effect of shuxietong joint cytidine diphosphate choline injection in treatment of acute cerebral infarction. Methods Forty patients with acute cerebral infarction were randomly divided into treatment group and control group,in conventional antiplatelet aggregation,fal blood pressure,fal hematic fat,such as treatment of foundation,the treatment group with shuxietong and cytidine diphosphate choline injection static drops of 14 days,the control group and salvia miltiorrhiza injection and pyrazole raschig and sodium chloride injection static drops of l4 days,observed the clinical effects of two groups,glasgow score,MMSE score changes,etc.Results Total effectiveness in treatment group(95.4%)was significantly higher than the control group(80.0%)(P< 0.05).Conclusion Shuxietong cytidine diphosphate choline injection in treatment of acute cerebral infarction clinical curative effect,can significantly improve the patients' consciousness,inteligence and prognosis.

  8. Adhesive threads of extraintestinal pathogenic Escherichia coli

    Directory of Open Access Journals (Sweden)

    Antão Esther-Maria

    2009-12-01

    Full Text Available Abstract The ability to adhere to host surfaces is by far the most vital step in the successful colonization by microbial pathogens. Colonization begins with the attachment of the bacterium to receptors expressed by cells forming the lining of the mucosa. Long hair like extracellular appendages called fimbriae, produced by most Gram-negative pathogens, mediate specific attachment to the epithelial cell surface. Associated with the fimbriae is a protein called an adhesin, which directs high-affinity binding to specific cell surface components. In the last couple of years, an enormous amount of research has been undertaken that deals with understanding how bacterial pathogens adhere to host cells. E. coli in all probability is one of the best studied free-living organisms. A group of E. coli called Extraintestinal pathogenic E. coli (ExPEC including both human and animal pathogens like Uropathogenic E. coli (UPEC, Newborn meningitic E. coli (NMEC and Avian pathogenic E. coli (APEC, have been found to harbour many fimbriae including Type 1 fimbriae, P fimbriae, curli fibres, S fimbriae, F1C fimbriae, Dr fimbriae, afimbrial adhesins, temperature-sensitive haemagglutinin and many novel adhesin gene clusters that have not yet been characterized. Each of these adhesins is unique due to the recognition of an adhesin-specific receptor, though as a group these adhesins share common genomic organization. A newly identified putative adhesin temporarily termed ExPEC Adhesin I, encoded by gene yqi, has been recently found to play a significant role in the pathogenesis of APEC infection, thus making it an interesting candidate for future research. The aim of this review is to describe the role of ExPEC adhesins during extraintestinal infections known till date, and to suggest the idea of investigating their potential role in the colonization of the host gut which is said to be a reservoir for ExPEC.

  9. Deuterium incorporation into Escherichia-coli proteins

    DEFF Research Database (Denmark)

    Lederer, H.; May, R. P.; Kjems, Jørgen

    1986-01-01

    Neutron small-angle scattering studies of single protein subunits in a protein-DNA complex require the adjustment of the neutron scattering-length densities of protein and DNA, which is attainable by specific deuteration of the protein. The neutron scattering densities of unlabelled DNA and DNA...... of the degree of deuteration and match point of any E. coli protein from the D2O content of the growth medium, taking the 2H incorporation into RNA polymerase amino acids to be representative for all amino acids in E. coli proteins. The small-angle scattering results, on which the calculation of the degree...

  10. Hydrogen production by recombinant Escherichia coli strains

    Science.gov (United States)

    Maeda, Toshinari; Sanchez‐Torres, Viviana; Wood, Thomas K.

    2012-01-01

    Summary The production of hydrogen via microbial biotechnology is an active field of research. Given its ease of manipulation, the best‐studied bacterium Escherichia coli has become a workhorse for enhanced hydrogen production through metabolic engineering, heterologous gene expression, adaptive evolution, and protein engineering. Herein, the utility of E. coli strains to produce hydrogen, via native hydrogenases or heterologous ones, is reviewed. In addition, potential strategies for increasing hydrogen production are outlined and whole‐cell systems and cell‐free systems are compared. PMID:21895995

  11. Deuterium incorporation into Escherichia-coli proteins

    DEFF Research Database (Denmark)

    Lederer, H.; May, R. P.; Kjems, Jørgen;

    1986-01-01

    Neutron small-angle scattering studies of single protein subunits in a protein-DNA complex require the adjustment of the neutron scattering-length densities of protein and DNA, which is attainable by specific deuteration of the protein. The neutron scattering densities of unlabelled DNA and DNA...... of the degree of deuteration and match point of any E. coli protein from the D2O content of the growth medium, taking the 2H incorporation into RNA polymerase amino acids to be representative for all amino acids in E. coli proteins. The small-angle scattering results, on which the calculation of the degree...

  12. Escherichia coli Pathotypes Occupy Distinct Niches in the Mouse Intestine

    OpenAIRE

    Jessica P Meador; Caldwell, Matthew E.; Cohen, Paul S.; Conway, Tyrrell

    2014-01-01

    Since the first step of the infection process is colonization of the host, it is important to understand how Escherichia coli pathogens successfully colonize the intestine. We previously showed that enterohemorrhagic O157:H7 strain E. coli EDL933 colonizes a niche in the streptomycin-treated mouse intestine that is distinct from that of human commensal strains, which explains how E. coli EDL933 overcomes colonization resistance imparted by some, but not all, commensal E. coli strains. Here we...

  13. Lipocalin 2 is protective against E. coli pneumonia

    DEFF Research Database (Denmark)

    Wu, Hong; Santoni-Rugiu, Eric; Ralfkiaer, Elisabeth

    2010-01-01

    Lipocalin 2 is a bacteriostatic protein that binds the siderophore enterobactin, an iron-chelating molecule produced by Escherichia coli (E. coli) that is required for bacterial growth. Infection of the lungs by E. coli is rare despite a frequent exposure to this commensal bacterium. Lipocalin 2...... is an effector molecule of the innate immune system and could therefore play a role in hindering growth of E. coli in the lungs....

  14. Metabolic engineering for the high-yield production of isoprenoid-based C₅ alcohols in E. coli.

    Science.gov (United States)

    George, Kevin W; Thompson, Mitchell G; Kang, Aram; Baidoo, Edward; Wang, George; Chan, Leanne Jade G; Adams, Paul D; Petzold, Christopher J; Keasling, Jay D; Lee, Taek Soon

    2015-06-08

    Branched five carbon (C5) alcohols are attractive targets for microbial production due to their desirable fuel properties and importance as platform chemicals. In this study, we engineered a heterologous isoprenoid pathway in E. coli for the high-yield production of 3-methyl-3-buten-1-ol, 3-methyl-2-buten-1-ol, and 3-methyl-1-butanol, three C5 alcohols that serve as potential biofuels. We first constructed a pathway for 3-methyl-3-buten-1-ol, where metabolite profiling identified NudB, a promiscuous phosphatase, as a likely pathway bottleneck. We achieved a 60% increase in the yield of 3-methyl-3-buten-1-ol by engineering the Shine-Dalgarno sequence of nudB, which increased protein levels by 9-fold and reduced isopentenyl diphosphate (IPP) accumulation by 4-fold. To further optimize the pathway, we adjusted mevalonate kinase (MK) expression and investigated MK enzymes from alternative microbes such as Methanosarcina mazei. Next, we expressed a fusion protein of IPP isomerase and the phosphatase (Idi1~NudB) along with a reductase (NemA) to diversify production to 3-methyl-2-buten-1-ol and 3-methyl-1-butanol. Finally, we used an oleyl alcohol overlay to improve alcohol recovery, achieving final titers of 2.23 g/L of 3-methyl-3-buten-1-ol (~70% of pathway-dependent theoretical yield), 150 mg/L of 3-methyl-2-buten-1-ol, and 300 mg/L of 3-methyl-1-butanol.

  15. Influence of the temperature in the uranyl sorption in zirconium diphosphate modified with salicylic acid; Influencia de la temperatura en la sorcion de uranilo en difosfato de circonio modificado con acido salicilico

    Energy Technology Data Exchange (ETDEWEB)

    Garcia G, N.; Solis C, D. A. [Universidad Autonoma del Estado de Mexico, Facultad de Quimica, Paseo Colon y Paseo Tollocan s/n, 50000 Toluca, Estado de Mexico (Mexico); Ordonez R, E., E-mail: nidgg@yahoo.com.mx [ININ, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (MX)

    2011-11-15

    In this work the experimental conditions were established to evaluate the uranium (Vi) sorption to 20 and 40 C on the surface of the zirconium diphosphate (ZrP{sub 2}O{sub 7}) modified with a solution of salicylic acid 0.1 M. The modification of the ZrP{sub 2}O{sub 7} was produced during the hydrate process, taking advantage that these are formed complexes between the carboxyl and hydroxyl groups of salicylic acid and amphoteric species of the interface solid/liquid. The method is used by lots to elaborate the isotherms that explain the behavior of this sorption in different ph conditions and temperature, the quantity of the uranium reaction is analyzed with the fluorescence technique. The results indicated that in the temperature increases the uranium sorption on the material and is more efficient to low ph values. (Author)

  16. Strong Correlation Between Concentrations of Tenofovir (TFV) Emtricitabine (FTC) in Hair and TFV Diphosphate and FTC Triphosphate in Dried Blood Spots in the iPrEx Open Label Extension: Implications for Pre-exposure Prophylaxis Adherence Monitoring.

    Science.gov (United States)

    Gandhi, Monica; Glidden, David V; Liu, Albert; Anderson, Peter L; Horng, Howard; Defechereux, Patricia; Guanira, Juan V; Grinsztejn, Beatriz; Chariyalertsak, Suwat; Bekker, Linda-Gail; Grant, Robert M

    2015-11-01

    Self-reported adherence to pre-exposure prophylaxis (PrEP) has limitations, raising interest in pharmacologic monitoring. Drug concentrations in hair and dried blood spots (DBS) are used to assess long-term-exposure; hair shipment/storage occurs at room temperature. The iPrEx Open Label Extension collected DBS routinely, with opt-in hair collection; concentrations were measured with liquid chromatography/tandem mass spectrometry. In 806 hair-DBS pairs, tenofovir (TFV) hair levels and TFV diphosphate (DP) in DBS were strongly correlated (Spearman coefficient r = 0.734; P hair TFV/DBS emtricitabine (FTC) triphosphate (TP) (r = 0.781; P hair FTC/DBS TFV-DP (r = 0.74; P hair FTC/DBS FTC-TP (r = 0.587; P Hair TFV/FTC concentrations correlate strongly with DBS levels, which are predictive of PrEP outcomes.

  17. Transport of Escherichia coli in saturated porous media

    NARCIS (Netherlands)

    Foppen, J.W.A.

    2007-01-01

    Over de manier waarop de bacterie en tevens meest bekende fecale indicator soort Escherichia coli getransporteerd wordt in grondwater is relatief weinig bekend. In deze studie wordt de verwijdering van E. coli uit grondwater ten gevolge van E. coli - sediment interacties bestudeerd en modelmatig

  18. 77 FR 9888 - Shiga Toxin-Producing Escherichia coli

    Science.gov (United States)

    2012-02-21

    ... Food Safety and Inspection Service Shiga Toxin-Producing Escherichia coli in Certain Raw Beef Products... manufacturing trimmings for six non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45..., non-intact product, that are contaminated with Shiga toxin-producing Escherichia coli (STEC) O26,...

  19. Transport of Escherichia coli in saturated porous media

    NARCIS (Netherlands)

    Foppen, J.W.A.

    2007-01-01

    Over de manier waarop de bacterie en tevens meest bekende fecale indicator soort Escherichia coli getransporteerd wordt in grondwater is relatief weinig bekend. In deze studie wordt de verwijdering van E. coli uit grondwater ten gevolge van E. coli - sediment interacties bestudeerd en modelmatig ge

  20. E. coli survival in waters: applicability of the Arrhenius equation

    Science.gov (United States)

    E. coli is an important microorganism indicator used to show the presence of pathogens and fecal contamination in waters. Knowing E. coli survival rates is important for assessing the severity of contamination that has occurred and making appropriate management evaluations. E. coli survival rates ...

  1. WGS accurately predicts antimicrobial resistance in Escherichia coli

    Science.gov (United States)

    Objectives: To determine the effectiveness of whole-genome sequencing (WGS) in identifying resistance genotypes of multidrug-resistant Escherichia coli (E. coli) and whether these correlate with observed phenotypes. Methods: Seventy-six E. coli strains were isolated from farm cattle and measured f...

  2. Transport of Escherichia coli in saturated porous media

    NARCIS (Netherlands)

    Foppen, J.W.A.

    2007-01-01

    Over de manier waarop de bacterie en tevens meest bekende fecale indicator soort Escherichia coli getransporteerd wordt in grondwater is relatief weinig bekend. In deze studie wordt de verwijdering van E. coli uit grondwater ten gevolge van E. coli - sediment interacties bestudeerd en modelmatig ge

  3. Escherichia coli mediated urinary tract infections: are there distinct uropathogenic E. coli (UPEC) pathotypes?

    Science.gov (United States)

    Marrs, Carl F; Zhang, Lixin; Foxman, Betsy

    2005-11-15

    A variety of virulence genes are associated with Escherichia coli mediated urinary tract infections. Particular sets of virulence factors shared by bacterial strains directing them through a particular pathogenesis process are called a "pathotype." Comparison of co-occurrence of potential urinary tract infection (UTI) virulence genes among different E. coli isolates from fecal and UTI collections provides evidence for multiple pathotypes of uropathogenic E. coli, but current understanding of critical genetic differences defining the pathotypes is limited. Discovery of additional E. coli genes involved in uropathogenesis and determination of their distribution and co-occurrences will further define UPEC pathotypes and allow for a more detailed analysis of how these pathotypes might differ in how they cause disease.

  4. Control of Ribosome Synthesis in Escherichia coli

    DEFF Research Database (Denmark)

    Molin, Søren; Meyenburg, K. von; Måløe, O.

    1977-01-01

    The rate of ribosome synthesis and accumulation in Escherichia coli during the transition after an energy source shift-down was analyzed. The shift was imposed on cultures of stringent and relaxed strains growing in glucose minimal medium by the addition of the glucose analogue {alpha}-methylgluc...

  5. Infectious endocarditis caused by Escherichia coli

    DEFF Research Database (Denmark)

    Lauridsen, Trine Kiilerich; Arpi, Magnus; Fritz-Hansen, Thomas

    2011-01-01

    -spectrum intravenous antibiotics. Transthoracic and transoesophageal echocardiography revealed a severe mitral endocarditis. E. coli DNA was identified from the mitral valve and the vegetation, and no other pathogen was found. The case was further complicated by spondylodiscitis and bilateral endophthalmitis. Extra...

  6. E. Coli: Preventing Outbreaks at Camp.

    Science.gov (United States)

    McKinney, Mary D.

    1996-01-01

    One strain of E. coli is not usually found in foods, but has been related to consumption of undercooked ground beef. Symptoms are stomach cramps and diarrhea, and 2-7% of infections lead to hemolytic uremic syndrome, which is life threatening. Camps can prevent outbreaks by avoiding uncooked meat on overnight campouts and requiring appropriate…

  7. ECMDB: the E. coli Metabolome Database.

    Science.gov (United States)

    Guo, An Chi; Jewison, Timothy; Wilson, Michael; Liu, Yifeng; Knox, Craig; Djoumbou, Yannick; Lo, Patrick; Mandal, Rupasri; Krishnamurthy, Ram; Wishart, David S

    2013-01-01

    The Escherichia coli Metabolome Database (ECMDB, http://www.ecmdb.ca) is a comprehensively annotated metabolomic database containing detailed information about the metabolome of E. coli (K-12). Modelled closely on the Human and Yeast Metabolome Databases, the ECMDB contains >2600 metabolites with links to ∼1500 different genes and proteins, including enzymes and transporters. The information in the ECMDB has been collected from dozens of textbooks, journal articles and electronic databases. Each metabolite entry in the ECMDB contains an average of 75 separate data fields, including comprehensive compound descriptions, names and synonyms, chemical taxonomy, compound structural and physicochemical data, bacterial growth conditions and substrates, reactions, pathway information, enzyme data, gene/protein sequence data and numerous hyperlinks to images, references and other public databases. The ECMDB also includes an extensive collection of intracellular metabolite concentration data compiled from our own work as well as other published metabolomic studies. This information is further supplemented with thousands of fully assigned reference nuclear magnetic resonance and mass spectrometry spectra obtained from pure E. coli metabolites that we (and others) have collected. Extensive searching, relational querying and data browsing tools are also provided that support text, chemical structure, spectral, molecular weight and gene/protein sequence queries. Because of E. coli's importance as a model organism for biologists and as a biofactory for industry, we believe this kind of database could have considerable appeal not only to metabolomics researchers but also to molecular biologists, systems biologists and individuals in the biotechnology industry.

  8. Synergistic effects in mixed Escherichia coli biofilms

    DEFF Research Database (Denmark)

    Reisner, A.; Holler, B.M.; Molin, Søren

    2006-01-01

    the pathways governing development of more complex heterogeneous communities. In this study, we established a laboratory model where biofilm-stimulating effects due to interactions between genetically diverse strains of Escherichia coli were monitored. Synergistic induction of biofilm formation resulting from...

  9. Escherichia Coli--Key to Modern Genetics.

    Science.gov (United States)

    Bregegere, Francois

    1982-01-01

    Mid-nineteenth century work by Mendel on plant hybrids and by Pasteur on fermentation gave birth by way of bacterial genetics to modern-day molecular biology. The bacterium Escherichia Coli has occupied a key position in genetic studies leading from early gene identification with DNA to current genetic engineering using recombinant DNA technology.…

  10. Progressive segregation of the Escherichia coli chromosome

    DEFF Research Database (Denmark)

    Nielsen, Henrik Jørck; Youngren, Brenda; Hansen, Flemming G.

    2006-01-01

    We have followed the fate of 14 different loci around the Escherichia coli chromosome in living cells at slow growth rate using a highly efficient labelling system and automated measurements. Loci are segregated as they are replicated, but with a marked delay. Most markers segregate in a smooth...

  11. Leaner and meaner genomes in Escherichia coli

    DEFF Research Database (Denmark)

    Ussery, David

    2006-01-01

    A 'better' Escherichia coli K-12 genome has recently been engineered in which about 15% of the genome has been removed by planned deletions. Comparison with related bacterial genomes that have undergone a natural reduction in size suggests that there is plenty of scope for yet more deletions....

  12. Escherichia coli O157:H7.

    Science.gov (United States)

    Mead, P S; Griffin, P M

    1998-10-10

    Escherichia coli O157 was first identified as a human pathogen in 1982. One of several Shiga toxin-producing serotypes known to cause human illness, the organism probably evolved through horizontal acquisition of genes for Shiga toxins and other virulence factors. E. coli O157 is found regularly in the faeces of healthy cattle, and is transmitted to humans through contaminated food, water, and direct contact with infected people or animals. Human infection is associated with a wide range of clinical illness, including asymptomatic shedding, non-bloody diarrhoea, haemorrhagic colitis, haemolytic uraemic syndrome, and death. Since laboratory practices vary, physicians need to know whether laboratories in their area routinely test for E. coli O157 in stool specimens. Treatment with antimicrobial agents remains controversial: some studies suggest that treatment may precipitate haemolytic uraemic syndrome, and other studies suggest no effect or even a protective effect. Physicians can help to prevent E. coli O157 infections by counselling patients about the hazards of consuming undercooked ground meat or unpasteurised milk products and juices, and about the importance of handwashing to prevent the spread of diarrhoeal illness, and by informing public-health authorities when they see unusual numbers of cases of bloody diarrhoea or haemolytic uraemic syndrome.

  13. Engineering Escherichia coli for methanol conversion.

    Science.gov (United States)

    Müller, Jonas E N; Meyer, Fabian; Litsanov, Boris; Kiefer, Patrick; Potthoff, Eva; Heux, Stéphanie; Quax, Wim J; Wendisch, Volker F; Brautaset, Trygve; Portais, Jean-Charles; Vorholt, Julia A

    2015-03-01

    Methylotrophic bacteria utilize methanol and other reduced one-carbon compounds as their sole source of carbon and energy. For this purpose, these bacteria evolved a number of specialized enzymes and pathways. Here, we used a synthetic biology approach to select and introduce a set of "methylotrophy genes" into Escherichia coli based on in silico considerations and flux balance analysis to enable methanol dissimilation and assimilation. We determined that the most promising approach allowing the utilization of methanol was the implementation of NAD-dependent methanol dehydrogenase and the establishment of the ribulose monophosphate cycle by expressing the genes for hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloisomerase (Phi). To test for the best-performing enzymes in the heterologous host, a number of enzyme candidates from different donor organisms were selected and systematically analyzed for their in vitro and in vivo activities in E. coli. Among these, Mdh2, Hps and Phi originating from Bacillus methanolicus were found to be the most effective. Labeling experiments using (13)C methanol with E. coli producing these enzymes showed up to 40% incorporation of methanol into central metabolites. The presence of the endogenous glutathione-dependent formaldehyde oxidation pathway of E. coli did not adversely affect the methanol conversion rate. Taken together, the results of this study represent a major advancement towards establishing synthetic methylotrophs by gene transfer.

  14. Compaction of isolated Escherichia coli nucleoids

    NARCIS (Netherlands)

    Wegner, Anna S.; Wintraecken, Kathelijne; Spurio, Roberto; Woldringh, Conrad L.; Vries, de Renko; Odijk, Theo

    2016-01-01

    Escherichia coli nucleoids were compacted by the inert polymer polyethylene glycol (PEG) in the presence of the H-NS protein. The protein by itself appears to have little impact on the size of the nucleoids as determined by fluorescent microscopy. However, it has a significant impact on the nucle

  15. E. Coli: Preventing Outbreaks at Camp.

    Science.gov (United States)

    McKinney, Mary D.

    1996-01-01

    One strain of E. coli is not usually found in foods, but has been related to consumption of undercooked ground beef. Symptoms are stomach cramps and diarrhea, and 2-7% of infections lead to hemolytic uremic syndrome, which is life threatening. Camps can prevent outbreaks by avoiding uncooked meat on overnight campouts and requiring appropriate…

  16. Escherichia coli as a bioreporter in ecotoxicology.

    Science.gov (United States)

    Robbens, Johan; Dardenne, Freddy; Devriese, Lisa; De Coen, Wim; Blust, Ronny

    2010-11-01

    Ecotoxicological assessment relies to a large extent on the information gathered with surrogate species and the extrapolation of test results across species and different levels of biological organisation. Bacteria have long been used as a bioreporter for genotoxic testing and general toxicity. Today, it is clear that bacteria have the potential for screening of other toxicological endpoints. Escherichia coli has been studied for years; in-depth knowledge of its biochemistry and genetics makes it the most proficient prokaryote for the development of new toxicological assays. Several assays have been designed with E. coli as a bioreporter, and the recent trend to develop novel, better advanced reporters makes bioreporter development one of the most dynamic in ecotoxicology. Based on in-depth knowledge of E. coli, new assays are being developed or existing ones redesigned, thanks to the availability of new reporter genes and new or improved substrates. The technological evolution towards easier and more sensitive detection of different gene products is another important aspect. Often, this requires the redesign of the bacterium to make it compatible with the novel measuring tests. Recent advances in surface chemistry and nanoelectronics open the perspective for advanced reporter based on novel measuring platforms and with an online potential. In this article, we will discuss the use of E. coli-based bioreporters in ecotoxicological applications as well as some innovative sensors awaited for the future.

  17. Incorporation of tetravalent actinides in three phosphated matrices: britholite, monazite/brabandite and thorium phosphate diphosphate ({beta}-TPD); Incorporation d'actinides tetravalents dans trois matrices phosphatees: britholite, monazite/brabantite et phosphate - diphosphate de thorium ({beta}-PDT)

    Energy Technology Data Exchange (ETDEWEB)

    Terra, O

    2005-03-01

    Three phosphate based ceramics were studied for the immobilization of tri- and tetravalent actinides: britholite Ca{sub 9}Nd{sub 1-x}An{sub x}{sup IV}(PO{sub 4}){sub 5-x}(SiO{sub 4}){sub 1+x}F{sub 2}, monazite/brabantite solid solutions Ln{sub 1-2x}{sup III} Ca{sub x}An{sub x}{sup IP}O{sub 4} and Thorium Phosphate Diphosphate ({beta}-TPD) Th{sub 4-}xAn{sub x}{sup IV}(PO{sub 4}){sub 4}P{sub 2}O{sub 7}. For each material, the incorporation of thorium and uranium (IV) was studied as a surrogate of plutonium. This work was the early beginning of the incorporation of {sup 239}Pu and/or {sup 238}Pu in order to evaluate the effects of {alpha}-decay on the three crystallographic structures. The incorporation of tetravalent cations was carried out by dry chemistry methods, using mechanical grinding to improve the reactivity of the initial mixture then the homogeneity of final solid prepared after calcination at high temperature (1200-1400 deg C). For britholites, the thorium incorporation was complete for weight loading up to 20 wt.%, leading to the preparation of homogeneous and single phase solid solutions when using the coupled substitution (Nd{sup 3+}, PO{sub 4}{sup 3-}) {r_reversible} (Th{sup 4+}, SiO{sub 4}{sup 4-}). Due to redox problems, the incorporation of uranium was limited to 5 to 8 wt.% and always led to a two-phase mixture of U-britholite and CaU{sub 2}O{sub 5+y}. The preparation of homogeneous solid solutions of {beta}-TUPD and of brabantites containing thorium and uranium samples was successfully obtained using three steps of mechanical grinding/calcination. For each matrix, dense pellets were prepared prior to the study of their chemical behaviour during leaching tests. The chemical durability of brabantites and {beta}-TUPD were found to be close to that reported in literature. The formation of neo-formed phases was also evidenced onto the surface of Th-britholite samples. (author)

  18. Escherichia coli in chronic inflammatory bowel diseases: An update on adherent invasive Escherichia coli pathogenicity

    Institute of Scientific and Technical Information of China (English)

    Margarita; Martinez-Medina; Librado; Jesus; Garcia-Gil

    2014-01-01

    Escherichia coli(E. coli), and particularly the adherent invasive E. coli(AIEC) pathotype, has been increasingly implicated in the ethiopathogenesis of Crohn’s disease(CD). E. coli strains with similar pathogenic features to AIEC have been associated with other intestinal disorders such as ulcerative colitis, colorectal cancer, and coeliac disease, but AIEC prevalence in these diseases remains largely unexplored. Since AIEC was described one decade ago, substantial progress has been made in deciphering its mechanisms of pathogenicity. However, the molecular bases that characterize the phenotypic properties of this pathotype are still not well resolved. A review of studies focused on E. coli populations in inflammatory bowel disease(IBD) is presented here and we discuss about the putative role of this species on each IBD subtype. Given the relevance of AIEC in CD pathogenesis, we present the latest research findings concerning AIEC host-microbe interactions and pathogenicity. We also review the existing data regarding the prevalence and abundance of AIEC in CD and its association with other intestinal diseases from humans and animals, in order to discuss the AIEC disease- and hostspecificity. Finally, we highlight the fact that dietarycomponents frequently found in industrialized countries may enhance AIEC colonization in the gut, which merits further investigation and the implementation of preventative measures.

  19. Production of glycoprotein vaccines in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Ihssen Julian

    2010-08-01

    Full Text Available Abstract Background Conjugate vaccines in which polysaccharide antigens are covalently linked to carrier proteins belong to the most effective and safest vaccines against bacterial pathogens. State-of-the art production of conjugate vaccines using chemical methods is a laborious, multi-step process. In vivo enzymatic coupling using the general glycosylation pathway of Campylobacter jejuni in recombinant Escherichia coli has been suggested as a simpler method for producing conjugate vaccines. In this study we describe the in vivo biosynthesis of two novel conjugate vaccine candidates against Shigella dysenteriae type 1, an important bacterial pathogen causing severe gastro-intestinal disease states mainly in developing countries. Results Two different periplasmic carrier proteins, AcrA from C. jejuni and a toxoid form of Pseudomonas aeruginosa exotoxin were glycosylated with Shigella O antigens in E. coli. Starting from shake flask cultivation in standard complex medium a lab-scale fed-batch process was developed for glycoconjugate production. It was found that efficiency of glycosylation but not carrier protein expression was highly susceptible to the physiological state at induction. After induction glycoconjugates generally appeared later than unglycosylated carrier protein, suggesting that glycosylation was the rate-limiting step for synthesis of conjugate vaccines in E. coli. Glycoconjugate synthesis, in particular expression of oligosaccharyltransferase PglB, strongly inhibited growth of E. coli cells after induction, making it necessary to separate biomass growth and recombinant protein expression phases. With a simple pulse and linear feed strategy and the use of semi-defined glycerol medium, volumetric glycoconjugate yield was increased 30 to 50-fold. Conclusions The presented data demonstrate that glycosylated proteins can be produced in recombinant E. coli at a larger scale. The described methodologies constitute an important step

  20. Escherichia coli Eyelid Abscess in a Patient with Alcoholic Cirrhosis

    Directory of Open Access Journals (Sweden)

    Matthew Stratton

    2015-01-01

    Full Text Available Escherichia coli (E. coli is a rare cause of ocular infections and has not yet been reported as a cause of an ocular abscess. We describe the case of a 47-year-old woman with a history of alcoholic cirrhosis who presented with painful left lower eyelid swelling that did not improve with oral antibiotics. The abscess was drained and cultures were positive for E. coli. Patients with cirrhosis are at increased risk for developing E. coli bacterial infections, but to our knowledge this is the first case of an E. coli eyelid abscess reported in the literature.

  1. {sup 99m}Technetium labelled Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Diniz, S.O.F.; Cardoso, V.N. [Radioisotope Laboratory, Faculty of Pharmacy/UFMG, Belo Horizonte (Brazil); Resende, B.M.; Nunan, E.A. [Biological Control Laboratory, Faculty of Pharmacy/UFMG, Belo Horizonte (Brazil); Simal, C.J.R. [Laboratory Nuclear Medicine, Faculty of Medicine/UFMG, Belo Horizonte (Brazil)

    1999-07-01

    Samples of a culture of unlabeled Escherichia coli were incubated with different concentrations of stannous chloride for various time periods. {sup 99m}Tc (26.0 MBq) was added to each preparation and the results showed a labelling yield of 98% for E. coli. Since the bacterial viability of {sup 99m}Tc-E. coli and E. coli did not show any statistical differences, these results demonstrate that labelling of E. coli with {sup 99m}Tc does not modify the bacterial viability, and the radiolabelled bacteria may be a good model to study bacterial translocation.

  2. Global gene expression in Escherichia coli biofilms

    DEFF Research Database (Denmark)

    Schembri, Mark; Kjærgaard, K.; Klemm, Per

    2003-01-01

    in expression have no current defined function. These genes, as well as those induced by stresses relevant to biofilm growth such as oxygen and nutrient limitation, may be important factors that trigger enhanced resistance mechanisms of sessile communities to antibiotics and hydrodynamic shear forces.......It is now apparent that microorganisms undergo significant changes during the transition from planktonic to biofilm growth. These changes result in phenotypic adaptations that allow the formation of highly organized and structured sessile communities, which possess enhanced resistance...... to antimicrobial treatments and host immune defence responses. Escherichia coli has been used as a model organism to study the mechanisms of growth within adhered communities. In this study, we use DNA microarray technology to examine the global gene expression profile of E. coli during sessile growth compared...

  3. Genes under positive selection in Escherichia coli

    DEFF Research Database (Denmark)

    Petersen, Lise; Bollback, Jonathan P; Dimmic, Matt

    2007-01-01

    We used a comparative genomics approach to identify genes that are under positive selection in six strains of Escherichia coli and Shigella flexneri, including five strains that are human pathogens. We find that positive selection targets a wide range of different functions in the E. coli genome......, including cell surface proteins such as beta barrel porins, presumably because of the involvement of these genes in evolutionary arms races with other bacteria, phages, and/or the host immune system. Structural mapping of positively selected sites on trans-membrane beta barrel porins reveals...... that the residues under positive selection occur almost exclusively in the extracellular region of the proteins that are enriched with sites known to be targets of phages, colicins, or the host immune system. More surprisingly, we also find a number of other categories of genes that show very strong evidence...

  4. Automatic tracking of Escherichia coli bacteria.

    Science.gov (United States)

    Xie, Jun; Khan, Shahid; Shah, Mubarak

    2008-01-01

    In this paper, we present an automatic method for estimating the trajectories of Escherichia coli bacteria from in vivo phase-contrast microscopy videos. To address the low-contrast boundaries in cellular images, an adaptive kernel-based technique is applied to detect cells in sequence of frames. Then a novel matching gain measure is introduced to cope with the challenges such as dramatic changes of cells' appearance and serious overlapping and occlusion. For multiple cell tracking, an optimal matching strategy is proposed to improve the handling of cell collision and broken trajectories. The results of successful tracking of Escherichia coli from various phase-contrast sequences are reported and compared with manually-determined trajectories, as well as those obtained from existing tracking methods. The stability of the algorithm with different parameter values is also analyzed and discussed.

  5. Production of recombinant avidin in Escherichia coli.

    Science.gov (United States)

    Airenne, K J; Sarkkinen, P; Punnonen, E L; Kulomaa, M S

    1994-06-24

    A recombinant avidin (re-Avd), containing amino acids (aa) 1-123 of the native chicken egg-white Avd, was produced in Escherichia coli. When cells were grown at 37 degrees C production was over 1 microgram/ml, due to altering the codon preference of the first ten codons. The re-Avd was recovered as a soluble protein from cells grown at 25 or 30 degrees C, whereas at 37 degrees C it was mostly insoluble in inclusion bodies. Our results indicated that, despite the potentially harmful biotin-binding activity of Avd, it is possible to produce biologically active Avd in E. coli which then can easily be purified by affinity chromatography on a biotin column in a single step.

  6. Escherichia coli growth under modeled reduced gravity

    Science.gov (United States)

    Baker, Paul W.; Meyer, Michelle L.; Leff, Laura G.

    2004-01-01

    Bacteria exhibit varying responses to modeled reduced gravity that can be simulated by clino-rotation. When Escherichia coli was subjected to different rotation speeds during clino-rotation, significant differences between modeled reduced gravity and normal gravity controls were observed only at higher speeds (30-50 rpm). There was no apparent affect of removing samples on the results obtained. When E. coli was grown in minimal medium (at 40 rpm), cell size was not affected by modeled reduced gravity and there were few differences in cell numbers. However, in higher nutrient conditions (i.e., dilute nutrient broth), total cell numbers were higher and cells were smaller under reduced gravity compared to normal gravity controls. Overall, the responses to modeled reduced gravity varied with nutrient conditions; larger surface to volume ratios may help compensate for the zone of nutrient depletion around the cells under modeled reduced gravity.

  7. Immunopurification of adenomatous polyposis coli (APC) proteins

    OpenAIRE

    Elliott, Kerryn L.; Catimel, Bruno; Church, Nicole L; Janine L Coates; Antony W Burgess; Layton, Meredith J.; Faux, Maree C.

    2013-01-01

    Background The adenomatous polyposis coli (APC) tumour suppressor gene encodes a 2843 residue (310 kDa) protein. APC is a multifunctional protein involved in the regulation of β-catenin/Wnt signalling, cytoskeletal dynamics and cell adhesion. APC mutations occur in most colorectal cancers and typically result in truncation of the C-terminal half of the protein. Results In order to investigate the biophysical properties of APC, we have generated a set of monoclonal antibodies which enable puri...

  8. Escherichia coli necrotizing fasciitis in Hirschsprung's disease

    Directory of Open Access Journals (Sweden)

    Manal A. Alsaif

    2015-04-01

    Full Text Available Necrotizing fasciitis is a rare post-operative complication of Hirschsprung's disease. Very recently the only previous case of necrotizing fasciitis following a Soave procedure was reported with the etiologic agent being Pseudomonas aeruginosa. Here we are reporting the second case of necrotizing fasciitis following a Soave procedure caused by an extended spectrum beta lactamase harboring strain of Escherichia coli which is a rare pathogen in type II necrotizing fasciitis.

  9. Homology requirements for recombination in Escherichia coli.

    OpenAIRE

    Watt, V M; Ingles, C J; Urdea, M S; Rutter, W J

    1985-01-01

    The DNA sequence homology required for recombination in Escherichia coli has been determined by measuring the recombination frequency between insulin DNA in a miniplasmid pi VX and a homologous sequence in a bacteriophage lambda vector. A minimum of approximately equal to 20 base pairs in a completely homologous segment is required for significant recombination. There is an exponential increase in the frequency of recombination when the length of homologous DNA is increased from 20 base pairs...

  10. Silver nanoparticle-E. coli colloidal interaction in water and effect on E. coli survival.

    Science.gov (United States)

    Dror-Ehre, A; Mamane, H; Belenkova, T; Markovich, G; Adin, A

    2009-11-15

    Silver nanoparticles exhibit antibacterial properties via bacterial inactivation and growth inhibition. The mechanism is not yet completely understood. This work was aimed at elucidating the effect of silver nanoparticles on inactivation of Escherichia coli, by studying particle-particle interactions in aqueous suspensions. Stable, molecularly capped, positively or negatively charged silver nanoparticles were mixed at 1 to 60microgmL(-1) with suspended E. coli cells to examine their effect on inactivation of the bacteria. Gold nanoparticles with the same surfactant were used as a control, being of similar size but made up of a presumably inert metal. Log reduction of 5log(10) and complete inactivation were obtained with the silver nanoparticles while the gold nanoparticles did not show any inactivation ability. The effect of molecularly capped nanoparticles on E. coli survival was dependent on particle number. Log reduction of E. coli was associated with the ratio between the number of nanoparticles and the initial bacterial cell count. Electrostatic attraction or repulsion mechanisms in silver nanoparticle-E. coli cell interactions did not contribute to the inactivation process.

  11. Long term effects of Escherichia coli mastitis.

    Science.gov (United States)

    Blum, Shlomo E; Heller, Elimelech D; Leitner, Gabriel

    2014-07-01

    Escherichia coli is one of the most frequently diagnosed causes of bovine mastitis, and is typically associated with acute, clinical mastitis. The objective of the present study was to evaluate the long term effects of intramammary infections by E. coli on milk yield and quality, especially milk coagulation. Twenty-four Israeli Holstein cows diagnosed with clinical mastitis due to intramammary infection by E. coli were used in this study. Mean lactation number, days in milk (DIM) and daily milk yield (DMY) at the time of infection was 3.3 ± 1.3, 131.7 days ± 78.6 and 45.7 L ± 8.4, respectively. DMY, milk constituents, somatic cells count (SCC), differential leukocytes count and coagulation parameters were subsequently assessed. Two patterns of inflammation were identified: 'short inflammation', characterized by 15% decrease in DMY and >30 days to reach a new maximum DMY (n = 19). The estimated mean loss of marketable milk during the study was 200 L/cow for 'short inflammation' cases, and 1,500 L/cow for 'long inflammation' ones. Significant differences between 'short' and 'long inflammation' effects were found in almost all parameters studied. Long-term detrimental effects on milk quality were found regardless of clinical or bacteriological cure of affected glands.

  12. Cyclomodulins in urosepsis strains of Escherichia coli.

    Science.gov (United States)

    Dubois, Damien; Delmas, Julien; Cady, Anne; Robin, Frédéric; Sivignon, Adeline; Oswald, Eric; Bonnet, Richard

    2010-06-01

    Determinants of urosepsis in Escherichia coli remain incompletely defined. Cyclomodulins (CMs) are a growing functional family of toxins that hijack the eukaryotic cell cycle. Four cyclomodulin types are actually known in E. coli: cytotoxic necrotizing factors (CNFs), cycle-inhibiting factor (Cif), cytolethal distending toxins (CDTs), and the pks-encoded toxin. In the present study, the distribution of CM-encoding genes and the functionality of these toxins were investigated in 197 E. coli strains isolated from patients with community-acquired urosepsis (n = 146) and from uninfected subjects (n = 51). This distribution was analyzed in relation to the phylogenetic background, clinical origin, and antibiotic resistance of the strains. It emerged from this study that strains harboring the pks island and the cnf1 gene (i) were strongly associated with the B2 phylogroup (P, urosepsis origin (P, urosepsis groups, suggesting that the pks island is more important for the colonization process and the cnf1 gene for virulence. pks- or cnf1-harboring strains were significantly associated with susceptibility to antibiotics (amoxicillin, cotrimoxazole, and quinolones [P, <0.001 to 0.043]). Otherwise, only 6% and 1% of all strains harbored the cdtB and cif genes, respectively, with no particular distribution by phylogenetic background, antimicrobial susceptibility, or clinical origin.

  13. Environmental Escherichia coli: Ecology and public health implications - A review

    Science.gov (United States)

    Jang, Jeonghwan; Hur, Hor-Gil; Sadowsky, Michael J.; Byappanahalli, Muruleedhara; Yan, Tao; Ishii, Satoshi

    2017-01-01

    Escherichia coli is classified as a rod-shaped, Gram-negative bacterium in the family Enterobacteriaceae. The bacterium mainly inhabits the lower intestinal tract of warm-blooded animals, including humans, and is often discharged into the environment through feces or wastewater effluent. The presence of E. coli in environmental waters has long been considered as an indicator of recent fecal pollution. However, numerous recent studies have reported that some specific strains of E. coli can survive for long periods of time, and potentially reproduce, in extra-intestinal environments. This indicates that E. coli can be integrated into indigenous microbial communities in the environment. This naturalization phenomenon calls into question the reliability of E. coli as a fecal indicator bacterium (FIB). Recently, many studies reported that E. coli populations in the environment are affected by ambient environmental conditions affecting their long-term survival. Large-scale studies of population genetics provide the diversity and complexity of E. coli strains in various environments, affected by multiple environmental factors. This review examines the current knowledge on the ecology of E. coli strains in various environments in regards to its role as a FIB and as a naturalized member of indigenous microbial communities. Special emphasis is given on the growth of pathogenic E. coli in the environment, and the population genetics of environmental members of the genus Escherichia. The impact of environmental E. coli on water quality and public health is also discussed.

  14. Cytotoxic Necrotizing Factor 1 Contributes to Escherichia coli Meningitis

    Directory of Open Access Journals (Sweden)

    Ming-Hsien Wang

    2013-11-01

    Full Text Available E. coli is the most common Gram-negative bacteria causing neonatal meningitis, and E. coli meningitis continues to be an important cause of mortality and morbidity throughout the world. Recent reports of E. coli meningitis caused by antimicrobial resistant strains are a particular concern. These findings indicate that a novel strategy is needed to identify new targets for prevention and therapy of E. coli meningitis. Cytotoxic necrotizing factor 1 (CNF1 is a bacterial virulence factor associated principally with E. coli strains causing urinary tract infection and meningitis. We have shown that CNF1 contributes to E. coli invasion of the blood-brain barrier and penetration into the brain, the essential step in the development of E. coli meningitis, and identified the host receptor for CNF1, 37-kDa laminin receptor precursor (37LRP. CNF1, however, is a cytoplasmic protein and its contribution to E. coli invasion of the blood-brain barrier requires its secretion from the bacterial cytoplasm. No signal peptide is found in the CNF1 sequence. CNF1 secretion is, therefore, a strategy utilized by meningitis-causing E. coli to invade the blood-brain barrier. Elucidation of the mechanisms involved in CNF1 secretion, as shown in this report with the involvement of Fdx and YgfZ provides the novel information on potential targets for prevention and therapy of E. coli meningitis by virtue of targeting the secretion of CNF1.

  15. Effect of E. coli coli on Anti-disease Activities of Scallops: Argopecten irradians

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The effect of acute E. coli challenge on the anti-disease activity of scallops Argopecten irradians is examined. The treatments of scallop from which hemolymph samples were taken for study included (1) control scallops, (2) sham-injected scallops, (3) PSW-injected scallops and (4) E. coli-injected scallops. From the beginning, the anti-disease activities of scallops are determined at 12 hr and 24 hr. The concentrations of circulating hemocytes, the total serum protein concentrations and the activities of alkaline phosphatase, acid phosphatase and superoxide dismutase in the scallops Argopecten irradians are determined. Injection with E. coli results in a significant elevation in the concentration of circulating hemocytes and in the alkaline phosphatase activity and a significant decline in the total serum protein concentration and in the superoxide dismutase activity at 24 hr postchallenge. It shows that metabolism of bay scallop is expedited to adopt the challenge.

  16. Evaluation of Petrifilm™ Select E. coli Count Plate medium to discriminate antimicrobial resistant Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jensen Lars

    2008-09-01

    Full Text Available Abstract Background Screening and enumeration of antimicrobial resistant Escherichia coli directly from samples is needed to identify emerging resistant clones and obtain quantitative data for risk assessment. Aim of this study was to evaluate the performance of 3M™ Petrifilm™ Select E. coli Count Plate (SEC plate supplemented with antimicrobials to discriminate antimicrobial-resistant and non-resistant E. coli. Method A range of E. coli isolates were tested by agar dilution method comparing the Minimal Inhibitory Concentration (MIC for eight antimicrobials obtained by Mueller-Hinton II agar, MacConkey agar and SEC plates. Kappa statistics was used to assess the levels of agreement when classifying strains as resistant, intermediate or susceptible. Results SEC plate showed that 74% of all strains agreed within ± 1 log2 dilution when comparing MICs with Mueller-Hinton II media. High agreement levels were found for gentamicin, ampicillin, chloramphenicol and cefotaxime, resulting in a kappa value of 0.9 and 100% agreement within ± 1 log2 dilution. Significant variances were observed for oxytetracycline and sulphamethoxazole. Further tests showed that the observed discrepancy in classification of susceptibility to oxytetracycline by the two media could be overcome when a plate-dependent breakpoint of 64 mg/L was used for SEC plates. For sulphamethoxazole, SEC plates provided unacceptably high MICs. Conclusion SEC plates showed good agreement with Mueller-Hinton II agar in MIC studies and can be used to screen and discriminate resistant E. coli for ampicillin, cephalothin, streptomycin, chloramphenicol, cefotaxime and gentamicin using CLSI standardized breakpoints, but not for sulphamethoxazole. SEC plates can also be used to discriminate oxytetracycline-resistant E. coli if a plate-dependent breakpoint value of 64 mg/L is used.

  17. A putative, novel coli surface antigen 8B (CS8B) of enterotoxigenic Escherichia coli.

    Science.gov (United States)

    Njoroge, Samuel M; Boinett, Christine J; Madé, Laure F; Ouko, Tom T; Fèvre, Eric M; Thomson, Nicholas R; Kariuki, Samuel

    2015-10-01

    Enterotoxigenic Escherichia coli (ETEC) strains harbor multiple fimbriae and pili to mediate host colonization, including the type IVb pilus, colonization factor antigen III (CFA/III). Not all colonization factors are well characterized or known in toxin positive ETEC isolates, which may have an impact identifying ETEC isolates based on molecular screening of these biomarkers. We describe a novel coli surface antigen (CS) 8 subtype B (CS8B), a family of CFA/III pilus, in a toxin producing ETEC isolate from a Kenyan collection. In highlighting the existence of this putative CS, we provide the sequence and specific primers, which can be used alongside other ETEC primers previously described.

  18. Obscured phylogeny and possible recombinational dormancy in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Sawyer Stanley A

    2011-06-01

    Full Text Available Abstract Background Escherichia coli is one of the best studied organisms in all of biology, but its phylogenetic structure has been difficult to resolve with current data and analytical techniques. We analyzed single nucleotide polymorphisms in chromosomes of representative strains to reconstruct the topology of its emergence. Results The phylogeny of E. coli varies according to the segment of chromosome analyzed. Recombination between extant E. coli groups is largely limited to only three intergroup pairings. Conclusions Segment-dependent phylogenies most likely are legacies of a complex recombination history. However, E. coli are now in an epoch in which they no longer broadly share DNA. Using the definition of species as organisms that freely exchange genetic material, this recombinational dormancy could reflect either the end of E. coli as a species, or herald the coalescence of E. coli groups into new species.

  19. The chemoenzymatic synthesis of clofarabine and related 2′-deoxyfluoroarabinosyl nucleosides: the electronic and stereochemical factors determining substrate recognition by E. coli nucleoside phosphorylases

    Directory of Open Access Journals (Sweden)

    Ilja V. Fateev

    2014-07-01

    Full Text Available Two approaches to the synthesis of 2-chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyladenine (1, clofarabine were studied. The first approach consists in the chemical synthesis of 2-deoxy-2-fluoro-α-D-arabinofuranose-1-phosphate (12a, 2FAra-1P via three step conversion of 1,3,5-tri-O-benzoyl-2-deoxy-2-fluoro-α-D-arabinofuranose (9 into the phosphate 12a without isolation of intermediary products. Condensation of 12a with 2-chloroadenine catalyzed by the recombinant E. coli purine nucleoside phosphorylase (PNP resulted in the formation of clofarabine in 67% yield. The reaction was also studied with a number of purine bases (2-aminoadenine and hypoxanthine, their analogues (5-aza-7-deazaguanine and 8-aza-7-deazahypoxanthine and thymine. The results were compared with those of a similar reaction with α-D-arabinofuranose-1-phosphate (13a, Ara-1P. Differences of the reactivity of various substrates were analyzed by ab initio calculations in terms of the electronic structure (natural purines vs analogues and stereochemical features (2FAra-1P vs Ara-1P of the studied compounds to determine the substrate recognition by E. coli nucleoside phosphorylases. The second approach starts with the cascade one-pot enzymatic transformation of 2-deoxy-2-fluoro-D-arabinose into the phosphate 12a, followed by its condensation with 2-chloroadenine thereby affording clofarabine in ca. 48% yield in 24 h. The following recombinant E. coli enzymes catalyze the sequential conversion of 2-deoxy-2-fluoro-D-arabinose into the phosphate 12a: ribokinase (2-deoxy-2-fluoro-D-arabinofuranose-5-phosphate, phosphopentomutase (PPN; no 1,6-diphosphates of D-hexoses as co-factors required (12a, and finally PNP. The substrate activities of D-arabinose, D-ribose and D-xylose in the similar cascade syntheses of the relevant 2-chloroadenine nucleosides were studied and compared with the activities of 2-deoxy-2-fluoro-D-arabinose. As expected, D-ribose exhibited the best substrate

  20. Stimulation of Escherichia coli F-18Col- Type-1 fimbriae synthesis by leuX

    DEFF Research Database (Denmark)

    Newman, Joseph V.; Burghoff, Robert L.; Pallesen, Lars

    1994-01-01

    Escherichia coli F-18, a normal human fecal isolate, is an excellent colonizer of the streptomycin-treated mouse large intestine. E. coli F-18Col-, a derivative of E. coli F-18 which no longer makes the E. coli F-18 colicin, colonizes the large intestine as well as E. coli F-18 when fed to mice a...